Amended Safety Assessment of

Benzophenones as Used in Cosmetics

Status: Tentative Amended Report for Public Comment

Release Date: September 25, 2020
Panel Date: December 7-8, 2020

All interested persons are provided 60 days from the above release date (i.e., November 24, 2020) to comment on this
safety assessment and to identify additional published data that should be included or provide unpublished data which
can be made public and included. Information may be submitted without identifying the source or the trade name of
the cosmetic product containing the ingredient. All unpublished data submitted to the Cosmetic Ingredient Review
(CIR) will be discussed in open meetings, will be available at the CIR office for review by any interested party and
may be cited in a peer-reviewed scientific journal. Please submit data, comments, or requests to the CIR Executive
Director, Dr. Bart Heldreth

The Expert Panel for Cosmetic Ingredient Safety members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald
V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Lisa A. Peterson, Ph.D.; Ronald C. Shank,
Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. Previous Panel member involved in this
assessment: James G. Marks, Jr., M.D. The Cosmetic Ingredient Review (CIR) Executive Director is Bart Heldreth,
Ph.D. This report was prepared by Wilbur Johnson, Jr., M.S., Senior Scientific Analyst, CIR and Jinqiu Zhu, Ph.D.,
Toxicologist.

© Cosmetic Ingredient Review

1620 L Street, NW, Suite 1200 ◊ Washington, DC 20036-4702 ◊ Ph 202.331.0651 ◊ fax 202.331.0088 ◊ CIRINFO@cir-safety.org
ABSTRACT: The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of benzophenones in
cosmetic products; these ingredients are reported to function mainly as light stabilizers in cosmetic products. The
Panel reviewed relevant data relating to the safety of these ingredients in cosmetic formulations, and concluded that
Benzophenones-1, -2, -3, -4, -5, -6, -8, -9, -10, -11, and -12 are safe in cosmetics in the present practices of use and
concentration described in this safety assessment.

INTRODUCTION
The Expert Panel for Cosmetic Ingredient Safety (Panel) published a safety assessment of benzophenones with
the following conclusion in 1983: on the basis of the available animal data and clinical human experience presented
in this report, the Panel concludes that Benzophenones-1, -3, -4, -5, -9, and -11 are safe for topical application to
humans in the present practices of use and concentration in cosmetics.1 During the same year, the Panel also
published an addendum to this published safety assessment, having concluded that Benzophenones-2, -6, and -8 are
not mutagenic or genotoxic and that the published conclusion on Benzophenones-1, -3, -4, -5, -9, and -11 is
applicable to these 3 ingredients.2 In accordance with the Cosmetic Ingredient Review (CIR) Procedures & Support
to the Expert Panel for Cosmetic Ingredient Safety document, the Panel evaluates the conclusions of previously-
issued reports every 15 years. Thus, the Panel re-evaluated the conclusion, and in 2005, published re-review
summary that stated the Panel determined to not reopen the 1983 published safety assessment until results from
National Toxicology Program (NTP) carcinogenicity studies on benzophenones are available.3
The NTP carcinogenicity study on Benzophenone-3 was published in May 2020, and results from this study
are included in the current safety assessment for the Panel’s review. Other safety test data on Benzophenone-3, as
well as data on other benzophenones that have been identified in the published literature since the original safety
assessment was published in 1983, are also included. Data from the published CIR safety assessments on
benzophenones appear, in italics, at the beginning of sections in the report text. (This information is not included in
Summary section.) For complete and detailed information, please refer to the original documents, which are
available on the CIR website (https://www.cir-safety.org/ingredients).
According to the web-based International Cosmetic Ingredient Dictionary and Handbook (wINCI;
Dictionary), the benzophenones reviewed in this safety assessment function mainly as light stabilizers in cosmetic
products, but also as sunscreens (see Table 1).4 In the United States, sunscreens are active ingredients in over-the-
counter (OTC) drug products, and are not cosmetic ingredients (21 CFR 352.10). Furthermore, in the United States,
Benzophenone-3 functions as a light stabilizer, but not as a sunscreen, in cosmetic products.4 In Europe,
Benzophenone-3 is permitted in cosmetics at concentrations up to 0.5% to protect formulations from
photodegradation, and at concentrations up to 6% as a sunscreen ingredient.5 It is important to note that sunscreens
are classified as cosmetics in Europe, but not in the United States. In Hawaii, a law banning the sale of any
sunscreen containing Benzophenone-3 without a prescription issued by a licensed healthcare provider will go into
effect on January 1, 2021 (HI SB2571). The reason for the ban relates to purported environmental toxicity. It is
within the Panel’s purview to review cosmetic ingredients in relation to human health and safety, but not for
environmental safety.

The published data in this document were identified by conducting an exhaustive search of the world’s
literature from year 1983 forward. A list of the typical search engines and websites used, sources explored, and
endpoints that Panel evaluates, is available on the CIR website (https://www.cir-safety.org/supplementaldoc/
preliminary-search-engines-and-websites; https://www.cir-safety.org/supplementaldoc/cir-report-format-outline).
Unpublished data may be provided by the cosmetics industry, as well as by other interested parties. Dossiers for
Benzophenones-1, -3, -4, -8, and -12 were found on the European Chemicals Agency (ECHA) website.6-10 The
ECHA website provides summaries of information generated by industry, and it is those summary data that are
reported in this safety assessment when ECHA is cited.

CHEMISTRY
Definition and Structure
Benzophenones-1 to -12 are substituted derivatives of 2-hydroxybenzophenone.1 Substituents include hydroxy,
methoxy, octyloxy, sulfonyl, methyl, and chloride groups. Benzophenones may be mono-, di-, tri-, or tetra-
substituted.
 Definitions, CAS numbers, and individual structures of the benzophenones included in this report are presented
in Table 1.
Chemical Properties
An important property of benzophenones is their ability to absorb and dissipate ultraviolet (UV) radiation.1
Most benzophenones are solid at room temperature, soluble in organic solvents, and insoluble in water.
Properties of benzophenones are presented in Table 2.1,11
Method of Manufacture
The most common method of production of benzophenones is the Friedel-Crafts reaction.1 No further
manufacturing information, specific to the cosmetic ingredients, has been found in the published literature or
submitted as unpublished data.
Composition/Impurities
Values for the maximum moisture content of benzophenones have been reported as follows: Benzophenone-1
(2%), Benzophenone-2 (5%), Benzophenone-3 (13%), Benzophenone-4 (10% to 16%, trihydrate form),
Benzophenone-6 (0.5%), Benzophenone-8 (2%), Benzophenone-9 (5%), and Benzophenone-11 (5%).1
A maximum concentration of 1 ppm arsenic as an impurity has been recommended for Benzophenones-1, -2, -
3, -4, -6, -9, and -11.1 The following maximum concentrations for lead as an impurity in benzophenones have been
recommended: Benzophenone-1 (18 ppm), Benzophenone-2 (8 ppm), Benzophenone-3 (13 ppm), Benzophenone-4
(18 ppm), Benzophenone-6 (13 ppm), Benzophenone-9 (8 ppm), and Benzophenone-11 (13 ppm).

USE
Cosmetic
The safety of the cosmetic ingredients included in this report is evaluated based, in part, on data received from
the United States (US) Food and Drug Administration (FDA) and the cosmetics industry on the expected use of this
ingredient in cosmetics. Use frequencies of individual ingredients in cosmetics are collected from manufacturers
and reported by cosmetic product category in the FDA Voluntary Cosmetic Registration Program (VCRP)
database.12 Use data are submitted by the cosmetics industry in response to surveys, conducted by the Personal Care
Products Council (Council), of maximum reported use concentrations by product category.13 The concentration of
use survey on benzophenones was conducted for ingredient use as a light stabilizer, but not as a sunscreen.
In the 1983 original report, Benzophenone-2 was the benzophenone with the highest reported use frequency
(229 uses total).1 In 2020, Benzophenone-4 was the benzophenone with the highest reported use frequency (2259
uses total).10 The use frequency of Benzophenone-2 (299 uses total) in the 1983 original report decreased to a value
of 103 in 2020. The use frequency of Benzophenone-4 (240 uses) in the 1983 original report increased substantially
to a value of 2259 in 2020. Of the ingredients reviewed in the1983 report, Benzophenone-4 had the highest use
concentration (≤ 10% in suntan gels, creams and liquids (leave-on products)).1 In 2020, Benzophenone-4 is the
benzophenone with the highest reported use concentration, and is used at substantially lower concentrations of up to
1.6% in other non-coloring hair preparations (leave-on products).13 Frequency and concentration of use data are
presented in Table 3.
According to VCRP and Council survey data, the following 6 ingredients are not currently in use in cosmetic
products: Benzophenone-6, Benzophenone-8, Benzophenone-10, Benzophenone-11, and Benzophenone-12.
Cosmetic products containing benzophenones may be applied to the skin or, incidentally, may come in contact
with the eyes (e.g., Benzophenone-4 in eye makeup preparations at concentrations up to 0.2%). Benzophenone-3 is
used in products that come in contact with mucous membranes during product use (maximum ingredient use
concentrations of 0.05 to 0.5% in bath soaps and detergents). Additionally, Benzophenone-3 could be incidentally
ingested (maximum use concentrations up to 0.5% in lipstick). In baby products, Benzophenone-3 is being used at
maximum concentrations up to 0.25% (in baby lotions, oils, and creams (not powder)). Products containing
benzophenones may be applied as frequently as several times per day and may come in contact with the skin for
variable periods following application. Daily or occasional use may extend over many years.
Benzophenone-3 is being used in aerosol hair spray (maximum concentration of 0.014%), pump hair spray
(maximum concentration of 0.05%), and in pump deodorant spray (at maximum concentration of 0.08%). A higher
concentration (0.48%) is stated for perfumes, but not all perfumes are spray products. Thus, there is uncertainty that
the 0.48% concentration is for a spray product. Benzophenone-4 is also being used in aerosol hair spray (maximum
concentration of 0.015%) and pump hair spray (maximum concentrations of 0.001% to 0.1%). In practice, 95% to
99% of the droplets/particles released from cosmetic sprays have aerodynamic equivalent diameters > 10 µm, with
propellant sprays yielding a greater fraction of droplets/particles below 10 µm, compared with pump sprays.14-17
Therefore, most droplets/particles incidentally inhaled from cosmetic sprays would be deposited in the
nasopharyngeal and bronchial regions and would not be respirable (i.e., they would not enter the lungs) to any
appreciable amount.14,15 There is some evidence indicating that deodorant spray products can release substantially
larger fractions of particulates having aerodynamic equivalent diameters in the range considered to be respirable.15
However, the information is not sufficient to determine whether significantly greater lung exposures result from the
use of deodorant sprays, compared to other cosmetic sprays. Benzophenone-3 is also being used in face powders
(use concentrations unknown). Conservative estimates of inhalation exposures to respirable particles during the use
of loose powder cosmetic products are 400-fold to 1000-fold less than protective regulatory and guidance limits for
inert airborne respirable particles in the workplace.18-20
Benzophenone-3, Benzophenone-4, and Benzophenone-5, but not the other benzophenones in this safety
assessment, are included on the European Union’s list of ultraviolet light (UV) filters allowed in cosmetic products.5
A maximum concentration of 6 % Benzophenone-3 (as a UV filter) is allowed in ready for use preparations. Not
more than 0.5% Benzophenone-3 is allowed to protect the product formulation. Benzophenone-4 and
Benzophenone-5 are allowed in ready for use cosmetic preparations at concentrations up to 5% (as acid).
Non-Cosmetic
According to the US FDA, the following benzophenone are allowed in sunscreens as active ingredients within
the concentration specified for each ingredient: Benzophenone-3 (a.k.a. oxybenzone, up to 6%), Benzophenone-4
(a.k.a. sulisobenzone, up to 10%), and Benzophenone-8 (a.k.a. dioxybenzone, up to 3%) (21 CFR 352.10). on
February 26, 2019, the FDA published a proposed rule to establish final monograph regulations for over-the-counter
(OTC) sunscreen drug products (84 FR (38) 6204).21 The rule proposes that the following 3 benzophenones would
be excluded from the final monograph because there are insufficient data to determine whether they are generally
recognized as safe and effective (GRASE): Benzophenone-3, Benzophenone-4, and Benzophenone-8. Particularly,
given the available data showing significant transdermal absorption and systemic availability of Benzophenone-3, as
well as the potential for endocrine activity, FDA proposes that Benzophenone-3 is not GRASE for use in sunscreens
without further data. FDA has determined that the following data on Benzophenone-3 are needed: human
absorption data (including metabolite study in humans); non-clinical safety studies (toxicokinetics, dermal
carcinogenicity, and systemic carcinogenicity); developmental and reproductive toxicity (if developmental and
reproductive toxicity (DART) studies do not resolve the concerns raised in the literature relating to potential
endocrine disruption, it may be possible to resolve these concerns through additional testing); and FDA is seeking
input on whether additional studies or contraindication are necessary to support the safety of sunscreens containing
Benzophenone-3 for children under 2 years of age. FDA has determined that the following data on Benzophenone-4
and Benzophenone-8 are needed: dermal irritation and sensitization testing; phototoxicity and photoallergenicity
testing; human maximal use bioavailability studies; post-marketing adverse event reports; dermal carcinogenicity;
systemic carcinogenicity; DART; toxicokinetics; and additional testing when data suggest a concern about other
long-term effects, such as endocrine effects.
According to the proposed rule, FDA expects that a systemic carcinogenicity study would not be needed to
support a GRASE determination for a sunscreen active ingredient if an adequately conducted human
pharmacokinetic maximal use trial (MUsT) resulted in a steady state blood level less than 0.5 ng/ml, and an
adequately conducted toxicology program did not reveal any other safety signals for the ingredient or any known
structurally similar compound indicating the potential for adverse effects at lower levels. The threshold value of 0.5
ng/ml is based on the assessment that the level would approximate the highest plasma level below which the
carcinogenic risk of any unknown compound would be less than 1 in 100,000 after a single dose.
Benzophenone-3 is among the substances listed by FDA as indirect food additives (substances for use as basic
components of single and repeated use food contact surfaces) (21CFR177.1010). Furthermore, Benzophenone-12
may be safely used as an antioxidant and/or stabilizer in polymers used in the manufacture of articles or components
of articles intended for use in producing, manufacturing, packing, processing, preparing, treating, packaging,
transporting, or holding food, subject to the following limitations (21CFR178.2010): For use only at levels not to
exceed 0.5% by weight of olefin copolymers complying with section 177.1520 (c).
 TOXICOKINETIC STUDIES
Dermal Penetration
In Vitro
Benzophenone-3
Sunscreen products were applied to excised human epidermis in Franz diffusion cells, with the amount
penetrating into and across the human epidermis assessed by high performance liquid chromatography (HPLC) for 8
h following application.22 The sunscreen agents in the products were: Benzophenone-3, octyl methoxycinnamate,
octyl salicylate, and octocrylene. All sunscreen agents investigated penetrated into the skin (0.25 g/m2 or 14% of
applied dose). Only Benzophenone-3 penetrated human skin to the receptor phase (0.08 g/m2 or 10% of applied
dose) after the 8-h study period.
The penetration of Benzophenone-3 across excised human epidermis and high-density polyethylene (HDPE)
membrane was measured using in vitro Franz-type diffusion cells.23 Human epidermal tissue (abdominal region of 1
female) was obtained by blunt dissection of full-thickness skin and heat separation. The tissue was mounted
between the donor and receptor chambers of the diffusion cell, and the surface area available for diffusion was 1.18
cm2. The receptor chamber volume was 3.4 ml, and the receptor fluid was bovine serum albumin (4%) in
phosphate-buffered saline. Both penetration and epidermal retention were measured following application of
infinite and finite (epidermis only) doses of Benzophenone-3 (2%) in the following 5 vehicles: liquid paraffin (LP),
coconut oil (CO), 50:50 ethanol:coconut oil (50:50 EC), aqueous cream (AC), and oily cream (OC). For the infinite
dose studies, an aliquot (200 mg/cm2) of each formulation was applied to the epidermal surface under occlusion.
For the finite dose studies, an aliquot of each formulation (20 mg/cm2) was applied without occlusion.
Benzophenone-3 remaining in the epidermis (Rs, µg) was extracted twice with methanol and quantified using HPLC.
The highest Benzophenone-3 skin retention was observed for the 50:50 EC combination. Maximal and minimal
Benzophenone-3 fluxes were observed from liquid paraffin and coconut oil, respectively.
In the infinite dose study, statistically significant differences existed between all 5 formulations with respect to
penetration of Benzophenone-3 across HDPE membrane, after application of an infinite dose in a range of
formulation vehicles. The order of flux (highest to lowest) was: LP > OC > 50:50 EC > CO. For Benzophenone-3
penetration across epidermal membrane, LP was greater than 50:50 EC; however, the difference between the 2
vehicles was not statistically significant. For the remaining vehicles, the order of flux (high to low) was OC > AC >
CO. Statistically significant differences (p < 0.05) existed between the Benzophenone-3 fluxes across the epidermis
for these formulation vehicles.
In the finite dose study (mimicking the real-life situation), the percentage of applied Benzophenone-3 dose
absorbed ranged between 1.97% from CO and 9.97% from LP. A comparison of the maximum amount of
Benzophenone-3 that penetrated indicated that LP and CO statistically significantly differed from each other and the
remaining formulations. The highest Benzophenone-3 skin retention was observed for 50:50 EC. The alcohol-
based vehicle showed low Benzophenone-3 release from the vehicle, but high skin penetration and retention. The
authors concluded that sunscreen chemicals applied to the skin are substantially retained in the superficial layers of
the stratum corneum. They also noted that the results of this study also indicated that the release and skin
penetration of Benzophenone-3 was influenced by the formulation vehicle in which it was applied to the membrane.
A study was performed to investigate whether long-wavelength UV (UVA; maximum wavelength from lamp =
365 nm) and mid-wavelength UV (UVB; maximum wavelength from lamp = 312 nm) affect the absorption of
Benzophenone-3 through the skin.24 The dorsal skin of female nude mice (ICR-Foxn/nu strain) was subjected to
UVA (24 and 39 J/cm2) or UVB (150, 200, and 250 mJ/cm2) irradiation. UVA irradiation was performed every
other day, and each mouse was exposed 3 times over a 5-d period. UVB irradiation was performed once a day for 5
d. The interval between each UVB irradiation was 24 h. Irradiated skin was excised from the mouse (back)
immediately after the last UV exposure. Senescent skin (24 wk old) was used for comparative purposes. In vitro
skin absorption was evaluated using a Franz cell. The donor compartment was filled with Benzophenone-3 (3.5
mg/ml in 30% ethanol/double distilled water). The receptor was loaded with 30% ethanol in pH 7.4 buffer. The
duration of the experiment was 48 h. When compared to intact skin, a negligible change in skin absorption after
UVA exposure was found, though there was a slight increase in flux at a dose of 24 J/cm2. UVB exposure resulted
in a decrease in skin deposition of Benzophenone-3 (statistically significant (p < 0.05) at dose of 250 mJ/cm2); no
statistically significant decrease was detected at doses of 150 and 200 mJ/cm2. UVB exposure at doses of 200 and
250 mJ/cm2 caused a slight, but statistically significant (p < 0.05) enhancement of Benzophenone-3 flux. The values
for Benzophenone-3 flux were: 11.92 ± 0.74 µg/cm2/h (normal), 14.05 ± 0.17 µg/cm2/h (UVA at 24 J/cm2), 12.02 ±
0.11 µg/cm2/h (UVA at 34 J/cm2), 8.04 ± 1.40 µg/cm2/h (UVB at 150 mJ/cm2), 13.98 ± 0.06 µg/cm2/h (UVB at 200
mJ/cm2), and 20.73 ± 0.03 µg/cm2/h (UVB at 250 mJ/cm2). The skin absorption parameters of intrinsically aged
skin and young skin were comparable.
Benzophenone-3 (10% in water-in -oil or oil-in-water emulsion) was evaluated in a skin penetration study
involving full-thickness pig ear skin in vitro.25 The skin permeation of Benzophenone-3 (in water-in-oil emulsion)
was described as rapid, i.e., after 1 h of skin exposure to 2 mg/cm2. After 1 h, skin permeation was ≥ the limit of
quantification (0.615µg/cm2). Approximately 0.5% of the applied dose passed into the receptor fluid (phosphate-
buffered saline). The absorption rate was higher from the water-in-oil emulsion than from the oil-in-water emulsion.
After 24 h of skin exposure, the amount of Benzophenone-3 that passed through the frozen-stored skin was 27.2 ±
1.3 µg/cm2 (from water-in-oil emulsion) and 22.1 ± 1.1 µg/cm2 (oil-in-water emulsion). Additionally, after 24 h of
exposure, the amount of Benzophenone-3 that passed through fresh skin was 22.4 ± 0.9 µg/cm2 (from water-in-oil
emulsion) and 17.6 ± 0.8 µg/cm2 (from oil-in-water emulsion).
Benzophenone-3 and Benzophenone-4
Static diffusion cells were used to evaluate the skin penetration of Benzophenone-3 and Benzophenone-4 in
vitro.26 Human skin from abdominal or breast surgery was used. The mean amount found in the receptor fluid was
1.0 ± 0.4 µg/cm2 for Benzophenone-3, compared to 1.1 ± 0.8 µg/cm2 for caffeine (known as a good penetrating
compound). The amount of Benzophenone-4 in the receptor fluid was below the limit of detection.
The percutaneous absorption of Benzophenone-3 and Benzophenone-4 (each in an oil-in-water emulsion) was
evaluated in vitro using fresh human skin of women who had undergone breast or abdominal surgery.27 The skin
(epidermal side up) was positioned on the lower part of the diffusion cell, and 3 mg/cm2 of test formulation applied.
Exposure times of 30 min and 16 h were observed. For Benzophenone-3, there was no difference between the
mean quantity found in the stratum corneum after 30 min or 16 h. These results indicate that Benzophenone-3
penetrated very quickly and saturated the stratum corneum in less than 30 min. For Benzophenone-4, the quantity
found after 30 min (2.1 ± 1.3 µg/cm2) was statistically significantly less than that found after 16 hours (4.0 ± 1.8
µg/cm2). Benzophenone-4 was found in the skin and receptor fluid.
Animal
Benzophenone-3
A study was performed to investigate whether UVA (maximum wavelength from lamp = 365 nm) and UVB
(maximum wavelength from lamp = 312 nm) affect the absorption of Benzophenone-3 through the skin.24 The
dorsal skin of female nude mice (ICR-Foxn/nu strain, groups of 4) was subjected to UVA (24 and 39 J/cm2) or UVB
(150, 200, and 250 mJ/cm2). UVA irradiation was performed every other day, and each mouse was exposed 3 times
over a 5-d period. UVB irradiation was performed once a day for 5 d. The interval between each UVB irradiation
was 24 h. A glass cylinder (available diffusion area = 0.785 cm2) was glued onto the mouse dorsal region. A 0.2 ml
aliquot of Benzophenone-3 (3.5 mg/ml in 30% ethanol/double distilled water) was pipetted into the cylinder. The
application time was 6 h, after which the skin was excised. Senescent skin (24 wk old) was treated similarly for
comparative purposes. When compared to non-irradiated skin, a statistically significant (p < 0.05) reduction in the
in vivo skin deposition of Benzophenone-3 was observed following UVB irradiation. There were no statistically
significant differences in Benzophenone-3 uptake between non-irradiated skin and skin irradiated with UVA.
Benzophenone-3 uptake levels between young and old skin were comparable.
Human
Benzophenone-3
The skin penetration of Benzophenone-3 was evaluated in vivo using 6 healthy volunteers (mean age = 37.3 ±
7.7 years) who were free of any dermatological disorders.28 In the first step, the percentage absorption was
measured using an occlusive and difference method. A solution consisting of 0.5 mg of Benzophenone-3 in 10 µl of
acetone (2190 nmol) was applied. Following Benzophenone-3 application, any residual formulation was washed
off, and the amount removed and analyzed. In the second step, the tape stripping method (a useful procedure for
selectively removing the skin’s outermost layer, the stratum corneum, and measuring the stratum corneum
adsorption) was performed. Benzophenone-3 [1000 nmol in 20 µl of ethylene glycol:triton X100 (90:10 v/v)] was
applied to the surface of the skin. The human skin permeation of Benzophenone-3 over a period of 4 h was near
35% of the applied dose with the occlusive method. The amount of topically applied Benzophenone-3 found in the
stratum corneum after 30 minutes of exposure using the stripping procedure was evaluated at 4% of the applied
dose.
A human study performed (5 males, 7 females) was a crossover design with sunscreen application to the face
or back on day 1, followed by application to the other side on day 8 of the study.23 A sunscreen lotion with the
following composition was applied at a rate of 2 mg/cm2 to an equal-sized area (112 cm2) on the face or back of the
volunteers: 8% (w/v) homosalate, 7.5% (w/v) octyl methoxycinnamate, 6% (w/v) Benzophenone-3, and 5% (w/v)
octyl salicylate. The sunscreen lotion remained occluded for 8 h before it was removed by washing. An area of the
skin was immediately tape-stripped using clear tape (3 cm x 1.9 cm). The stratum corneum was sequentially
stripped 16 times on the back and 6 times on the face. Sunscreen content in all samples was analyzed. Urine
output over 48 h post-application was collected. Blood samples were obtained before and after application. A
substantial amount of all sunscreen chemicals in the stratum corneum of the back was noted after 8h. Greater
amounts of sunscreen were present in the superficial layers (ranging from ~4% to 10% of the applied dose) than in
the deeper layers. Approximately 2 to 4 times the amount of sunscreen was present in the superficial stratum
corneum layers of the face, when compared to the back. The difference in absorption between the anatomical sites
was statistically significant for Benzopeonone-3, octyl salicylate, and homosalate only. The percentage of applied
dose in the 6 superficial layers of the stratum corneum was ~10%, 18%, 18%, and 25% for homosalate, octyl
methoxycinnamate, Benzophenone-3, and octyl salicylate, respectively. Sunscreens were not detected in the plasma
or urine samples.

Benzophenone-4
Benzophenone-4 (in water; 6 mg/ml) was deposited on the skin of each of 21 healthy women (22 to 34 years
old; mean age = 25 ± 3 years).29 Twenty µl of solution were applied. Skin strippings were performed at 1 to 7 h
after treatment. The stratum corneum was removed (with transparent adhesive tape) by a series of 6 strippings.
After 1 h, and for the first strip, 70% of the Benzophenone-4 remained at the level of the stratum corneum
(compared to 40% for PEG-25 PABA [para-aminobenzoic acid]). At 7 hours, 40% of the Benzophenone-4
remained at the level of the stratum corneum (compared to 20% for PEG-25 PABA).
Absorption, Distribution, Metabolism, and Excretion (ADME)
In Vitro
Benzophenone-2
The fate of Benzophenone-2 was studied in human and zebrafish in vitro cell models.30 In the human in vitro
cell models, Benzophenone-2 was metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar
patterns of Benzophenone-2 biotransformation were observed among zebrafish models (primary hepatocytes, ZFL,
and ZELH-zfER cell lines). Metabolic patterns in the zebrafish models and human hepatic cell line HepaRG shared
many similarities, while biotransformation rates in the cell lines MELN (human female cancer (invasive ductal
carcinoma) cell line) and T47D-KBLuc (human female cancer (mammary gland breast/duct) cell line) were
quantitatively low and qualitatively different.
Benzophenone-3
Benzophenone-3 (0.1 µmol) was incubated for 15 min with liver microsomes from untreated Sprague-Dawley
rats in the presence of NADPH (1 µmol).31 2,5-Dihydroxy-4-methoxybenzophenone, metabolite of Benzophenone-
3, was formed. Another metabolite, 2,4-dihydroxybenzophenone (Benzophenone-1, the 4-desmethylated
metabolite), was also formed. The amount of 2,5-dihydroxy-4-methoxybenzophenone formed in vitro was
approximately the same as 2,4-dihydroxybenzophenone. Data on the specific amount of each metabolite were not
included.
The metabolism of Benzophenone-3 by rat and human liver microsomes was studied.32 When Benzophenone-
3 (10 µM) was incubated for 15 min with rat liver microsomes in the presence of NADPH, the following metabolites
resulted: 2,4,5-triydroxybenzophenone; 3-hydroxylated benzophenone-3; 5-hydroxylated benzophenone-3;
Benzophenone-1; and 2,3,4-trihydroxybenzophenone. Benzophenone-3 was also metabolized by human liver
microsomes, yielding Benzophenone-1 and 5-hydroxylated benzophenone-3.
Animal
Dermal
Benzophenone-2
A study was performed, using groups of 10 male Wistar rats, to determine the concentrations of
Benzophenone-2 in the rat brain after topical administration.33 Benzophenone-2 was dissolved in a small amount
(volume not stated) of ethanol and olive oil and formulated with Hascobase. The test substance was then applied to
shaved skin at a dose of 100 mg/kg for 4 wk. Hascobase, with a small amount of ethanol and olive oil, was applied
to the skin of control rats. Blood and tissue Benzophenone-2 concentrations in the frontal cortex and hippocampus
were determined. After dermal application (24 h after last dose at 4 wk), the blood level of Benzophenone-2 was
~300 ng/ml. Liver and adipose tissue concentrations were 1354 ng/g wet tissue and 823 ng/g wet tissue,
respectively. In the brain structures studied, the Benzophenone-2 concentration ranged from 5 to 19 ng/g tissue. In
the hippocampus, the Benzophenone-2 concentration was approximately 3.5-fold lower in the frontal cortex.

To assess the concentration of total Benzophenone-2 (parent compound and its metabolites – glucuronide and
sulfate), the liver was homogenized and plasma was mixed with 1 M ammonium acetate buffer. Prior to incubation
of the homogenate for 6 h, freshly prepared enzyme mixtures (β-glucuronidase and sulfatase) were added. After
hydrolysis with β-glucuronidase and sulfatase, the Benzophenone-2 peak was significantly higher than in the same
serum and liver samples before hydrolysis. Calculation of the Benzophenone-2 concentration from the calculated
standard curves revealed that the test compound was present in the plasma of treated animals at concentrations
ranging from 164 to 648 ng/ml (average = 324 ng/ml; 1.3 µM). After hydrolysis, the Benzophenone-2 concentration
was 2218 ng/ml (9 µM). These results indicated that, in the blood, the there was more of the Benzophenone-2
metabolites than the parent compound. Additionally, in the liver, the Benzophenone-2 concentration after
hydrolysis was much higher (3758 ng/g) when compared to the free form of the compound (1482 ng/g).
Benzophenone-2 concentrations in all examined tissues in control animals were below the detection limit. The
authors noted that the results of this study indicate that Benzophenone-2 passes through the blood-brain barrier, but
that its concentration in the brain structures is much lower than in the blood.
Benzophenone-3
A study was performed to characterize the skin permeation and tissue disposition of Benzophenone-3 (in
ethanol) in rats (groups of 10; 5 males and 5 females per group).34 The test solution was applied (volume = 100 µl;
dose = 5 mg/kg [312.5 µg/cm2]) topically to a 4 cm2 area on the back, daily, for 30 d. Two negative control groups
received topical applications of 0.9% saline and 70% ethanol solution for 30 d. The positive control group received
an intraperitoneal (i.p.) dose (25 mg/kg) of acrylamide for 10 d. Tape stripping was used to recover the application
dose that permeated into skin layers. Benzophenone was recovered in appreciable amounts from the application
sites. Quantifiable amounts of Benzophenone-3 were detected in plasma samples, indicating systemic absorption
from the skin. Benzophenone-3 was also detected in the brain and liver (the only tissues collected). The authors
noted that Benzophenone-3 primarily undergoes metabolism in the liver and is subsequently excreted in the urine.
The elimination half-life of Benzophenone-3 was estimated to be 7.9 ± 1.7 h. It was measurable 24 h after skin
application. The authors concluded that the results of this study indicate that Benzophenone-3 penetrated across the
skin after a 30-d topical application, and that systemic absorption was correlative among skin, plasma, and tissue
samples.
The percutaneous absorption of Benzophenone-3 and its metabolite (Benzophenone-1) was studied using
female Sprague-Dawley rats and their offspring.35 Benzophenone-3 (10% in cream; dose = 100 mg/kg) was
administered dermally (shaved skin on back) twice daily to adult female rats during the prenatal period and
adulthood. Control female rats were treated with cream without Benzophenone-3. At 21 d after birth, the offspring
(male and female) were divided into groups of 5 males and groups of 5 females. From 43 to 56 d age, the test
substance was administered dermally to the male offspring. Cream without Benzophenone-3 was applied to control
offspring. The calculation of Benzophenone-3 concentrations from the standard calibration curves revealed that the
test substance was present in the plasma of treated animals at a concentration of 215.9 ± 38.5 ng/ml. The
concentration of Benzophenone-3 in the liver was 96.81 ± 17.3 ng/g wet tissue. Higher concentrations of
Benzophenone-1 (main metabolite, 196.4 ± 67.5 ng/g wet tissue) were also detected in the liver. Only the parent
compound was detected in the frontal cortex and hippocampus of the brain at concentrations of 50.6 ± 11.0 and 46.7
± 14.4 ng/g wet tissue, respectively. The authors stated that the results of this experiment showed that
Benzophenone-3 is absorbed through rat skin and passes through the blood brain barrier.
Benzophenone-3 (10%), at a dose of 100 mg/kg, was applied to the backs of mated Sprague-Dawley rats
(number not stated) twice daily.36 A cream without Benzophenone-3 was applied to control rats. At 21 d after birth,
the offspring were weaned and organized into groups of 5 (males separated from females). From 43 to 56 d of age,
the female offspring of test animals received dermal applications of Benzophenone-3 (10%). A cream without
Benzophenone-3 was applied to control offspring. At 24 h after the last test substance application, the animals were
killed, and the brains and livers were excised. In the plasma of all control rats, the concentration of Benzophenone-3
was below the limit of detection. In the plasma of test animals, Benzophenone-3 was detected in the range of 70 to
220 ng/ml (average = 169 ng/ml). A much higher concentration of the main Benzophenone-3 metabolite,
Benzophenone-1, was detected in the liver (156 ng/g wet tissue), when compared to Benzophenone-3 (25 ng/g wet
tissue). After dosing with Benzophenone-3, the concentration in the frontal cortex was 26 ng/g and the
hippocampus had a concentration of 40 ng/g. Benzophenone-1 was also detected in the frontal cortex and
hippocampus. In the control group, the concentration of Benzophenone-3 in the hippocampus was above the
detection limit in only one female rat. Benzophenone-3 was not detected in the frontal cortex and liver.
The metabolism and disposition of [14C]Benzophenone-3 (formulated in different vehicles) was evaluated
using Harlan Sprague-Dawley rats (groups of 5) and B6C3F1/N mice (groups of 5).37 The vehicles used were as
follows: paraffin oil, lotion, coconut oil, ethanol:coconut oil, and ethanol. In rats, a single dose of the test substance
(0.1, 1, 10, or 15 mg/kg; dose volume = 0.5 to 1 ml/kg) was administered in most of the vehicles. When the lotion
(olive oil:emulsifying wax:water formulation) vehicle was used, the dose volume was ≈ 100 µl. Application (using
syringe equipped with needle) was made to an area of skin that was not less than 4 cm2. A foam or steel isolator was
used to protect the dermal dosing site. In mice, the dose volume was ≈ 2 ml/kg. Urine and feces were collected for
up to 72 h. The absorbed dose varied depending on the vehicle. After application of [14C]Benzophenone-3 to male
rats, the percent dose absorbed in all vehicles was high (64 % to 80%), except in the lotion vehicle where absorption
was moderate (46%). The % dose absorbed was similar following application of 0.1 mg/kg (73%) or 10 mg/kg
(80%) [14C]Benzophenone-3 formulated in paraffin oil. The absorption of [14C]Benzophenone-3 was lower in
female rats (30%, 15 mg/kg dose) than in male rats (46%, 10 mg/kg dose) after application of [14C]Benzophenone-3
in the lotion vehicle. The absorbed dose was excreted mainly via the urine (including cage rinse) (18% to 48%) and
feces (15% to 22%), with ~3% to 10% of the absorbed dose remaining in the tissues. Urinary metabolites included
Benzophenone-3, Benzophenone-3-glucuronide, Benzophenone-1, Benzophenone-1-glucuronide, and
Benzophenone-1-sulfates. Novel minor dihydroxy metabolites, including 2,5-dihydroxy-4-methoxybenzophenone,
were also detected.
The distribution of [14C]Benzophenone-3 radioactivity in tissues and excreta following dermal application to
male mice was similar between the vehicles at 10 mg/kg with the exception of acetone showing higher tissue levels.
[14C]Benzophenone-3 absorption in female mice following dermal application at 10 mg/kg lotion or 10 mg/kg
ethanol was similar to that seen in males. The unabsorbed dose in female mice was ~41%, with the majority of
radioactivity recovered in urine and feces at a 10 mg/kg dose in lotion.
Oral
Benzophenone-3 and Benzophenone-12
An absorption study on Benzophenone-3 involving rats, and absorption studies on Benzophenone-12 involving
rats and rabbits were performed.1 When ingested, absorbed benzophenones were primarily conjugated and
excreted in the urine, while the unabsorbed material passed out with the feces.
Benzophenone-2
A dose-response experiment involving 5 doses (10, 33, 100, 333, or 1000 mg/kg) of Benzophenone-2 was
performed using female Sprague-Dawley adult, ovariectomized (ovx) rats (groups of 5).38 Doses were administered
(by gavage) once per day for 5 d. Additionally, the time-dependent metabolism and excretion of Benzophenone-2
were analyzed in a kinetic experiment, for further identification of metabolites. In this kinetic experiment, urine and
serum samples were analyzed after Helix pomatia glucuronidase-/sulfatase (HPG) hydrolysis. Serum concentrations
of Benzophenone-2 after dosing ranged from 0.1 µg/ml (after 10 mg/kg dose) to 1.1 µg/ml (after 1000 mg/kg dose).
After hydrolysis with HPG, the serum concentrations of total Benzophenone-2 ranged from 1 to 62 µg/ml.
Benzophenone-2 was metabolized to glucuronide- and sulfate-conjugates. In the serum, the maximum
concentrations of Benzophenone-2, benzophenone-2-glucuronide, and benzophenone-2-sulfate were observed at 30
min post-dosing. The highest concentrations of Benzophenone-2 and its metabolites in the urine were measured at
20 min post-dosing. It was suggested that this biotransformation occurs via a first-pass effect in the gut wall or the
liver.
Benzophenone-3
The toxicokinetics and metabolism of Benzophenone-3 was evaluated using groups of 7 male Sprague-Dawley
rats.39 Benzophenone-3 (in corn oil) was administered orally at a dose of 100 mg/kg (dose volume = 4 ml/kg).
Blood samples were collected at various time points up to 24 h after dosing. Benzophenone-3 was converted into
Benzophenone-1, which was formed via o-demethylation. Benzophenone-1 was subsequently converted to
2,3,4-trihydroxybenzophenone. Benzophenone-3 was also metabolized to 2,2'-dihydroxy-4-methoxybenzophenone,
which was formed via the aromatic hydroxylation of Benzophenone-3. After a single oral dose of Benzophenone-3,
the toxicokinetics curve showed a peak concentration (Cmax) of 21.21 ± 11.61 µg/ml within 3 h (Tmax), and then
declined rapidly. The concentrations of the metabolites in rat blood decreased much more slowly over time, when
compared to the parent compound.
Groups (5 animals per group) of mated female Sprague-Dawley rats were fed the following Benzophenone-3
concentrations (in low-phytoestrogen chow) from gestation day 6 until weaning on postnatal day 23: 1000; 3000;
10,000; 25,000; or 50,000 ppm.40 Serum concentrations of Benzophenone-3 and its metabolites were measured.
The limit of detection for Benzophenone-3, Benzophenone-1, and Benzophenone-8 was < 0.005 µg/ml. The limit of
detection for 2,3,4-trihydroxybenzophenone was < 0.1 µg/ml or 0.05 µg/ml. Both Benzophenone-8 and
2,3,4-trihydroxybenzone were below the limits of detection. Therefore, only serum concentrations of
Benzophenone-3 and Benzophenone-1 (metabolite) were reported. In the 1000 ppm group, the mean values (on
postnatal day 23) for Benzophenone-3 and Benzophenone-1 were 0.0072 ± 0.0008 µg/ml and 0.0382 ± 0.0122
µg/ml, respectively. In the 50,000 ppm group, the mean values (on postnatal day 23) for Benzophenone-3 and
Benzophenone-1 were 0.6886 ± 0.2447 µg/ml and 1.0066 ± 0.3874 µg/ml, respectively.
The metabolism and disposition of [14C]Benzophenone-3 were evaluated using Harlan Sprague-Dawley rats
(groups of 5) and B6C3F1/N mice (groups of 5).37 A mixture of Benzophenone-3 and [14C]Benzophenone-3 (in
corn oil) was administered orally (by gavage) at a single target doses of 10, 100, or 500 mg/kg (male mice and rats),
and a single target dose of 100 mg/kg (female mice and rats). The dose volume was 5 ml/kg in rats and 10 ml/kg in
mice. The animals were killed and the following tissues and organs were collected for analysis: adrenals, brain,
lung, heart, spleen, pancreas, kidneys, testes or uterus and ovaries, liver, thyroid, thymus, small intestine, cecum,
large intestine, urinary bladder, and adipose and muscle samples. The distribution of radioactivity was reported for
tissues/organs collectively, and individually for the rat liver and kidney. In male rats, the radioactivity in tissues
increased with the increasing dose. In general, the male rat livers had a higher tissue:blood ratio (2.27 to 4.93) when
compared to the kidney (1.26 to 3.53) at 72 h post-dosing. Values for total radioactivity in the tissues of male rats at
2 h, 24 h, and 72 h after dosing with 100 mg/kg were 27.5%, 3.1%, and < 0.5%, respectively. These results suggest
that Benzophenone-3 was distributed to the tissues, but was not retained in the tissues. No sex differences (rats) in
the disposition of [14C]Benzophenone-3 following oral administration were apparent. The total dose of
[14C]Benzophenone-3 recovered in male and female rats was > 94%. After dosing with [14C]Benzophenone-3 (100
and 500 mg/kg) in male mice, it was excreted mainly in the urine (40 to 41%, including cage rinse) and feces (24 to
39%) within 72 h. The tissues with the most radioactivity in male mice were the thymus and thyroid in both 100 and
500 mg/kg dose groups. The disposition was similar in female mice 72 h following a single 100 mg/kg gavage
administration of [14C]Benzophenone-3, with ~34% and ~24% in the urine and feces, respectively. The total
radioactivity recovered in the 500 mg/kg dose group for male mice was lower (~69%) than in 100 mg/kg dose
groups for male mice (~89%) and female mice (~76%).
Overall, [14C]Benzophenone-3 was well absorbed and excreted mainly in the urine (39% to 57%) and feces
(24 % to 42%) in male and female rats and mice. The distribution of Benzophenone-3 in tissues was minimal in rats
(0.36%) and mice (< 0.55%). In male and female rats and mice, urinary metabolites included Benzophenone-3,
Benzophenone-3-glucuronide, Benzophenone-1, Benzophenone-1-glucuronide, and Benzophenone-1-sulfates.
Novel minor dihydroxy metabolites, including 2,5-dihydroxy-4-methoxybenzophenone, were also detected.
Benzophenone-12
Groups of 6 male rats (Carworth Farms Elias strain) were fed Benzophenone-12 at dietary concentrations of
1.25% and 5% daily for 35 d, in accordance with Organization for Economic Co-operation and Development
(OECD) Test Guideline (TG) 417.6 Results indicated that Benzophenone-12 had low absorption after oral feeding.
The daily recovery of unchanged material from the feces was ~90%. The conjugation and urinary excretion of the
test substance (metabolized to glucuronide conjugate) in rats fed both dietary levels was ~10% of the dose over the
35-d test period. The authors concluded that Benzophenone-12 did not have any bioaccumulation potential in this
study.
Human
Solid-phase microextraction, combined with gas chromatography-quadrupole ion trap mass spectrometry, was
used to identify Benzophenone-3 and its metabolites in human urine.41 A urine specimen was collected from a
subject after a sunscreen containing Benzophenone-3 (~ 8 ml) was applied topically to the body. The results
indicated the presence of Benzophenone-3 and its metabolite, 2,4-dihydroxybenzophenone.
Benzophenone-1, Benzophenone-2, Benzophenone-3, Benzophenone-4
Benzophenone-6, and Benzophenone-8
Eleven subjects applied a sun protecting lotion containing 4% Benzophenone-3 (40 g for average body area of
2 m2).42 The lotion was applied to most of the body, and the subjects were instructed to shower only once (i.e., after
12 h during the 48-h period). During the 48 h after application, urine samples were collected. The data indicated
that application of the lotion resulted in excretion of Benzophenone-3 for as long as 48 h post-application. The
average total amount of Benzophenone-3 excreted was 11 mg (median = 9.8 mg), which is approximately 0.4% of
the amount applied.
The systemic absorption of Benzophenone-3 was evaluated in a 2-wk, single-blinded study involving 32
healthy volunteers (15 males, 17 postmenopausal females).43 The subjects served as their own control. During the
first week, a basic cream formulation without Benzophenone-3 was applied topically (whole-body application, 2 mg
per cm2) daily for 4 d. This dose corresponded to 40 g for an average body area of 2 m2. The protocol for the
second week was the same, and involved topical application of 10% Benzophenone (in cream). Benzophenone-3
was absorbed through the skin and detected in the urine. The maximum concentration of Benzophenone-3 in the
urine was 200 ng/ml in women and 300 ng/ml in men. Results from this study also indicated that serum follicle
stimulating hormone (FSH) and luteinizing hormone (LH) in both men and women were unchanged, but statistically
significant differences in testosterone levels (decreased) were observed in men and women during the 2 wk study.
Minor differences in serum estradiol and inhibin B levels were observed in men only. It was determined that the
differences in hormone levels observed were unrelated to Benzophenone-3 exposure.
Twenty-five subjects applied a sunscreen containing Benzophenone-3 (4%), morning and night, for 5 d.44 The
25 subjects were randomly divided into 2 groups (Groups A and B). The sunscreen was applied to most of the body
(in both groups), and the subjects were allowed to shower once per day (prior to next application). Unlike Group A,
the application sites of Group B subjects were irradiated after test substance application (time varied between times
9 h and 15 h). From days 1 to 5, the UVA doses ranged from 60 J/cm2 to 100 J/cm2. The 60 J/cm2 irradiation was
for 34 min and 17 min on each side of the body. The total dose of UVA varied among participants, i.e., between
400 and 707 J/cm2. For UVB irradiation, the sites of subjects were irradiated according to Fitzpatrick skin type
(types I to III). The UVB dose was ~ 195 mJ/cm2 for 90 s, and the total UVB dose varied among participants from
0.46 to 2 J/cm2. In both groups, urine samples were obtained daily for 5 d and for 5 d after the last application. The
subjects excreted 1.2% to 8.7% (mean = 3.7%) of the total amount of Benzophenone-3 applied, and there was no
statistically significant difference between the 2 groups. The authors concluded that a large amount of
Benzophenone-3 was absorbed, and that Benzophenone-3 accumulated in the body as the subjects excreted
Benzophenone-3 five d after the last application.
A sunscreen cream containing Benzophenone-3 was applied (2 mg/cm2) to 32 subjects (15 males and 17
females), and this amount corresponded to 40 g over an average body area of 2 m2.45 Application of the sunscreen
formulation was described as a daily whole-body topical application of 10% (w/w) Benzophenone-3 for 4 d.
Showering, bathing, and swimming were not allowed until 4 h after the daily application. Blood concentrations
were measured at 0, 1, 2, 3, 4, 24, and 96 h. Urine concentrations were measured at 0, 24, 48, 72, and 96 h. Prior to
the first application, the sunscreen was not detected in the plasma or urine. The maximum median plasma
concentration of Benzophenone-3 was 187 ng/ml in females, and 238 ng/ml in males. The level of Benzophenone-3
in the urine of females was 44 ng/ml, and was 81 ng/ml in the urine of males.
Serum samples were obtained from 2 volunteers after topical application of a sunscreen cream containing 5%
Benzophenone-3.46 The cream (20 g) was applied to 1 volunteer, and the other volunteer received a 30 g
application. Each volunteer applied the cream all over the body. Blood samples were collected before and after
application at different time intervals for a period of 24 h. After application, the amount of Benzophenone-3 in the
serum increased significantly and reached a maximum concentration ranging between 6 h (200 µg/l, after 20 g dose)
and 9 h (304 µg/l, after 30 g dose). The amount of Benzophenone-3 in the serum then decreased slowly. At 24 h
after cream application, high amounts of Benzophenone-3 were present in the serum (84 µg/l, after 20 g dose; 206
µg/l after 30 g dose). Formation of the Benzophenone-8 metabolite occurred at a very small extent. The
Benzophenone-1 metabolite was detectable from the first hour after cream application, and the increase was slightly
more pronounced during the first 6 h. At 24 h post-application, the amount of Benzophenone-1 in the serum was 34
µg/l (after 20 g dose) and 102 µg/l (after 30 g dose).
A dermal absorption study on a sunscreen containing 5% Benzophenone-3 was performed using 9 adult
subjects.47 The sunscreen was applied (8 g) to the skin (arms and legs) using a glue bottle with a cotton gauze head.
Urine was collected within the next 12 h after application. Urine samples were mixed with acetate buffer solution
containing β-glucuronidase. Using HPLC, Benzophenone-3 and the following 3 metabolites were detected in the
urine: Benzophenone-1; 2,3,4-trihydroxybenzophenone; and 2,2'-dihydroxymethoxybenzophenone. The limits of
detection for Benzophenone-3 and its metabolites were 0.5 to 1 µg/ml in urine sample solution and, except for the
baseline samples, the concentrations in all samples were far above the limits.
A study was performed to determine whether active sunscreen ingredients are absorbed into the systemic
circulation.48 The study involved groups of 6 subjects (1 group per product). Study participants were enrolled from
July to August of 2018. None of the participants were using any of the sunscreen products tested in the study or
products containing any of the listed active ingredients. The sunscreen formulations containing Benzophenone-3
applied were: spray product #1 (6% Benzophenone-3), spray product #2 (5% Benzophenone-3), and lotion (4%
Benzophenone-3). Each product was applied (2 mg/cm2) to 75% of the body surface area 4 times per day for 4 d.
The subjects remained in the clinic for up to 7 d, during which time they were not exposed to direct sunlight. In
each group, 30 blood samples per person were collected over 7 d. The application of each product containing
Benzophenone-3 resulted in plasma Benzophenone-3 concentrations that exceeded 20 ng/ml on day 7. For all
participants, plasma concentrations exceeded 0.5 ng/ml within 2 h after a single application on day 1. Geometric
mean maximum plasma concentrations of Benzophenone-3 reported following product application were as follows:
209.6 ng/ml (for 6% Benzophenone spray product), 194.9 ng/ml (for 5% Benzophenone-3 spray product), and 169.3
ng/ml (for 4% Benzophenone-3 lotion). The relationship between recent, self-reported personal care product use
and ingredient (Benzophenone-3 included) concentrations in the urine was evaluated in 100 adolescent girls.49
Study participants were recruited in May to July of 2013. The use of sunscreen was associated with 57.8% higher
urinary concentrations of Benzophenone-3.
The systemic absorption and pharmacokinetics of Benzophenone-3 in sunscreen products were studied using
38 healthy participants.50 The study was conducted between January and February of 2019. The protocol and
product types were similar to that in the preceding study. Product application was described as 2 mg/cm2 to 75% of
the body surface area at 0 h on day 1, and 4 times on day 2 through day 4 at 2-h intervals. Thirty-four blood samples
were collected from each participant over 21 d. The maximum plasma concentrations of Benzophenone-3 were
258.1 ng/ml (from 4% Benzophenone-3 sunscreen lotion) and 180.1 ng/ml (from 6% Benzophenone-3 aerosol
spray). The authors concluded that Benzophenone-3 was systemically absorbed.
The dermal uptake of Benzophenone-3 from clothing was studied using 3 subjects.51 Cotton shirts (purchased
in May of 2016) were exposed to Benzophenone-3 at an elevated concentration (final concentration = 4.4 µg/m3 for
32 d). The 3 subjects wore the exposed shirts for 3 h. After the exposure period, they wore their usual clothing
during the collection of urine samples for 48 h. The rate of urinary excretion of the sum of Benzophenone -3 and
Benzophenone-1 (metabolite of Benzophenone-3) increased for all 3 subjects during and following the 3-h exposure.
The summed mass of Benzophenone-1 and Benzophenone-3 that was excreted during the first 24 h (attributable to
wearing the exposed t-shirts) were 12, 9.9, and 82 µg for the first, second, and third participant, respectively. The
authors noted that the analysis of these results, taken together with predictions of steady-state models, suggest that
dermal uptake of Benzophenone-3 from clothing could meaningfully contribute to overall body burden.
Benzophenone-3 absorption (over a 4-h period) after application of a sunscreen containing 6% Benzophenone-
3 was calculated.52 The calculation appears below:
60 g (amount of product applied/4h) x 0.06 (6% Benzophenone-3 in product) /75 kg (average weight of
women) = 0.048 g/kg or 48 mg/kg or 48 ppm/exposure
48 ppm/exposure x 0.08 (8% Benzophenone-3 absorbed topically) = 3.84 ppm or 3840 ppb absorbed over 4
h (i.e., 1 day’s exposure).
 The ratio of fetal to maternal blood levels after just 2 applications over a 4-h period of the sunscreen was 384
ppb/3840 ppb (at 10% fetal exposure) and 2880 ppb/3840 ppb (at 75% fetal exposure).
Biomonitoring Studies
Human
Benzophenone-1, Benzophenone-2, and Benzophenone-3
Benzophenones were detected in urine samples that were obtained in a study involving 20 male subjects.53 The
detection methodology was dispersive liquid-liquid microextraction (DLLME), followed by ultra-high-performance
liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Regarding method validation in terms of
linearity, a concentration range from the minimal quantified amount (limit of quantification) to 40 ng/ml was
selected for the benzophenones. Urinary concentrations were described as follows: The free form of
4-hydroxybenzophenone was detected and quantified in all samples in a range of 0.9 to 2.0 ng/ml. The conjugated
form of 4-hydroxybenzophenone was detected in 60% of the samples in concentrations ranging from 0.1 to 0.9
ng/ml. The conjugated form of Benzophenone-2 was detected and quantified in 85% of the samples in
concentrations ranging from 0.1 to 7.1 ng/ml. The free form of Benzophenone-2 was detected in all samples and
quantified in 65% of the samples, at concentrations ranging from 0.5 to 2.2 ng/ml. The conjugated form of
Benzophenone-3 was detected and quantified in almost all samples (n = 19/20) at concentrations ranging from 0.6 to
44 ng/ml. The free form of Benzophenone-3 was detected in 100% of the samples, but it was not quantified in any
of them. For Benzophenone-1, the conjugated form was detected and quantified in 100% of the samples at
concentrations ranging from 0.1 to 25 ng/ml. The free form of Benzophenone-1 was detected in 95% and quantified
in 90% of the samples in a concentration range of 1.2 to 5.7ng/ml. The authors noted that there seemed to have been
a relationship between the presence of Benzophenone-3 and Benzophenone-1 because, in all samples in which
Benzophenone-3 was present, Benzophenone-1 was also present. Furthermore, they noted that this observation is
suggestive of the possible conversion of Benzophenonoe-3 to Benzophenone-1 and that the content of
Benzophenone-1 may be due to human metabolism to and not direct exposure.
Spot urine samples (157 total) obtained from a segment of the general German population (59 females, 39
males, and 59 children) were analyzed.54 Urine samples were obtained between October of 2007 and February of
2009. Benzophenone-1 and Benzophenone-3 had high detection rates (26%). Urinary concentrations (µg/l) of the
two benzophenones were not reported. High detection rates were also reported for bisphenol A (95%) and triclosan
(45%).
A study was performed to investigate the exposure of human embryos and fetuses to UV filters.55 Placentas
(12) from volunteer mothers in Spain were collected at delivery. The presence of UV filters was analyzed by liquid
chromatography-tandem mass spectrometry (LC-MS/MS). Benzophenone-1 was detected in all samples, and at
concentrations below the method limit of quantification (MLOQ, between 0.02 and 0.07 ng/g fresh weight).
Benzophenone-3 was not detected in any sample. 4-Hydroxybenzophenone, metabolite of Benzophenone-1 and
Benzophenone-3, was detected in 3 of the 12 placental samples at a concentration (0.07 ng/g fresh weight) that was
below the MLOQ.
Urinary concentrations of benzophenones were measured in 34 Tunisian women.56 Chemical analyses were
performed using dispersive liquid-liquid microextraction and UHPLC-MS/MS. Benzophenone-1 and
Benzophenone-3 were found in 91.2% and 64.7% of the analyzed samples, respectively. Geometric mean
concentrations of Benzophenone-1 and Benzophenone-3 were 1.3 and 1.1 ng/ml, respectively.
Benzophenone-1 and Benzophenone-3 were detected in the urine of reproductive-aged women.57 A total of
143 women provided 509 spot urine samples collected across 2 months of study (3 to 5 samples per woman).
Urinary concentrations were measured and biomarker variability was characterized using the intraclass correlation
coefficient (ICC). The ICC is defined as the ratio of between-subject variance to total variance, with 95% CI. ICC
values close to 0 indicate little to no reproducibility, while values close to 1 indicate perfect reproducibility, where
most of the variance is attributed to differences between individuals as opposed to within-person differences.
Geometric mean urinary concentrations of Benzophenone-3 and Benzophenone-1 were 4.3 µg/l and 3.3 µg/l,
respectively. ICCs for Benzophenone-3 and Benzophenone-1 were 0.66 and 0.55, respectively.
A prospective study involving 200 pregnant women was performed.58 The study involved simultaneously-
collected, paired samples, of amniotic fluid and maternal serum and urine. Additionally, samples of human fetal
blood (n = 4) were obtained during cordogenesis; cord blood (n = 23) was obtained at the time of delivery. Samples
were collected from September of 2012 to August of 2014. The following benzophenones were all detectable in
amniotic fluid and cord blood, and, except for 4-hydroxybenzophenone, also in fetal blood: Benzophenone-1,
Benzophenone-3, 4-methylbenzophenone, and 4-hydroxybenzophenone. Benzophenone-1 and Benzophenone-3
were detected at ~ 10 times lower concentrations in fetal and cord blood, when compared to maternal serum, and at
a 1000 times lower concentration when compared to maternal urine concentrations. Therefore, Benzophenone-1 and
Benzophenone-3 were only detectable in the fetal circulation in cases of high maternal exposure, indicating some
protection by the placental barrier. 4-Methoxybenzophenone appeared to pass into fetal and cord blood more freely,
with a median 1:3 ratio between cord blood and maternal serum levels. The women appeared to have been most
exposed to Benzophenone-3, and this was the only benzophenone in which the measured concentrations in the
maternal urine and serum correlated with concentrations measured in amniotic fluid. Based on these data, the
authors determined that for Benzophenone-3, but not the other benzophenones, maternal urinary concentrations
seem to be a valid proxy for fetal exposure.
Benzophenone-1, Benzophenone-2, Benzophenone-3, Benzophenone-4
Benzophenone-6, and Benzophenone-8
Urinary concentrations of the following benzophenone derivatives were evaluated in a national sample of the
South Korean population (1576 subjects): Benzophenone-1, Benzophenone-2, Benzophenone-3, Benzophenone-4,
Benzophenone-8, and 4-hydroxybenzophenone.59 The urine samples were collected from July to September in 2010
and 2011. Liquid chromatography-mass spectrometry was used for analysis. The detection rates for Benzophenone-
1 and 4-hydroxybenzophenone were 56% (limit of detection = 0.59 ng/ml) and 88% (limit of detection = 0.04
ng/ml), respectively. The geometric means of urinary Benzophenone-1 and 4-hydroxybenzophenone concentrations
were 1.24 ng/ml and 0.45 ng/ml, respectively. The detection rate for the following benzophenones was below 25%:
Benzophenone-2, Benzophenone-3, Benzophenone-4, and Benzophenone-8.
Benzophenones have been identified in human menstrual blood of 25 subjects in Southern Spain.60 Of the 6
benzophenones, Benzophenone-3 was detected most frequently ( in 24 of 25 subjects), followed by Benzophenone-
6 (in 17 of 25 subjects), and Benzophenone-1 (in 11 of 25 subjects). Neither Benzophenone-2 nor Benzophenone-8
was detected in any of the samples. Maximum concentrations were very similar for Benzophenone-1,
Benzophenone-3, and Benzophenone-6 (3.1 to 3.7 ng/ml).
A study with data on benzophenones in the urine was performed from 2015 to 2016, using 441 adult pre-
menopausal females in South Korea.61 The detection frequencies of benzophenones in urine samples were:
Benzophenone-1 (98.4%), Benzophenone-3 (74.6%), and Benzophenone-8 (22.9%). The authors noted that
Benzophenone-1 is a major urinary metabolite of Benzophenone-3.

Benzophenone-3
Urine samples (166 total) were collected from children and adults in the US and China.62 In the US, urine
samples were collected from children in 2012, and from adults during May to July of 2011. In China, urine samples
were collected from children in March and April of 2002, and from adults during August and September of 2010.
The samples were analyzed for free and total forms (free + conjugated) of Benzophenone-3 as well as the following
4 of its metabolic derivatives: 4-hydroxbenzophenone; Benzophenone-1; Benzophenone-2; and Benzophenone-8.
Benzophenone-3 was detected in practically all urine samples from the US and China. The concentrations of
Benzophenone-3 in children (geometric mean = 9.97 ng/ml) and adults (geometric mean: 15.7 ng/ml) from the US
were statistically significantly higher when compared to children (geometric mean = 0.622 ng/ml) and adults
(geometric mean = 0.099 ng/ml) from China. A statistically significant positive relationship was found between the
concentrations of urinary Benzophenone-3 and its derivatives. The profiles of Benzophenone-3 derivatives in the
urine suggested that demethylation was a major route of Benzophenone-3 metabolism. A statistically significantly
lower percentage of the free form of Benzophenone-3 was found in urine from the US population than in the
Chinese population.
The urinary excretion of ingredients in personal care products over a 6-d period was studied using 8 subjects.63
The participants identified their usual personal care products. A total of 352 individual urine samples was collected
over a 6-day period. Benzophenone-3 was frequently detected, i.e., in 70% of the total urine samples. The authors
noted that exposure to Benzophenone-3 likely also occurred via the food pathway or other unknown sources.
Human adipose fat samples were collected from 20 subjects during years 2003 to 2004.64 High concentrations
of Benzophenone-3 (maximum of 4940 ng/g wet weight) were detected. These results suggest that adipose tissue is
an important repository for Benzophenone-3 in the human body.
 Postmortem brain material (hypothalamus and white-matter tissue) obtained from up to 24 individuals was
analyzed for the presence of Benzophenone-3.65 The limit of detection was 0.18 ng/g. In the hypothalamus, the
mean amount (n = 24) of Benzophenone-3 was below the limit of detection. In the white-matter, the mean amount
(n = 10) of Benzophenone was 0.32 ng/g. A study on human UV filters in human breast milk was performed, and
involved 79 breast milk samples from mothers in Spain.66 Milk samples were provided from the first day up to 31
months after childbirth. Between April and October of 2014, 79 individual breast milk samples were obtained from
71 primaparous and 8 multiparous (up to 4) nursing mothers. Most samples were collected in the period 4-6 months
after delivery. The percentage of samples that contained UV filters was 24%, and two of the major contributors
were Benzophenone-3 (779.9 ng/g milk) and its metabolite, 4,4'-dihydroxybenzophenone (73.3 ng/g milk).
Additionally, the plastic containers for the milk had high concentrations (up to 10.6 µg/g plastic) of Benzophenone-3
and 4,4'-dihydroxybenzophenone. Concentrations of UV filters in breast tissue (3 serial locations within) from 40
women undergoing mastectomy for breast cancer were measured.67 Tissue samples were collected between 2005
and 2008. Benzophenone-3 was measured in 83 of 120 (69%) tissue samples and at least 1 breast region for 33 of
40 women (range: 0 to 26 ng/g tissue). Spearman’s analyses showed statistically significant positive correlations
between concentrations of Benzophenone-3 in each of the 3 breast regions. For ethical reasons, cancerous tissue
was not available, but the location of the cancer was known. Mann-Whitney U-tests were used to investigate any
link between chemical concentration and whether or not a tumor was present in that region. In the lateral region,
more Benzophenone-3 was measured when a tumor was present (P = 0.007).

TOXICOLOGICAL STUDIES
Acute Toxicity Studies
Acute toxicity studies are summarized in Table 4.
Dermal
Benzophenones-3, -4, -8, and -12
In studies on Benzophenones-3, -4, -8, and -12 there was no toxicity at doses of 5 g/kg and greater .1 The
highest dose administered in these studies was 16 g/kg.
Benzophenones-3 and -12
A sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was evaluated for dermal toxicity in a
study involving 24 Wistar albino rats (12 males, 12 females).68 The authors concluded that the acute dermal LD50 of
the sunscreen formulation was greater than 2000 mg/kg in male and female rats. The same sunscreen formulation
(0.6% to 0.9% Benzophenone-3) was applied to the skin of 6 male New Zealand rabbits.68 Systemic toxicity was
not observed. The acute dermal toxicity of Benzophenone-12 was evaluated using 5 albino rabbits.6 The LD50 was
> 10,000 mg/kg.
Oral
Benzophenones-1, -2, -3, -4, -6, -8, -9, -11, and -12
Benzophenones-1, -2, -3, -4, -6, -8, -9, -11, and -12 were practically nontoxic to slightly toxic (Benzophenones-
2, -4, and -11) when administered orally to rats at doses up to 16 g/kg.1
Benzophenones-1 and -3
The acute oral toxicity of Benzophenone-1 was evaluated using rats (number and strain not stated).7 The LD50
was 8600 mg/kg. The acute oral toxicity of a sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was
evaluated using 10 female Wistar albino rats.68 The LD50 for the sunscreen formulation was > 2000 mg/kg.
Benzophenone-4
Benzophenone-4 was evaluated for acute oral toxicity using 20 rats (strain not stated).1 Doses of the test
substance (in agar/tween) ranging from 1250 to 10,000 mg/kg were administered orally by gavage. Dosing was
followed by a 7- to 14-d observation period. Clinical signs (ataxia) were observed. An LD50 of 3530 mg/kg was
reported.
Benzophenones-8 and -12
The acute oral toxicity of Benzophenone-8 was evaluated using 6 female Wistar rats of the CLR:(WI) strain.9
The LD50 was > 2000 mg/kg. Benzophenone-12 was evaluated for acute oral toxicity using 10 male rats of the CF
Nelson strain.6 The LD50 was > 10,000 mg/kg.
Short-Term Toxicity Studies
Repeated dose toxicity studies (short-term and subchronic toxicity) are summarized in Table 5.
Dermal
Benzophenone-3
In a 2-wk dermal toxicity study, B6C3F1 mice (5 males and 5 females per group) received topical applications
in amounts of 0.5 to 8 mg of Benzophenone-3 in an acetone or lotion vehicle.69 The only effects noted were
minimal, variable increases in liver and kidney weights. In another 2-wk dermal toxicity study, F344/N rats (5 males
and 5 females per group) received topical applications of 1.25 to 20 mg of Benzophenone-3 in an acetone or lotion
vehicle.69 The only effects noted were small and variable increases in liver and kidney weights. Benzophenone-3
(in ointment base) was applied to the skin of male Sprague-Dawley rats (groups of 4 to 6 animals; weights = 300 g)
at a dose of 100 mg/kg, twice daily for 4 wk.70 The results of this study suggest that Benzophenone-3 is not toxic to
rats when applied dermally at a dose of 100 mg/kg for 2 wk. The short-term dermal toxicity of Benzophenone-3 was
studied using female Sprague-Dawley rats and their offspring.35 Benzophenone-3 (10% in cream; dose = 100
mg/kg) was administered dermally (shaved skin on back) twice daily to adult female rats from the first to the last
day of pregnancy (~22 to 23 days). At 21 d after birth, the offspring (male and female) were divided into groups of
5 males and groups of 5 females. From 43 to 56 d age, the test substance was administered dermally to the male
offspring. The dosing of adult pregnant females did not significantly alter their body weight or cause any apparent
adverse effects, when compared to control rats. No significant differences in body weight and sex-ratio were
observed in the offspring.
Oral
Benzophenones-3 and -8
When rats were fed Benzophenone-3 at concentrations up to 1% in the diet for 27 d, no toxic effects were
observed.1 A no-effect-level of 2.5% was reported in a study in which rats were fed Benzophenone-8 in the diet at
concentrations up to 10% for 36 d. Groups of mice received Benzophenone-8 (in corn oil, 50 to 5000 mg/kg) by
gavage daily for 2 d.2 No toxic signs or deaths were observed after dosing with 50 mg/kg. Signs of toxicity were
observed at doses of 166 to 5000 mg/kg. At doses of 1666.6 and 5000 mg/kg, abnormal gait and a very low mortality
incidence were reported. In another experiment, groups of mice were dosed, by gavage, with 1500 mg/kg
Benzophenone-8 (2 doses, 24 h apart). Body drop, decreased activity, and abnormal gait were observed.
Benzophenones-3, -4, and -12
In a 2-wk oral toxicity study, B6C3F1 mice (5 males and 5 females per group) received feed containing 0,
3125, 6250, 12,500, 25,000, or 50,000 ppm Benzophenone.69 The no-observed-adverse-effect level (NOAEL) for
microscopic lesions was 6250 ppm Benzophenone-3 in the diet for mice. In another 2-wk oral toxicity study,
F344/N rats (5 males and 5 females per group) received diets containing 0, 3125, 6250, 12,500, 25,000, or 50,000
ppm Benzophenone-3.69 The NOAEL for microscopic lesions was 6250 ppm Benzophenone-3 in the diet for rats. A
short-term oral toxicity study on Benzophenone-4 was performed using groups of 26 Wistar rats (13 males, 13
females/group).8 The test substance was administered orally (in corn oil, by gavage) at doses of 750, 1000, and
1250 mg/kg/d. Male rats were treated 2 wk before mating and thereafter for a total of 48 dosing days. Female rats
were treated 2 wk before mating, and during mating, gestation, and lactation, for a total of approximately 63 d of
dosing. The NOAEL (systemic toxicity) for Benzophenone-4 in this study was established at 1250 mg/kg/d for
male and female rats. Groups of 6 male rats (Carworth Farms Elias strain) were fed Benzophenone-12 at dietary
concentrations of 1.25% and 5% daily for 35 d, in accordance with OECD TG 417.6 There were no lesions of the
liver or kidneys at histological examination. The repeated dose toxicity of Benzophenone-12 was evaluated in
Wistar rats.6 The test substance (in 0.5% carboxymethylcellulose suspension in drinking water + 5 mg/100 ml
Tween 80) was administered by gavage to groups of Wistar rats (F0 animals: 12 males, 12 females/group) at doses
of 100, 300, and 1000 mg/kg/d. The duration of treatment was described as follows: 10-wk premating period
(males), 2-wk premating period (females), 2-wk mating period (both sexes), ~2 d post-mating (males), entire
gestation period, up to 30 d of lactation (corresponding to 21 d of lactation and up to 9 d post-weaning), and 35 d
post-mating (for sperm-negative females). Pups from the F1 litter were selected (F1 rearing animals) for specific
post-weaning examinations. A NOAEL of 1000 mg/kg/d for general, systemic toxicity was determined.
Developmental and reproductive toxicity data are included in that section of this safety assessment.
Subchronic Toxicity Studies
Oral
Benzophenones-1, -3, and -12
In subchronic (90-d) oral toxicity studies, Benzophenones-3 and -12, at 1% and 1.8% in the diet, respectively,
were nontoxic to rats.1 Benzophenones-1 and -12 elicited toxic effects (liver and kidney lesions) in rats at 0.6 and
1.9 g/kg, respectively, when fed for 90 d. In the same time period, Benzophenone-3, fed at 0.5% in the diet, and
Benzophenone-8, fed at 5%, produced toxic effects (degenerative nephrosis). In a 120-d feeding study,
Benzophenone-12 was nontoxic to dogs at a concentration of 0.6% in the diet.
Benzophenones-1 and -3
The subchronic oral toxicity of Benzophenone-1 was evaluated in a 90-d study involving male and female rats
(number and strain not stated).7 The NOAEL was 236 mg/kg body weight/d. In a 13-wk oral toxicity study,
B6C3F1 mice (10 males and 10 females per group) received feed containing 0, 3125, 6250, 12,500, 25,000, or
50,000 ppm Benzophenone.69 The NOAEL for microscopic lesions was 6250 ppm Benzophenone-3 in the diet for
mice. In another 13-wk oral toxicity study, F344/N rats (10 males and 10 females per group) received diets
containing 0, 3125, 6250, 12,500, 25,000, or 50,000 ppm Benzophenone-3.69 The NOAEL for microscopic lesions
was 6250 ppm Benzophenone-3 in the diet for rats. Results on subchronic oral toxicity are included in an NTP oral
carcinogenicity study on Benzophenone-3 involving male and female Sprague-Dawley rats.71 Groups of 10 male
and 10 female rats were exposed to 0 or 10,000 ppm Benzophenone-3 in the diet for 14 wk. In males, the absolute
and relative liver and right kidney weights were increased in the 10,000 ppm group compared to the control group.
In females, the absolute kidney weight was significantly decreased, and the relative liver weight was significantly
increased relative to the control group.

DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES

Developmental and reproductive toxicity studies are summarized in Table 6.
Embryo/Ovary Cultures
Benzophenone-3
The embryotoxicity of Benzophenone-3 was evaluated in the fish embryotoxicity test using zebrafish
embryos.72 The applied number of zebrafish embryos was 40 at each concentration in 4 replicates. The experiment
was continued until 120 h post-fertilization, The following 6 concentrations of Benzophenone-3 (in
dimethylsulfoxide (DMSO)) were prepared: 0.116 mM, 0.0789 mM, 0.0523 mM, 0.0307 mM, 0.0219 mM, and
0.00535 mM. Benzophenone-3 decreased the number of hatched embryos after 96 h post-fertilization. The EC50
value was 0.0543 mM. Other malformations were observed, but their frequency was not concentration-dependent.
These included pericardial and yolk sac edema, deformed jaw and ventricle or dilated gut, and jaw deformity. The
effect of Benzophenone-3 on follicular assembly was studied using whole ovary cultures collected from Wistar
rats.73 Ovaries (n = 120) were collected from rats at birth (postnatal day 0). Pups from the same litters were
randomly assigned to different treatment groups so that each group contained ovaries of different pups from
different litters. The ovary cultures were treated for 7 d with the following Benzophenone-3 concentrations (in
(DMSO): 5.8 nM, 276 nM, 576 nM, and 876 nM. Even at the lowest concentration of Benzophenone-3 (5.8 nM),
stimulation of the process of germ cell nest breakdown and c a decrease in the reserve of total oocytes were
observed.
Animal
Dermal
Benzophenone-3
In a 13-wk dermal dosing study, B6C3F1 mice (10 males and 10 females per group) received topical doses of
22.75 to 364 mg/kg Benzophenone-3 in acetone.74 Epididymal sperm density was decreased at all 3 dose levels
evaluated (22.75, 91.0, and 364.0 mg/kg). In female mice, there was no significant difference in estrous cycle length
between the control group and each dose group. A study was performed to analyze whether dermal exposure to
Benzophenone-3 during pregnancy affects the outcome of a second pregnancy in mice.75 Pregnant mice (number
not stated) were exposed dermally to Benzophenone-3 (50 mg/kg/d) from GD 0 to 6. Dermal exposure to
Benzophenone-3 during early pregnancy resulted in an intrauterine growth restriction (IUGR) phenotype, disturbed
sex ratio, and alterations in the growth curve of the offspring in the mouse model.
Oral
Benzophenone-1, -2, -3, -4, and -12
The reproductive toxicity of Benzophenone-1 was evaluated using female rats (number and strain not stated).7
The test substance was administered orally for 3 d. A NOAEL of 10 mg/kg/d was reported. The developmental
toxicity of Benzophenone-2 (in 10% ethanol/90% corn oil vehicle) was evaluated using groups of 5 timed pregnant
C57BL/6NCr mice.76 Benzophenone-2 (6.25 mg) was administered via gavage on GD 12 through 17. In the test
group, 8 of 57 male fetuses had hypospadias (p = 0.0064, when compared to controls). The co-administration of
Benzophenone-2 with an estrogen receptor antagonist (10 µg in vehicle (subcutaneously (s.c.)) during gestation,
yielded normal genital tubercles; i.e., no hypospadias in 26 of 26 mice. The authors concluded that Benzophenone-2
may cause hypospadias via signaling through the estrogen receptor. Benzophenone-3 (administered in feed) was
tested for its effects on fertility and reproduction in Swiss CD-1 mice, according to the continuous breeding
protocol.77 Based on the results of a dose-finding study, 1.25%, 2.5%, and 5.0% (w/w) were chosen to investigate
effects on fertility and reproduction. Male and female mice were continuously exposed for a 7-d precohabitation
and a 98-d cohabitation period. It was concluded that Benzophenone-3 caused systemic toxicity, but had minimal
effects on fertility and reproduction. In a 13-wk oral dosing study, B6C3F1 mice (10 males and 10 females per
group) received feed containing 0, 3125, 6250, 12,500, 25,000, or 50,000 ppm Benzophenone-3.69 Mice in the
highest dose group (50,000 ppm in feed) exhibited a decrease in epididymal sperm density and an increase in length
of the estrous cycle.
The effects of oral exposure to Benzophenone-3 on growth and morphology of the mammary gland and
anogenital distance was evaluated using 3 groups of mated BALB/c female mice.78 From pregnancy day 0 until the
day before weaning (lactational day 21), the females were dosed orally with Benzophenone-3 (in tocopherol-
stripped corn oil). The following doses were administered: 30 µg/kg/d, 212 µg/kg/d, and 3000 µg/kg/d. In males,
the anogenital index was reduced after exposure to 30 and 212 µg/kg/d at postnatal day 21 and in puberty. In adult
males, no differences in anogenital distance were observed. In females, the anogenital index was unaffected at
postnatal day 21, but decreased (at 212 µg/kg/d) when measured at puberty. No effects on female anogenital index
were observed in adulthood. In a 13-wk oral dosing study, F344/N rats (10 males and 10 females per group)
received diets containing 0, 3125, 6250, 12,500, 25,000, or 50,000 ppm Benzophenone-3.69 Rats receiving a diet
with 50,000 ppm Benzophenone-3 showed markedly lower epididymal sperm density and an increase in the length
of the estrous cycle at the end of the study. A study was performed to determine the effects of maternal and
lactational exposure to Benzophenone-3 on the development of offspring.40 Groups (7 to 8 animals per group) of
mated female Sprague-Dawley rats were fed the following Benzophenone-3 concentrations (in low-phytoestrogen
chow) from GD 6 until weaning on postnatal day 23: 1000; 3000; 10,000; 25,000; or 50,000 ppm. There were no
statistically significant differences in the mean number of implantation sites/litter, mean resorptions per litter, %
litters with resorptions, number and weights of live fetuses, or sex ratios between the control and Benzophenone-3
dose groups.
Groups of 25 pregnant Sprague-Dawley rats were fed low-phytoestrogen chow containing 3000 or 30,000 ppm
Benzophenone-3 from GD 6 until postnatal day 21.79 The male offspring evaluated in this study were weaned on
postnatal day 28, and then dosed with the same concentrations of Benzophenone-3 (in chow and milk). The animals
were killed on postnatal day 30. Rats exposed perinatally to 30,000 ppm Benzophenone-3 had statistically
significantly lower weights of the paired-testis, paired-epididymis, and prostate. Results relating to developmental
toxicity are included in an NTP oral carcinogenicity study on Benzophenone-3 involving male and female Sprague-
Dawley rats.71 On GD 6, groups of 42, 35, 35, and 43 F0 time-mated female rats were fed diets containing 0, 1000,
3000, and 10,000 ppm Benzophenone-3, respectively, for 39 d. Groups of 50 (1000 and 3000 ppm) or 60 (0 and
10,000 ppm) F1 rats per sex continued on study after weaning, and were fed diets containing the same exposure
concentrations for 105 wk; 10 F1 rats per sex from the 0 and 10,000 ppm groups were evaluated at 14 wk. The
administration of Benzophenone-3 had no effects on the percentage of mated females producing pups, litter size,
pup sex distribution, or numbers of male or female pups. Benzophenone-3 was evaluated for developmental toxicity
in accordance with OECD TG 414, using groups of 25 mated Wistar rats of the Crl:WI (Han) strain.10
Benzophenone-3 (in corn oil) was administered at doses of 40, 200, and 1000 mg/kg/d (once daily, by gavage) on
days 6 through 19 post-coitum. The NOAEL for Benzophenone-3 was 200 mg/kg/d. In a study involving groups of
26 Wistar rats (13 males, 13 females/group), Benzophenone-4 was administered orally (in corn oil, by gavage) at
doses of 750, 1000, and 1250 mg/kg/d.8 Male rats were treated 2 wk before mating and thereafter for a total of 48 d
of dosing. Female rats were treated 2 wk before mating, during mating, during gestation and during lactation, for a
total of approximately 63 d of dosing. The NOAEL (reproductive toxicity) for Benzophenone-4 in this study was
established at 1250 mg/kg/d.
The developmental toxicity of Benzophenone-12 (in 0.5% carboxymethylcellulose suspension in drinking
water + 5 mg/100 ml Tween 80) was evaluated using groups of 50 Wistar rats (25 males (for mating), 25 females).6
The test substance was administered by gavage to mated females at doses of 100, 300, and 1000 mg/kg/d. The
groups were dosed daily, from implantation to one day prior to the expected day of parturition (GD 6 to 19 The
NOAEL for maternal and prenatal developmental toxicity was 1000 mg/kg/d. Benzophenone-12 (in 0.5%
carboxymethylcellulose suspension in drinking water + 5 mg/100 ml Tween 80) was administered by gavage to
groups of Wistar rats (F0 animals: 12 males, 12 females/group) at doses of 100, 300, and 1000 mg/kg/d.6 The
duration of treatment was described as follows: 10-wk premating period (males), 2-wk premating period (females),
2-wk mating period (both sexes), ~2 d post-mating (males), entire gestation period, up to 30 d (corresponding to 21
d of lactation and up to 9 d post-weaning), and 35 d post-mating (for sperm-negative females). The NOAEL for
reproductive performance and fertility of the F0 parental rats and developmental toxicity in the offspring was 1000
mg/kg/d.

GENOTOXICITY STUDIES
Genotoxicity studies (in vitro and in vivo) are summarized in Table 7.
In Vitro
Benzophenones-1, -2, -3, -4, -6, -8, -9, and -11
Benzophenone-2 (up to 10,000 µg/plate), Benzophenone-6 (up to 1000 µg/plate), and Benzophenone-8 (up to
700 µg/plate) were reported to be weakly mutagenic with metabolic activation in the Ames test.1 Benzophenones-6
and -8 were mutagenic in one of the Salmonella typhimurium strains (TA1537) tested. Benzophenone-2 was weakly
mutagenic in the mouse lymphoma forward mutation assay (at doses of 24 and 32 µg/plate) and in a cytogenic assay
evaluating sister chromatid exchanges and chromosome aberrations (at doses of 100 and 200 µg/plate). These
effects in L5178Y mouse lymphoma cells were observed at the high end of the range of doses tested. All other
benzophenones (Benzophenones-1, -3, -4, -9, and -11) were non-mutagenic both with and without metabolic
activation in the Ames test.
In a modified Ames test, Benzophenone-2 and Benzophenone-6 (concentrations up to 1000 µg/ml) were not
genotoxic to Salmonella typhimurium strains with or without metabolic activation.2 Benzophenone-2 and
Benzophenone-6 did not induce unscheduled DNA synthesis in rat hepatocytes at concentrations up to 1000
nmol/ml. In a sister chromosome exchange assay, Benzophenone-8 was tested using Chinese hamster ovary cell
cultures. Without metabolic activation, there was no significant increase in sister chromatid exchanges at
concentrations ranging from 333 ng/ml to 10 µg/ml, but a slight increase was noted at 10 µg/ml. With metabolic
activation, there was no increase in sister chromatid exchanges at concentrations ranging from 3.1 to 50 µg/ml.
Benzophenone-8 was not genotoxic in a forward mutation assay involving Chinese hamster ovary cells, at
concentrations ranging from 2.2 to 66.6 µg/ml with or without metabolic activation.
Benzophenone-1, -3, -6, -8, and -12
The photo-genotoxicity of Benzophenone-1 (1 to 25 µg/ml, in culture medium) and apoptotic parameters were
evaluated using human keratinocytes (HaCaT cells).80 Results indicated that Benzophenone-1 photosensitized and
generated intracellular reactive oxygen species (2.02 folds) under sunlight/UV radiation. Decrease in cell viability
was recorded as 80.06%, 60.98%, and 56.24% under sunlight, UVA, and UVB, respectively. In the same study, the
genotoxicity potential of Benzophenone-1 (5 to 25 µg/ml, in culture medium) was confirmed through photo-
micronuclei and cyclobutene pyrimidine dimers (CPDs) formation. HaCaT cells treated with Benzophenone-1 in
the presence of UVB (1.08 J/cm2) caused cyclobutane CPD formation. Micronuclei formation was detected in
HaCaT cells treated with Benzophenone-1 (10 µg/ml) in the presence of UVB (1.08 J/cm2). Cells exposed to
different concentrations of Benzophenone-1 in the presence of UVA (2.7 J/cm2) exhibited statistically significant (p
> 0.01) DNA damage when compared to control cells. The genotoxicity of Benzophenone-1, Benzophenone-3,
Benzophenone-6, and Benzophenone-8 (doses up to 10 µg/well) was evaluated in the luminescent umu-test, using
Salmonella typhimurium strain TL210.81 Results indicated positive results for Benzophenone-3 and pseudo-positive
results for Benzophenone-1 and Benzophenone-8. In the same study, the genotoxicity of Benzophenone-1 (doses up
to 600 µg/plate), Benzophenone-3 (up to 200 µg/plate), Benzophenone-6 (up to 2000 µg/plate), and Benzophenone-
8 (up to 300 µg/plate) was evaluated in the Ames test using S. typhimurium strains TA98 and TA100 (with and
without metabolic activation).81 None of the test substances produced clear positive results with or without
metabolic activation.
The genotoxicity of Benzophenone-3 and Benzophenone-8 (each in seawater) was evaluated at doses of 4 to
10 µl per plate using S. typhimurium strain TA98 (without metabolic activation).82 Neither ingredient was
genotoxic. The genotoxicity of Benzophenone-3 and Benzophenone-8, each in chlorinated bromide-rich water
(artificial seawater), was also evaluated in the Ames test using S. typhimurium strain TA98 without metabolic
activation. Only Benzophenone-8 (1:10) had clear genotoxic activity that was dose-related (doses of 4, 6, 8, and 10
µl). A sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was evaluated for genotoxicity using the
following S. typhimurium strains: TA 98, TA100, TA1535 and TA1538.68 The formulation was tested at a dose of
5000 µg/plate with and without metabolic activation. Benzophenone-3 was not genotoxic. The cytogenetic effect of
Benzophenone-3 on human peripheral lymphocytes was evaluated using in vitro chromosomal aberrations and
micronuclei assays.83 Lymphocyte cultures were exposed to the following 5 concentrations of Benzophenone-3:
0.20 µg/ml, 0.10 µg/ml, 0.05 µg/ml, 0.025 µg/ml, and 0.0125 µg/ml. A statistically significant increase in
chromosomal aberrations and aberrant cell frequencies was observed at all test concentrations, when compared to
the solvent (DMSO) control. In the micronuclei test, Benzophenone-3 caused a statistically significant increase in
micronuclei formation at all test concentrations. The effect of Benzophenone-3 on DNA damage was studied using
human breast epithelial cells.84 Concentrations of 1 µM and 5 µM Benzophenone-3 increased DNA damage in a
manner that was similar to that of treatment with E2, and in an estrogen-receptor alpha (ERα)-dependent manner.
Benzophenone-3 was evaluated for genotoxicity using S. typhimurium strains TA98 and TA100, and Escherichia
coli strain uvrA pKM101.71 The test substance was evaluated at doses up to 6000 µg/plate with and without
metabolic activation. Benzophenone-3 was non-genotoxic.
A bacterial reverse mutation assay was used to evaluate the genotoxicity of Benzophenone-8 (in DMSO), using
S. typhimurium strain TA100 and E. coli (E. coli) strain WP2vurA.9 Strain TA100 was selected for testing at doses
up to 1500 µg/plate, and strain WP2vurA was selected for testing at doses up to 5000 µg/plate. Benzophenone-8
was negative for genotoxicity in this assay, with and without metabolic activation. The mutagenicity of
Benzophenone-8 (in ethanol) was evaluated in the Salmonella/mammalian microsome mutagenicity assay using the
following S. typhimurium tester strains: TA98, TA100, TA1535, TA1537, and TA1538.85 Benzophenone-8 test
concentrations ranged from 0.008 to 700 µg/plate. Benzophenone-8 caused a weak, but reproducibly significant
increase in the number of TA1537 revertants per plate. Benzophenone-8 (in ethanol) was tested in the L5178Y
TK+/- mouse lymphoma mutagenesis assay (with and without metabolic activation) at concentrations ranging from
13 to 56 µg/ml.86 Cultures treated in the presence of metabolic activation exhibited a significant increase in the
mutant frequencies, and a dose response was evident. Benzophenone-8 was genotoxic in this assay. The
genotoxicity of Benzophenone-12 in the mammalian cell gene mutation assay (mouse lymphoma L5178Y cells) was
evaluated.6 Benzophenone-12 (in DMSO) was tested at doses up to 50 µg/ml (with metabolic activation) and up to
52 µg/ml (without metabolic activation). Benzophenone-12 was non-genotoxic without metabolic activation, and
results were ambiguous with metabolic activation.
In Vivo
Benzophenone-1
The genotoxicity of Benzophenone-1 was evaluated in the micronucleus assay (OECD TG 474) using mouse
erythrocytes.7 The doses tested were not stated. Genotoxicity results were classified as inconclusive.
Benzophenones-2, -6, and -8
Benzophenone-2 and Benzophenone-6 did not induce sister chromatid exchanges in Chinese hamster bone
marrow cells from animals (species not stated) dosed orally (doses up to 500 mg/kg).2 In the micronucleus test, the
oral dosing of mice with Benzophenone-8 (1500 mg/kg) did not cause a significant increase the number of bone
marrow micronuclei.
Benzophenone-3
The genotoxicity of Benzophenone-3 was evaluated using the Drosophila somatic mutation and recombination
test (SMART) and the in vivo cytogenetics assay using rat bone marrow cells.87 In the SMART assay, larva from a
mating of “multiple wing hair” (mwh) females with heterozygous “flare” (flr) males were exposed to 0, 3000, or
3500 ppm Benzophenone-3. None of the Benzophenone-3-treated larva produced flies with significantly more
single or multiple wing spots than controls. An in vivo cytogenetic assay in rat bone marrow cells was conducted to
evaluate the clastogenicity of Benzophenone-3.87 Sprague-Dawley rats were treated by oral gavage with a single
administration of 0.0, 0.5, 1.67, or 5 g/kg Benzophenone-3, or a dose of 5 g/kg/d Benzophenone-3 for 5 consecutive
days. None of the Benzophenone-3 concentrations caused any significant increase in chromosomal aberrations. The
genotoxicity of a sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was evaluated in the
mammalian erythrocyte micronucleus test using groups of 10 Wistar albino rats.68 Doses of 0.5 g/kg, 1 g/kg, and 2
g/kg were administered dermally for 2 consecutive days. The sunscreen formulation as non-genotoxic. The same
sunscreen formulation (0.6% to 0.9% Benzophenone-3) was evaluated for genotoxicity in the mammalian bone
marrow chromosome aberration test using groups of 10 Wistar albino rats.68 Doses of the sunscreen, 0.5 g/kg, 1
g/kg, and 2 g/kg, were administered dermally for 2 consecutive days. The sunscreen formulation was non-
genotoxic. Ovariectomized mice (Balb/c female mice) were exposed to Benzophenone-3 at 10 d after the surgical
procedure.84 Eight mice were dosed orally with E2 and 12 mice were dosed orally with Benzophenone-3 daily for 4
d. Each mouse was administered 1 µl of tocopherol-stripped corn oil per gram of body weight to deliver E2 (250
µg/kg/d) or Benzophenone-3 (3000 µg/kg/d). Results indicated that R-loops and DNA damage were detected in
mammary epithelial cells of mice treated with Benzophenone-3.

CARCINOGENICITY STUDIES
In Vitro
Benzophenone-1
Effects of Benzophenone-1 on the proliferation and metastasis of MCF-7 human breast cancer cells expressing
estrogen receptors were studied.88 The underlying mechanisms for these effects was also studied, including the
study of alterations in transcriptional and translational levels of proliferation and metastasis-related markers (cyclin
D1, p21, and cathepsin D). Treatment of the cells with Benzophenone-1 (10-7 to 10-5 M) promoted the proliferation
of MCF-7 cells in a manner that was similar to the positive control (E2). The addition of Benzophenone-1 also
markedly induced the migration of MCF-7 cells in a manner that was similar to E2. Regarding underlying
mechanisms of action, an increase in the expression of cyclin D1 and cathepsin D, and a decrease in p21 (at both
transcriptional and translational levels) were reported. The authors concluded that Benzophenone-1 may accelerate
the growth of MCF-7 breast cancer cells by regulating cell cycle-related genes and promote cancer metastasis
through amplification of cathepsin D.
A wound healing assay and western blot assay were performed to show the effect of Benzophenone-1 on the
migration of BG-1 ovarian cancer cells and the protein expression of epithelial-mesenchymal transition (EMT)-
related genes.89 The EMT process is associated with cell migration. Benzophenone-1 (10-6 M) statistically
significantly enhanced the migration capability of BG-1 cells by reducing the wounded area in the cell monolayer
relative to the control, i.e., in a manner that was similar to E2 (10-9 M). The authors stated that the results of this
study indicate that Benzophenone-1 may have the ability to induce ovarian cancer metastasis via regulation of the
expression of EMT markers and migration of estrogen receptor-expressing BG-1 ovarian cancer cells.
Benzophenone-3
The effect of Benzophenone-3 (concentrations up to 150 µg/l) on cancer cell growth was studied using NCI-
H460 lung cancer cells.90 At concentrations of 50 µg/l, 100 µg/l, and 150 µg/l, Benzophenone-3 statistically
significantly increased colony formation of the NCI-460 cells, in both number and size. These observations indicate
that Benzophenone-3 has a cancer potentiating effect by enhancing anchorage-independent survival and growth of
lung cancer cells.
Animal
Oral
Benzophenone-3
The oral carcinogenicity of Benzophenone-3 was evaluated in a National Toxicology Program (NTP) study
using male and female Sprague-Dawley rats and male and female B6C3F1/N mice. 71 Groups of 50 male and 50
female mice were fed diets containing 0, 1000, 3000, or 10,000 ppm Benzophenone-3 in the diet (equivalent to
average daily doses of approximately 113, 339, and 1207 mg Benzophenone-3/kg body weight, respectively, for
male mice and 109, 320, and 1278 mg/kg, respectively, for female mice) for 104 (female mice) or 105 (male mice)
weeks. Survival of all exposed groups of male and female mice was not statistically significantly different from that
of the control groups. Mean body weights of 1000 and 3000 ppm males and females were within 10% of those of
the control groups throughout the study. Mean body weights of 10,000 ppm male and female mice were at least
10% lower than those of the control groups, generally after weeks 17 and 12, respectively. Feed consumption by
exposed groups of male and female mice was not statistically significantly different from that of the control groups.
The incidences of pigment in the bone marrow were statistically significantly increased in 10,000 ppm male
and female mice. The incidences of pigment in the spleen were statistically significantly increased in 10,000 ppm
male mice and 3000 ppm and 10,000 ppm female mice. In the liver, the incidence of hepatocyte syncytial alteration
was statistically significantly increased in all exposed groups of male mice. In the kidney, the incidence of renal
tubule cytoplasmic alteration was statistically significantly increased in 10,000 ppm male mice. The incidence of
osseous metaplasia was statistically significantly increased in 10,000 ppm female mice, when compared to the
control group.
In the same NTP carcinogenicity study, on GD 6, groups of 42, 35, 35, and 43 F0 time-mated female rats were
fed diets containing 0, 1000, 3000, and 10,000 ppm Benzophenone-3, respectively, for 39 d. Groups of 50 (1000 and
3000 ppm) or 60 (0 and 10,000 ppm) F1 rats per sex continued on study after weaning and were fed diets containing
the same exposure concentrations for 105 wk; 10 F1 rats per sex from the 0 and 10,000 ppm groups were evaluated
at 14 wk. Dietary concentrations of 1000, 3000, and 10,000 ppm resulted in average daily doses of approximately
58, 168, and 585 mg Benzophenone-3/kg body weight for males and 60, 180, and 632 mg/kg for females. Survival
of all exposed groups of F1 male and female rats was not statistically significantly different from that of the control
groups. Over the course of the study, mean body weights of F1 male rats and females in the 10,000 ppm exposure
groups were 10 - 25% lower than those of the control groups. After week 77, F1 female rat mean body weights in the
3000 ppm exposure group were 10% lower than those of the control group. Feed consumption by exposed groups of
F1 males and females was generally similar to that of the control group throughout the study.
In the brain, the occurrence of malignant meningiomas in male rats at the end of the 2-year study was 0/50
(control group), 1/50 (1000 ppm group), 3/50 (3000 ppm group), and 0/50 (10,000 ppm group). One male rat in the
3000 ppm group had a malignant meningioma in the spinal cord. In the thyroid gland, the incidence of C-cell
adenoma in 3000 ppm female rats was statistically significantly greater than that in the control group at the end of
the 2-year study. Only one female rat, in the 10,000 ppm group, had bilateral C-cell adenomas; the rest were
unilateral lesions. One animal in the 1000 ppm group had both a C-cell adenoma and a C-cell carcinoma (in the
opposite gland). There was no significant exposure concentration-related difference in the incidence of C-cell
adenomas in male rats (0 ppm (7/50); 1000 ppm (10/50); 3000 ppm (8/50); and 10,000 ppm (8/50)) when compared
to the control group.
In the uterus, the incidence of stromal polyps in 3000 ppm females was statistically significantly increased. A
statistically significantly increased incidence of atypical endometrium hyperplasia of the uterus also occurred at
3000 ppm; however, the incidence of adenocarcinoma was statistically significantly decreased in this group. In the
adrenal cortex, the incidences of focal hypertrophy were statistically significantly increased in 1000 and 3000 ppm
female rats at the end of the 2-year study. In the testes, the incidence of interstitial cell hyperplasia showed a
statistically significant positive trend, but there were no statistically significant pairwise comparisons of the exposed
groups to the control group. The incidence of fibrinoid necrosis of the arterioles was statistically significantly
increased in 10,000 ppm males when compared to the control group. In the pancreas, the incidence of chronic active
inflammation affecting the arterioles was statistically significantly increased in 1000 ppm males, when compared to
the control group at the end of the 2-year study. The incidences of mammary gland fibroadenoma and carcinoma
were statistically significantly decreased, relative to the control group, in 10,000 ppm females at the end of the 2-
year study (fibroadenomas: 32/50 (control), 30/50 (1000 ppm), 27/50 (3000 ppm), and 18/50 (10,000 ppm);
carcinomas: 7/50 (control), 5/50 (1000 ppm), 7/50 (3000 ppm), and 1/50 (10,000 ppm)).
Perinatal studies and 14-wk interim evaluations were also conducted in rats from the NTP carcinogenicity
study. Results from perinatal studies are included in the section on Developmental and Reproductive Toxicity.
Results from 14-wk interim evaluations are included in the Subchronic (Oral) Toxicity section.
The authors concluded that, under the conditions of these 2-year studies, there was equivocal evidence of
carcinogenic activity of Benzophenone-3 exposure in male Hsd:Sprague Dawley® SD® rats, based on the
occurrence of malignant meningiomas in the brain. There was equivocal evidence of carcinogenic activity in female
Hsd:Sprague Dawley® SD® rats, based on the increased incidence of thyroid C-cell adenomas and the increased
incidence of uterine stromal polyps. There was no evidence of carcinogenic activity in male or female B6C3F1/N
mice at exposure concentrations of 1000, 3000, and 10,000 ppm. It was noted that increases in the incidences of
non-neoplastic lesions of the testis in male rats and of the uterus and adrenal cortex in female rats occurred with
exposure to Benzophenone-3. Increases in the incidences of non-neoplastic lesions of the bone marrow (males and
females), spleen (males and females), kidney (males and females), and liver (males) in mice occurred with exposure
to Benzophenone-3.
In Vitro Cell Transformation Studies
Benzophenone-1
The xenoestrogenic effect of Benzophenone-1 on BG-1 human ovarian cancer cells expressing estrogen
receptors and relevant xenografted animals models, when compared to E2, was evaluated.91 In the in vitro cell
viability assay, Benzophenone-1 (10-8 to 10-5 M) statistically significantly increased BG-1 cell growth, as did E2.
The mechanism underlying BG-1 cell proliferation induced by Benzophenone-1 was shown to be related to the up-
regulation of cyclin D1, a cell cycle progressor. Both Benzophenone-1 and E2 induced cell growth and up-
regulation of cyclin D1 were reversed by co-treatment with an ER antagonist, suggesting that Benzophenone-1 may,
similar to E2, mediate the cancer cell proliferation via an estrogen receptor-dependent pathway. However, the
expression of p21 (regulator of cell cycle progression at G1 phase) was not altered by Benzophenone-1, though it
was down-regulated by E2.
In a second experiment, BG-1 cells (5 x 106) were injected s.c. into the backs of groups of 6 female mice of the
BALB/c nu/nu strain. The mice were monitored for tumor growth. Once the tumors reached a volume of 50 mm3,
the mice were surgically ovariectomized. One week after surgery, 6 mice were injected s.c. with E2 (20 µg/kg)
every 2 d for 8 wk, and another group of 6 mice was dosed s.c. with Benzophenone-1 (200 mg/kg). The vehicle
control group was dosed with corn oil. Benzophenone-1 or E2 treatment statistically significantly increased the
tumor mass formation (compared to corn oil vehicle) within 8 wk. At histopathological examination, the tumor
sections of the E2 or Benzophenone-1 group displayed extensive cell formations with high density and disordered
arrangement. These results were supported by the increased number of bromodeoxyuridine (BrdUrd) positive nuclei
and the over-expression of cyclin D1protein. The authors noted that the results of this study suggest that
Benzophenone-1 is an endocrine disrupting chemical that exerts xenoestrogenic effects by, like E2, stimulating the
proliferation of BG-1 ovarian cancer via the estrogen receptor signaling pathway associated with the cell cycle.
A study was performed to evaluate the effects of Benzophenone-1 on prostate cancer progression, including
cell proliferation and migration.92 Additionally, the alterations in protein expressions of cell cycle related genes, as
well as cathepsin D gene as a metastasis marker by Benzophenone-1, were investigated in an effort to explain the
underlying mechanism. To evaluate the effect on cell proliferation, the 3-(4-5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay (n = 3) was performed using LNCaP prostate cancer cell cultures. This
cell line was originally isolated from the lymph node of a patient with metastatic prostate cancer. LNCaP cells were
treated with Benzophenone-1 (10-8 to 10-5 M) for 4 d. The incubation medium was described as phenol-free
Dulbecco’s modified eagle medium (DMEM) supplemented with 1% DMSO. To demonstrate the connection
between Benzophenone-1 and the androgen receptor signaling pathway, LNCaP cells were co-treated with
Benzophenone-1 (10-6 M) and biclutamide (10-9 M, androgen receptor antagonist; n = 3). In the migration assay,
LNCaP cells were treated with 10% charcoal/dextran-treated fetal bovine serum (FBS) containing 10-6 M
Benzophenone-1 for 5 d (n =4). The Western blot analysis was used to measure protein expressions for c-fos, cyclin
E, p321, and cathepsin D. LNCaP cells were cultured with Benzophenone-1 for a fixed period of time. After
treatment, whole cell lysates of LNCaP cells were prepared (in buffer solution) in a time-dependent manner (0, 24,
and 48 h). The proteins were transferred to a polyvinylidene difluoride membrane, and the membranes were
incubated overnight with the following antibodies: rabbit polyclonal anti-cyclin E, anti-c-fos antibody, anti-
cathepsin D antibody, mouse monoclonal anti-p21, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
This experiment was repeated (n = 3).
Benzophenone-1 increased the viability of LNCaP cells at concentrations of 10-6 M and 10-7 M. In the MTT
assay, when the cells were co-treated with Benzophenone-1 (10-6 M) and biclutamide (10-9), the cell viability that
was increased by Benzophenone-1 alone was statistically significantly reduced. These results suggest that the
proliferative effects of Benzophenone-1 on LNCaP cells was mediated by the androgen receptor signaling pathway.
In the experiments relating to cell mobility, Benzophenone-1 (10-6 M) increased cell migration when compared to
the DMSO control. In parallel with the changes in cell viability levels, the migration activity of LNCaP cells
increased by Benzophenone-1 was statistically significantly reduced by co-treatment with biclutamide (10-9 M).
These results indicate that the stimulatory effects on LNCaP cell migration induced by Benzophenone-1 were
mediated via the androgen receptor signaling pathway.
Protein expression of cyclin E, one of the proteins required for cell cycle progression, was enhanced by
Benzophenone-1 at 24 and 48 h. The protein expression level of p21 (regulator of cell cycle progression) was
statistically significantly reduced by Benzophenone-1 at 24 h, when compared to DMSO. Protein expression of c-
fos was not statistically significantly induced by Benzophenone-1. For cathepsin D (metastasis marker), its protein
expression levels were statistically significantly increased by Benzophenone-1 (10-4 M) at 24 and 48 h, when
compared to the control. To determine whether or not the effects of Benzophenone-1 on the expressions of cyclin E,
p21, and cathepsin D were mediated by the androgen receptor signaling pathway, a Western blot analysis was
performed on protein samples isolated from LNCaP cells treated with Benzophenone-1 (10-6 M) in the presence of
biclutamide. The protein levels of cyclin E, p21, and cathepsin D were not changed at 24 and 48 h. These results
may suggest that the protein expressions of these genes are induced by Benzophenone-1 via the androgen receptor
signaling pathway. The authors concluded that the results of this study indicate that Benzophenone-1 may enhance
the progression of prostate cancer by regulating cell cycle and metastasis-related genes via the androgen receptor
signaling pathway.
Benzophenone-1, Benzophenone-3, Benzophenone-6, and Benzophenone-8
The second process in carcinogenesis, promotion, was studied using the Bhas promotion assay.81 This is a test
that is used to detect the formation of transformation foci, using Bhas 42 cells established from BALB/3T3 cells.
Benzophenone-1, Benzophenone-3, Benzophenone-6, and Benzophenone-8 were tested, and each was evaluated at
concentrations ranging from 2 to 100 µg/ml. On day 21 of incubation, the cells were fixed with methanol and dyed.
After air-drying, the number of transformation foci was counted using a stereoscopic microscope. The
transformation foci were identified using the following 5 criteria: (1) more than 50 cells in a focus area, (2) cells in
the focus area are spindle-shaped and different from surrounding cells, (3) cells in the focus area across each other in
a random sequence, (4) cells grow in a stacked manner, and (5) the cytoplasm is intensely dyed by basicity. 12-O-
Tetradecanoylphorbol-13-acetate (TPA) served as the positive control.
Dosing (all doses) with Benzophenone-3 and Benzophenone-6 did not result in any statistically significant
increase in the number of foci relative to the solvent controls, indicating negative promotion activity. Particularly,
Benzophenone-6 and Benzophenone-8 produced less foci than the solvent controls at concentrations of 5 µg/ml and
above 20 µg/ml, respectively. Cell survival rates declined at the concentrations at which the number of foci
decreased. Thus, the effect of cytotoxicity was believed to have been the cause of the decrease in the number of
foci. For testing with Benzophenone-1, there was no increase in the number of foci at concentrations below 5
µg/µl. However, at 10 µg/ml, there was a statistically significant increase to 6 ± 2.4 foci/well. This increase was
more than twice that of the number of foci in the solvent controls (2.2 ± 1.5 foci/well). However, the increase was
1.5% per gram when compared to the number of foci in the positive controls (50 ng/ml TPA, 20.2 ± 5.2 foci/well).
At a concentration of 20 µg/ml, the number of foci was comparable to that of the solvent controls, but the cell
survival rate was lower (31%), suggesting toxicity of the test substance. Benzophenone-1 was believed to have been
a tumor promoter (at 10 µg/ml), based on results indicating that it caused a statistically significant increase to more
than twice that of the controls. However, the tumor promotion potential of Benzophenone-1 was apparently weak
when compared to the level in the positive controls. The authors noted that the results of this study indicate that
none of the test substances resulted in a statistically significant increase in the number of foci (relative to the solvent
controls DMSO and methanol) over the range of concentrations tested, indicating negative promotion activity.

ANTI-CARCINOGENICITY STUDIES
Benzophenone-8 and Benzophenone-12
The in vivo antitumor activity of Benzophenone-8 and Benzophenonone-12 was evaluated using a two-stage
mouse skin carcinogenesis model.93 In this model, (±)-E-4-methyl-2-[-E-hydroxyamino]-5-nitro-6-methoxy-3-
hexamide (NOR-1) served as the inducer and TPA as the promoter. Groups of 15 pathogen-free, female hairless
mice of the HOS:HR-1 strain were used. Skin tumors were induced by a single dose of NOR-1 (390 nmol in 100 µl
of acetone). At 1-wk post-dosing, TPA (1.7 nmol in 100 µl of acetone) was applied to the skin twice weekly for 20
wk as a tumor promoter. Each test substance was administered at a concentration of 0.0025% to mice through
drinking water (ad libitum), beginning at 1 week prior to tumor initiation and ending at 1 week after tumor initiation.
All animals were examined weekly for the development of skin papillomas. When compared to the positive control
(NOR-1) group, the following observations were made for both test substances: 2-wk delay in tumor appearance,
statistically significant inhibition (p < 0.001) of tumor incidence (60% for Benzophenone-8; 50% for
Benzophenone-12), and statistically significant inhibition of tumor burden (papilloma inhibition per mouse: 70% for
Benzophenone-8 and 50% for Benzophenone-12). Benzophenone-8 was a more potent inhibitor of skin tumors
than Benzophenone-12.

OTHER RELEVANT STUDIES

Effect on Gene Expression
Benzophenone-3
A study was performed to determine whether Benzophenone-3 exposure alters gene expression profiling in the
prostate and testis.79 Groups of 25 pregnant Sprague-Dawley rats were fed low-phytoestrogen chow containing
3000 or 30,000 ppm Benzophenonoe-3 from GD 6 until postnatal day 21. The male offspring were then weaned on
postnatal day 28, and subsequently dosed with Benzophenone-3 via chow and milk. The offspring were killed on
postnatal day 30 and tissue samples were collected. RNA samples from the prostate and testis (1 male pup per litter;
5 litters per group) were extracted. Microarray gene expression profiling was performed on the tissue samples.
Results indicated that gene expression profiles of the prostate and testis were differentially affected by
Benzophenone-3 dose and duration of exposure. Tissue-specific alterations were also indicated. Microarray
analyses of prostate gene expression patterns of rats exposed perinatally to Benzophenone-3 identified significant
expression of 334 and 689 genes in the 3000 and 30,000 ppm exposure groups, respectively, when compared to the
controls (p < 0.05; fold change > 1.5). Seventy-six genes overlapped between the 2 Benzophenone-3 exposure
groups in the prostate. Microarray analyses of testis-gene expression patterns identified 239 and 1159 genes that
were significantly altered in the testis in animals of the 3000 ppm and 30,000 ppm Benzophenone-3 perinatally
exposed groups, respectively. Between the 2 Benzophenone-3 exposure groups, 220 genes overlapped in expression
profile in the testis. The authors noted that the gene expression changes observed in this study were only observed
at concentrations that exceed typical human exposure to Benzophenone-3.
Effect on Melanogenesis
Benzophenone-2
The dual action of Benzophenone-2 in the biosynthetic pathway of melanin has been identified.94 It has been
observed to act as a weak competitive inhibitor of tyrosinase (inhibition constant (Ki) = 2.02 ± 0.09 mM; half
maximal inhibitory concentration (IC50) = 3.82 ± 0.39 mM). Both forms of Benzophenone-2 (protonated and
deprotonated) interact with tyrosinase, the enzyme that catalyzes the production of melanin from tyrosine.
Benzophenone-2 (at 250 and 500 µM) also accelerated the conversion of dopachrome (intermediate in melanin
biosynthesis) to melanin.
Neurotoxicity
Benzophenone-2
A study was performed, using groups of 10 male Wistar rats, to determine apoptosis and oxidative stress
markers in the rat brain after topical administration of Benzophenone-2.33 The markers studied were: active form of
caspase-3, pro-apoptotic protein (Bax), and anti-apoptotic protein (Bcl-2). The effect of dosing on these markers
was studied to determine whether Benzophenone-2 may be involved in the induction or exacerbation of
neurodegenerative changes. Benzophenone-2 was dissolved in a small amount (volume not stated) of ethanol and
olive oil, and formulated with Hascobase. The test substance was then applied to shaved skin at a dose of 100
mg/kg for 4 wk. Hascobase with a small amount of ethanol and olive oil was applied to the skin of control rats. In
the hippocampus, where the Benzophenone-2 concentration was ~3.5-fold lower than in the frontal cortex, no
statistically significant changes in oxidative stress and apoptosis markers were observed. In the frontal cortex, there
was no change in apoptosis markers, but, unexpectedly, the oxidative stress markers were reduced. The authors
concluded that Benzophenone-2 did not exacerbate oxidative stress and apoptosis markers in the hippocampus and
frontal cortex. However, it did lower oxidative stress in the frontal cortex.
Benzophenone-2 and Benzophenone-3
The effect of Benzophenone-2 and Benzophenone-3 on the neuroblastoma (SH-SY5Y) cell line was evaluated,
by studying effects on cell viability and caspase-3 (main executive enzyme in programmed cell death) activity).95
The MTT reduction test and LDH release activity assay were used. After a 72-h incubation period, both
Benzophenone-2 and Benzophenone-3 produced a statistically significant cytotoxic effect at concentrations of 10-5
M and 10-4 M in both assays. Additionally, both Benzophenone-2 and Benzophenone-3 caused an increase in
caspase-3 activity at much lower concentrations (from 10-8 M to 10-7 M). The authors noted that the results of this
study indicate that Benzophenone-2 and Benzophenone-3 adversely affected the viability of nerve cells, most likely
by enhancing the process of apoptosis.
Benzophenone-3
The toxicity of Benzophenone-3 to primary cortical neurons and primary cortical astrocytes (cultured from E17
and E19 rat fetuses) was studied.34 Cultures were treated with the following 3 concentrations at culture durations of
24 h, 48 h, and 7 d: 0.1 µg/ml, 1 µg/ml, and 10 µg/ml. Cell viability was analyzed using the standard MTT assay.
The experiments were performed in triplicates on a minimum of 3 independent cultures. Untreated cultures served
as controls. No significant differences in astrocyte viability were observed for a 24-h or 48-h exposure when
compared to the control group. A 36% decrease in neuron viability was observed when cultures were exposed to
Benzophenone-3 (10 µg/ml) for 7 d.
A study was performed to determine the effects of Benzophenone-3 on apoptosis and the expression of
estrogen, androgen, and arylhydrocarbon receptors (AhR) in the rat frontal cortex and hippocampus.96 The test
substance was administered dermally to pregnant female Sprague-Dawley rats and to their male offspring through 6
and 7 wk of age. Benzophenone-3 (in a cream) was applied to a 25 cm2 (5 cm x 5 cm) area on the back, at a dose of
100 mg/kg twice daily. After birth, the offspring were observed for any abnormalities daily. The animals were
killed at 24 h after the last dose of Benzophenone-3. Brain structures (hippocampus and frontal cortex) were
removed. Benzophenone-3 in the frontal cortex induced the mitochondrial apoptosis pathway by increasing the
active forms of caspase-3 and caspase-9, thereby inducing the pro-apoptotic proteins Bax and Bak and increasing the
number of cells with apoptotic DNA fragmentation. In the hippocampus, an increase in caspase-9 and a downward
trend in the level of anti-apoptotic proteins were observed. In both regions of the brain, the contents of estrogen
receptor beta (ERβ) in the nuclear fraction and G protein-coupled receptor 30 (GPR30) in the membrane fraction
were statistically significantly reduced. Benzophenone-3 statistically significantly increased AhR in the cytosol of
the frontal cortex, but had no effect on the content of this receptor in the hippocampus. The authors noted that the
results of this study indicate that exposure to Benzophenone-3 induces the mitochondrial apoptosis pathway in the
rat frontal cortex.
Mouse neuronal cells (from neocortical and hippocampal tissues prepared from Swiss mouse embryos) were
used to evaluate the neurotoxicity of Benzophenone-3 (in DMSO).97 Primary neuronal cell cultures were exposed to
Benzophenone-3 (1 to 100 µM) for 24 h. A continuous 24-h exposure of neocortical cultures to Benzophenone-3
(25 to 100 µM) induced apoptosis in mouse neuronal cells. Hippocampal cells exhibited weaker vulnerability.
The neurotoxicity of Benzophenone-3 and its metabolite (Benzophenone-1) was studied using female Sprague-
Dawley rats and their offspring.35 Benzophenone-3 (10% in cream; dose = 100 mg/kg) was administered dermally
(shaved skin on back) twice daily to adult female rats (number not stated) during the prenatal period and adulthood.
Control female rats were treated with cream without Benzophenone-3. At 21 d after birth, the offspring (male and
female) were divided into groups of 5 males and groups of 5 females. From 43 to 56 d of age, the test substance was
administered dermally to the male offspring. Cream without Benzophenone-3 was applied to control offspring. In
brain structures, selected markers of brain damage were measured. Results indicated that dosing with
Benzophenone-3 raised oxidative stress and induced apoptosis in the brain. Benzophenone-3 increased the
concentration of extracellular glutamate in examined brain structures and changed the expression of glutamate
transporters. The results of this study indicated that dermal Benzophenone-3 exposure may cause damage to
neurons that might be associated with the increase in the level of extracellular glutamate. The authors noted that this
increase is most likely evoked by changes in expression of the glutamate transporters, glutamate transporter-1
(GLT-1) and cystine/glutamate antiporter (xCT).
Behavioral Toxicity
Benzophenone-3
A study was performed to characterize the skin permeation and tissue disposition of Benzophenone-3 (in
ethanol) in rats (groups of 10; 5 males and 5 females per group).34 The test solution was applied (volume = 100 µl;
dose = 5 mg/kg (312.5 µg/cm2)) topically to a 4 cm2 area on the back, daily for 30 d. Results relating to skin
permeation and tissue distribution are included in the section on Skin Penetration. In this study, various behavioral
testing protocols were used to assess the arousal (open field tests), locomotion (open field and ladder test),
habituation (open field test), and motor coordination (open field and ladder test) of the animals over the study
duration. Each rat was tested individually, 4 h after dosing on day 29, to assess behavioral changes from the topical
applications. Except for positive controls, all animals (test and negative (saline and vehicle (70% ethanol solution)
control groups) passed the 29-d study period without significant adverse effects. Visible impairment was observed in
the positive control (acrylamide) group.
Immunomodulatory Effects
Benzophenone-2
The in vitro effect of Benzophenone-2 on the production of interferon (IFN)-ℽ and interleukin (IL)-10 was
studied.98 IFN-ℽ and IL-10 are two cytokines representing the Th-1 lymphocyte and Th-2 lymphocyte response,
respectively, by activated murine splenocytes. Splenocytes were cultured in the presence of different concentrations
of Benzophenone-2 (10-8 to 10-5 M). Benzophenone-2 (10-5 M) shifted the Th1/Th2 balance toward a Th2 response
(lower IFN-ℽ production and higher IL-10). The authors noted that these results show that Benzophenone-2 at high
doses may possess immunomodulatory effects.
Benzophenone-2 (in ethanol) was administered dermally (100 mg/kg), twice daily for 4 wk, to 10 male Wistar
rats.99 Immunological parameters were assayed 24 h after the last administration. Dosing with Benzophenone-2 did
not change relative weights of the spleen and thymus, and was not toxic to splenocytes and thymocytes. However,
dosing did increase the proliferative activity of splenocytes, and also enhanced the metabolic activity and viability of
splenocytes and thymocytes. The authors concluded that dosing with Benzophenone-2 increased the activity and
function of the immune cells (thymocytes and splenocytes).
Benzophenone-4
The immunosuppressive activity of Benzophenone-4 (0.01%) was evaluated using human dendritic cells (e.g.,
CD14+ human monocytes).100 Cytokines can be released by dendritic cells and regulate the activation of T cells.
The culturing of monocytes with Benzophenone-4 (0.01%) did not induce significant morphological changes and
did not impair monocyte differentiation. The monocytic marker CD14 was unchanged. The effect of
Benzophenone-4 (0.01%) on the expression of surface molecules that are critical for dendritic cell function was also
investigated. Immature and mature dendritic cells were cultured with Benzophenone-4 (0.01%). Immature
dendritic cells generated with or without the test substance showed a similar expression profile. In mature dendritic
cells, treatment with the test substance led to down-regulation of HLA-DR (major histocompatibility complex
(MHC) molecule) and CD40 (cell surface receptor that belongs to tumor necrosis factor receptor family) expression.
Benzophenone-4 treatment also slightly decreased the secretion of IL-12, but this did not reach statistical
significance. Treatment with Benzophenonoe-4 did not impair the proliferation of lymphocytes. Thus, in this study,
Benzophenone-4 modulated the phenotype and function of monocyte-derived dendritic cells. CD40 expression was
reduced by Benzophenone-4. All of these features suggest that the treatment of dendritic cells with Benzophenone-4
favors an immature activation status that can regulate T cell responses.
Endocrine Activation
Benzophenone-1, Benzophenone-2, Benzophenone-3,
Benzophenone-4, and Benzophenone-8
A study was performed to investigate the thyroid-activation potential of benzophenones, using a rat pituitary
carcinoma cell line (GH3 cell line) and a rat thyroid follicular cell line (FRTL-5 cell line).101 Also, zebrafish
(Danio rerio) embryo exposure (up to day 6 post-fertilization) involved the benzophenones
(Benzophenones-1, -2, -3, -4, and -8) that were identified based on the transcriptional changes that were observed in
the cells. The test concentrations in GH3 cells were as follows: Benzophenone-1 (up to 6.9 mg/l (32 µM)),
Benzophenone-2 (up to 2.5 mg/l (10 µM)), Benzophenone-3 (up to 22.8 mg/l (100 µM)), Benzopheone-4 (up to 98.7
mg/l (320 µM), and Benzopenone-8 (up to 24.4 mg/l (100 µM)). In FRTL-5 cells, the test concentrations were:
Benzophenone-1 (up to 68.6 mg/l), Benzophenone-2 (up to 78.8 mg/l), Benzophenone-3 (up to 73 mg/l),
Benzophenone-4 (up to 98.7 mg/l), and Benzophenone-8 (up to 24.4 mg/l). The test concentrations in zebrafish
embryos were: Benzophenone-1 (up to 1000 µg/l), Benzophenone-3 (up to 320 µg/l), and Benzophenone-8 (up to
320 µg/l). Results indicated that, in GH3 cells, Benzophenone-1 (1 to 32 µM), Benzophenone-2 (0.32 to 10 µM),
Benzophenone-3 (at doses around 32 µM), and Benzophenone-8 (at doses around 32 µM), but not Benzophenone-4,
statistically significantly down-regulated the Tshβ, Trhr, and Trβ genes. For Benzophenone-4 (concentration not
stated), slight but significant down-regulation was observed only for the Trβ gene. Additionally, some of the
benzophenones (Benzophenones -1, -2, -3, and -4 (10 to 320 µM; Benzophenone-8 (3.2 to 100 µM) statistically
significantly upregulated the Nis and Tg genes, while down-regulating the Tpo gene in the FRTL-5 cells. Zebrafish
larvae treated with Benzopheonone-3 and Benzophenone-4 had a statistically significant decrease in
triiodothyronine (T3) levels, but not thyroxine (T4) levels, at test concentrations as low as 32 µg/l. However,
Benzophenone-1 statistically significantly decreased both T3 and T4 levels in fish larvae at 320 and 1000 µg/l. The
up-regulation of the dio1 and ugtr1ab genes in the zebrafish suggests that decreased thyroid hormones are caused by
changing metabolism of the hormones. The results of this study indicate that benzophenones can alter thyroid
hormone balances by influencing the central regulation and metabolism of hormones.
Benzophenone-2
The endocrine activation potential of Benzophenone-2 was evaluated using groups of 11 ovariectomized adult
Sprague-Dawley rats.102 The test groups were dosed orally (by gavage) with 250 mg/kg and 1000 mg/kg
Benzophenone-2 (1 ml) daily for 5 d. Another group was dosed with estradiol valerate (600 µg/kg) according to the
same procedure. Control animals were dosed with olive oil. Dosing was initiated 14 d after ovariectomy. Average
food intake was significantly reduced during the treatment period. However, there were no differences in liver,
spleen, nor adrenal weights between test and control groups. Dosing with estradiol valerate resulted in significantly
increased uterine weight. Both doses of Benzophenone-3 also had this effect on the uterus. Blood luteinizing
hormone levels were statistically significantly reduced after dosing with estradiol valerate and 1000 mg/kg
Benzophenone-2. There was no evidence of changes in mRNA levels of gonadotropin releasing hormone in the
preoptic area of the hypothalamus. A dose-dependent suppression of T4 concentration by Benzophenone-2 was
observed. T3 levels were also reduced.
A dose-response experiment involving 5 doses (10, 33, 100, 333, or 1000 mg/kg) of Benzophenone-2 was
performed using female Sprague-Dawley adult, ovariectomized (ovx) rats (groups of 5).38 Doses were administered
(by gavage) once per day for 5 d. Free levels of Benzophenone-2 in rat serum were sufficient to induce an
unequivocal estrogen-like effect in the uterus. When compared to the vehicle (olive oil) control group, mean uterine
weight was increased statistically significantly in the 333 mg/kg and 1000 mg/kg dose groups. A similar study
(groups of 12; same doses and protocol) involved ovariectomized rats of the same strain.103 Estradiol-valerate
served as the positive control. None of the animals showed clinical signs of toxicity. Benzophenone-2 exerted an
estrogenic effect on the following uterine parameters at the administered doses: wet weight, complement protein 3
(C3), insulin-like growth factor (IGF1), and estrogen receptor β (ERβ) gene expression. According to results from
another study, Benzophenone-2 acts as a n ERα and ERβ agonist mimicking the effects of estradiol benzoate (E2).104
Benzophenone-2 was evaluated for its effect on the hypothalamic-pituitary-thyroid (HPT) axis.99 The test
substance was dissolved in a small amount (volume not stated) of ethanol and olive oil and formulated with
Hascobase. The test substance was then applied to shaved skin of 10 male Wistar rats at a dose of 100 mg/kg for 4
wk. HPT activity was increased, i.e., the level of TSH was reduced and the free fraction of T3 and T4 in the blood
was increased.
Benzophenone interference with the thyroid hormone axis was studied.105 Whether or not Benzophenone-2
inhibits key reactions of thyroid hormone biosynthesis catalyzed by thyroid peroxidase was examined in this study.
A novel in vitro assay, based on human recombinant thyroid peroxidase stably transfected into the human follicular
thyroid carcinoma cell line FTD-238, was used. Benzophenone-2 (300 nmol/l) combined with the thyroid
peroxidase substrate hydrogen peroxide (10 µmol/l) inactivated human recombinant thyroid peroxidase.
Benzophenone-2 interference with thyroid function was also studied in vivo.105 Groups of 12 adult female
Sprague-Dawley rats were bilaterally ovariectomized and fed a soy-free diet containing iodide ad libitum. At 14 d
after ovariectomy, groups of 12 rats were dosed orally (by gavage, once per day) with Benzophenone-2 at the
following doses (dose volume = 1 ml): 10 mg/kg, 33 mg/kg, 100 mg/kg, 333 mg/kg, and 1000 mg/kg. The animals
were killed at day 5, and thyroid glands were excised. A dose-dependent decrease in total serum T4 levels was
observed, with statistically significant alterations at doses of 333 mg/kg and 1000 mg/kg. The small decrease in
total T3 was not statistically significant. TSH levels were increased at doses of 333 mg/kg and 1000 mg/kg, and this
increase was statistically significant at both doses. Thyroid peroxidase activities in the thyroid glands of treated
animals were measured ex vivo, but no statistically significant dose-dependent changes were observed. In the livers
of animals treated with 1000 mg/kg Benzophenone-2, type I 5'-deiodinase activity was decreased, and this decrease
was statistically significant. However, an increase in type I 5'-deiodinase activity was observed at a dose of 33
mg/kg.
Benzophenone-3
The effect of a sunscreen containing Benzophenone-3 (10%) on thyroid function was studied using 32 subjects
(15 men and 17 women).106 The product was applied daily as a whole-body topical application (2 mg/cm2) in 1
week. The daily amount of cream applied over 4 d was 40 ± 3 g (mean value for men) and 35 ±3 g (mean value for
women). Hormone levels were measured by commercially available automated immunoassay systems. No
biologically significant effects on hormone levels were observed. This indicates that absorbed Benzophenone-3 was
not capable of disturbing the homeostasis of thyroid hormones in adult humans. There was no effect on TSH levels,
and there was no increase in the level of T4 or T3 in males or females.
The estrogenic activity of Benzophenone-3 was evaluated in a reporter gene assay using the human cervical
epithelioid HeLa cell line as the host cell line for the generation of stable reporter cells for screening substances that
act via human estrogen receptor alpha (hERα) and β (hERβ).107 The following 3 reporter cell lines (all estrogen
receptor cell lines) were used: HELN, HELN ERα, and HELN ERβ. HELN ERα and HELN ERβ cell lines
exhibited transactivation of luciferase gene expression by E2. Luciferase (served as the reporter) assays were
performed at concentrations between 10-7 and 10-5 M. Cells were incubated with Benzophenone-3 for 16 h.
Benzophenone-3 activated ERα moderately and had almost no effect on ERβ. Benzophenone-3 was not considered
estrogenic at 10-5 M.
The effect of Benzophenone-3 on the secretory and proliferative activity of rat (adult female Wistar rats)
adrenocortical cells was investigated in vitro.108 Within 120 min of culture, Benzophenone-3 (10-12 to 10-8 M)
stimulated basal corticosterone production from dispersed adrenocortical cells. The chronic, 24-h exposure to
Benzophenone-3 (10-10 M) increased basal corticosterone secretion from cultured adrenocortical cells. The
proliferative activity of the cultured adrenocortical cells was unaffected by treatment with Benzophenone-3.
Benzophenone-3 was evaluated for estrogenic potential, both in vivo and in vitro.109 In MCF-7 breast cancer
cells incubated for 6 d, Benzophenone-3 increased cell proliferation, with a median effective concentration (EC50)
between 1.56 and 3.73 µM. In the uterotrophic assay, immature Long-Evans rats (ages not stated) received
Benzophenone-3 in powdered feed for 4 d. An increase in uterine weight (weak effect, active at dose of 1525
mg/kg/d) was reported.
The estrogen/antiestrogen and androgen/antiandrogen effects of Benzophenone-3 were evaluated using
Saccharomyces cerevisiae strains BLYES and BLYAS.72 Concentration-response curves were fitted by nonlinear
regression. In the estrogen assay, an EC50 (half maximal effective concentration) value of 6.44E-03 mM was
reported for Benzophenone-3. In the androgen assays, Benzophenone-3 did not increase the bioluminescence of the
BLYAS strain. Thus, the androgenicity of Benzophenone-3 was not proven. In antiestrogen assays, Benzophenone-
3 showed a sigmoidal concentration-response curve. In antiandrogen assays, the EC50 value for Benzophenone-3
was 0.0102 mM. The results of this study indicate that Benzophenone-3 has estrogenic and antiandrogenic
potential.
Effect on Hematological Parameters
Benzophenone-2
Benzophenone-2 was also evaluated for its effect on hematological parameters.99 The test substance was
dissolved in a small amount (volume not stated) of ethanol and olive oil and formulated with Hascobase.
Benzophenone-2 was then applied to shaved skin at a dose of 100 mg/kg for 4 wk. Dosing with Benzophenone-2
had no effect on the following: leukocyte count, erythrocyte count, platelet count, erythrocyte morphology, and
erythrocyte hemoglobin content.
Cytotoxicity
The cytotoxicity of a sunscreen formulation composed of polymeric nanocapsules loading Benzophenone-3
was evaluated using the L929 fibroblast (murine) cell line.110 The nanocapsules contained poly(ℇ-caprolactone),
carrot oil, and a non-ionic surfactant. Cell viability was studied using the MTT assay for the assessment of cell
metabolic activity. The nanocapsules were seeded at a concentration of 30 µg/ml. Non-loaded (blank) and
Benzophenone-3-loaded nanocapsules did not exhibit metabolic changes or cell death in the cell culture. Cell
viability above 70 wt % was recorded (91.12 wt % for non-loaded and 89.45% for Benzophenone-3-loaded
nanocapsules). It was noted that these data indicate that the sunscreen formulation was non-cytotoxic.
 Photoprotective Effect
The photoprotective effect of Benzophenone-3 (in vehicle consisting of isopropyl myristate and SD alcohol)
against UVA radiation was evaluated using 30 female, Hartley albino guinea pigs.111 Applications were made to the
dorsal lumbar area (depilated skin). The erythema grade increased with increasing concentrations of Benzophenone-
3. At the vehicle control site, a mean erythema grade of 1.5 ± 0.11 was reported. Concentrations of 0.1% and 0.3%
produced erythema grades greater than 1+, and provided very little photoprotection. Significant photoprotection
was noted after the application of 1%, 3%, and 6% solutions (p ≤ 0.01, 0.001, and 0.001, respectively), with
erythema grades less than 1+ for the latter two treatments. The 6% solution resulted in greater photoprotection than
the 3% solution (p ≤ 0.001).
Phototoxicity Mechanism
Benzophenone-3 and Benzophenone-8
Benzophenone-3 (10 µM) significantly increased phosphodiesterase 4B (PDE4B) expression UVB (20
mJ/cm2)-irradiated normal human keratinocytes (from neonatal foreskins) in vitro.112 PDE4B has a well-established
role in inflammatory responses in immune cells. Additionally, upon UVB irradiation, Benzophenone-3 upregulated
the expression of pro-inflammatory factors such as prostaglandin endoperoxide synthase 2, tumor necrosis factor α,
IL-8, and S100A7. Benzophenone-3 downregulated the level of cornified envelope associated proteins, which are
important in the development of the epidermal permeability barrier. Benzophenone-8 (10 µM), which shares the
2-hydroxy-methoxyphenyl methanone moiety with Benzophenone-3, also upregulated PDE4B expression in normal
human keratinocytes. The Benzophenone-3 and UVB co-stimulation-induced PDE4B upregulation and its
association with the upregulation of pro-inflammatory mediators and the downregulation of epidermal
differentiation markers were confirmed in a reconstituted three-dimensional human epidermis model. The authors
concluded that PDE4B has a role in the mechanism of Benzophenone-3-induced phototoxicity.

DERMAL IRRITATION AND SENSITIZATION STUDIES

Dermal irritation and sensitization studies are summarized in Table 8.
Irritation
In Vitro
Benzophenones-3 and -4
The hen’s egg-chorioallantoic membrane test (HET-CAM) was used to evaluate the irritation potential of a
sunscreen formulation composed of polymeric nanocapsules loading Benzophenone-3.110 The nanocapsules
contained poly(ℇ-caprolactone), carrot oil, a non-ionic surfactant, and Benzophenone-3 (0.005 wt%). The
formulation was non-irritating to the embryonated hen’s egg membrane. The dermal corrosion potential of
Benzophenone-4 was determined using a three-dimensional human epidermis model,.8 Approximately 25 mg of
solid test article was evenly applied to the apical surface of each tissue. The exposure period for the test article was
up to 60 min, and the MTT assay was performed on exposed tissue samples. Benzophenone-4 was considered
corrosive to the skin.
Animal
Benzophenones-1, -2, -3, -4, -6, -9, and -11
At concentrations up to 16%, Benzophenones-1, -4, and -6 were non- to minimally irritating, and
Benzophenone-11 was non-irritating, to rabbit skin.1 Benzophenone-2 and Benzophenone-3 (both at 100%) were
non-irritating to rabbit skin. Benzophenone-9 was non-irritating to rabbit skin at concentrations up to 10.72%.
Results from a cumulative skin irritation test indicated that Benzophenone-4 was capable of causing minimal
irritation in rabbits at a concentration of 10%.
Benzophenones-3, -8, and -12
A sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was tested in a study involving 24 Wistar
albino rats (12 males, 12 females).68 The formulation (2000 mg/kg) was applied to a 2" x 2", 4-ply gauze pad, and
the patch was applied to hairless, dorsal skin for 24 h. There were no signs of erythema or edema. The skin
irritation potential of this sunscreen formulation (0.6% to 0.9% Benzophenone-3) was also evaluated using 6 male
New Zealand rabbits 68 The formulation was applied for 72 h to a 25 cm2 area of dorsal skin, using a 2" x 3", 4-ply
gauze pad. There was no evidence of erythema or edema. Benzophenone-8 was evaluated for skin irritation
potential using 3 New Zealand white rabbits.9 The test substance (0.5 g in water (0.5 ml)) was applied to the skin
for 4 h using a semi-occlusive patch. Benzophenone-8 was classified as a non-irritant. A skin irritation test on
Benzophenone-12 (ground to fine powder) was performed using 3 male New Zealand white rabbits.6 The test
substance was applied (0.5 g, abraded and intact skin of back) for 4 h under an occlusive patch. Benzophenone-12
was classified as non-irritating to the skin.
Human
Benzophenones-1, -2, -3, -4, -6, -8, -9, -11, and -12
Benzophenones-1, -2, -3, and -6 were nonirritating to the skin of human subjects at concentrations up to 16%.1
Benzophenone-1 and Benzophenone-6 were also nonirritating at a much higher concentration of 100%.
Benzophenone-4 was irritating at a concentration of 16% in one test, but nonirritating at concentrations of 5% and
25% in other tests. Benzophenone-11 was also irritating at a concentration of 16%, but nonirritating at 4%, 8%, or
20%. Benzophenone-3 and Benzophenone-12 were nonirritating at a concentration of 25%, but mild to no irritation
was observed at a lower concentration of 3% Benzophenone-3. Benzophenone-8 was irritating at a concentration of
25%, but nonirritating at 2%. Benzophenone-9 was non-irritating at concentrations up to 10.72%.
Benzophenone-4
The frequency of irritant reactions to Benzophenone-4 was studied using 80 subjects.113 Benzophenone-4 was
tested on each subject at concentrations of 2%, 5%, and 10% in petrolatum. Each test concentration of
Benzophenone-4 (20 µl) was applied to an 8-mm diameter Finn chamber. Patches were applied for 2 d to the upper
back. Benzophenone-4 (5% in petrolatum) induced skin irritation in 4 subjects. Benzophenone-4 (10% in
petrolatum) induced skin irritation in 6 subjects.
Sensitization
In Vitro
Benzophenone-8
The in vitro antioxidant response element (ARE)-nuclear erythroid 2-related factor 2 (Nrf2) Luciferase test
method was used to evaluate the skin sensitization potential of Benzophenone-8.9 The test substance was evaluated
at concentrations up to 200 mM in DMSO using the KeratinoSens cell line. Benzophenone-8 was classified as
positive in the KeratinoSens assay. The authors stated that further testing is required, having noted that this test is
part of a tiered strategy for the evaluation of skin sensitization potential.
Animal
Benzophenone-3
Benzophenone-3 was evaluated for skin sensitization potential using the Kligman guinea pig maximization
test.1 Induction involved intradermal injection of 5% Benzophenone-3 in corn oil or 50% Benzophenone-3 in
aqueous Freund’s Adjuvant. This was followed by challenge with 2.5% Benzophenone-3 in petrolatum. Results
were negative.
The skin sensitization potential of a sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was
tested in a study involving 10 adult male guinea pigs.68 The formulation was loaded on a 2 cm x 4 cm filter paper
that was secured with an occlusive dressing. Observations relating to challenge reactions were assessed after 24 h of
the induction, and reactions were scored. The sunscreen formulation was classified as a non-sensitizer. The local
lymph node assay was used to evaluate the sensitization potential of Benzophenone-3.10 Groups of 4 female mice of
the CBA strain were used, and the test substance was applied at concentrations of 12.5%, 25%, and 50%.
Applications were made to the dorsum of each ear lobe on 3 consecutive days. Benzophenone-3 was classified as a
non-sensitizer. The maximization test was used to assess the cutaneous allergenic potential of Benzophenone-12.114
Ten female albino guinea pigs were tested. The intradermal induction of sensitization in the test group was
performed with a 15% dilution of Benzophenone-12. The epidermal induction of sensitization was conducted for 48
h under occlusion with the test substance (at 40% in PEG 300). Two weeks after epidermal injection, the control
and test animals were challenged (24 h) with Benzophenone-12 (at 40% in PEG 300). Seven of 9 surviving test
animals had sensitization reactions. Benzophenone-12 was evaluated for skin sensitization potential in the
maximization test using 20 guinea pigs (10 males, 10 females) of the Pirbright white (Tif:DHP) strain.6
Benzophenone-12 was applied at a concentration of 5% during the first week of induction, and at a concentration of
30% during the second week. The challenge phase (week 5; i.e., 2 wk after induction) consisted of a single, 24-h
application of Benzophenone-12 (20% in petrolatum (w/w)). Results indicated that 65% and 60% of the animals
were sensitized to Benzophenone-12 at 24 h and 48 h after challenge, respectively.
Human
Benzophenones-1, -2, -3, -4, -6, -8, and -11
Benzophenone-1 was nonsensitizing at a concentration of 1% in human subjects.1 Evidence of fatiguing,
possible sensitization at 5%, and no sensitization at 2.5% were noted after testing with Benzophenone-2.
Benzophenone-3, Benzophenone-4, and Benzophenone-11 were nonsensitizing at a concentration of 10%.
Benzophenone-3 was also nonsensitizing at 3% in one test, but minimum sensitization at this concentration was
observed in another test. Benzophenone -4 also did not induce sensitization at a concentration of 5%.
Benzophenone-11 was a non-sensitizer at a higher concentration of 20%. Benzophenone-8 induced skin
sensitization at a concentration of 10%, but not at 2%. At a concentration of 100%, Benzophenone-6 did not induce
sensitization.

Photosensitization/Phototoxicity
Animal
Benzophenone-8 (3%) and Benzophenone-3 (6%) were non-phototoxic in guinea pigs and rabbits,
respectively.1
Human
Benzophenones-2, -3, and -4
Cosmetic products containing Benzophenones-2, -3, or -4 (0.l% to 3.5%) were evaluated for phototoxicity
using human subjects.1 Products containing Benzophenones-2, -3, and -4 were non-phototoxic in all studies;
however, a number of subjects experienced slight irritation (usually a 1 + response) to the test material. Cosmetic
products containing up to 3.5% Benzophenone-3 were tested for photoallergenicity potential in human subjects.
The products were non-photoallergenic in all studies; however, a number of subjects experienced irritation or
sensitization to the test material.

OCULAR IRRITATION STUDIES

In Vitro
Benzophenone-4
The ocular irritation potential of Benzophenone-4 was evaluated using the MatTek EpiOcular™ model, in
accordance with OECD TG 492.8 The viability of normal human-derived keratinocytes in the 3-dimensional human
tissue model following exposure to the test substance was determined via the MTT cytotoxicity assay. The 3-
dimensional tissue construct models the corneal epithelium, with progressively stratified, but not cornified, cells.
Tissues were exposed to Benzophenone-4 (solid, 50 mg) for ~ 6 h. The mean % tissue viability of Benzophenone-4
was determined to be 3.6%. Based on the results of this test, Benzophenone-4 was classified as irritating to the
human eye.
Benzophenone-8
An ocular irritation study on Benzophenone-8 was performed using the bovine corneal opacity and
permeability test (OECD TG 437).9 Corneas from 3 animals were exposed to the test substance (20% w/v in
paraffin oil; volume = 750 µl) for 4 h. The test substance was then removed from the front opening of the anterior
chamber and the epithelium was rinsed. For the evaluation of corneal permeability, the passage of sodium
fluorescein dye was measured using ultraviolet-visible (UV/Vis) spectrophotometry. Benzophenone-8 did not cause
corneal opacity or permeability, resulting in a mean in vitro irritancy score of 1 after 4 h of exposure. Based on
these results, the authors concluded that Benzophenone-8 was not a severe irritant or corrosive agent in the bovine
corneal opacity and permeability test.
Animal
Benzophenones-1, -2, -3, -4, -6, -8, -9, -11, and -12
Most of the ocular irritation tests indicated that Benzophenones-1, -2, -3, -6, -9, -11, and -12 were non-
irritating to the eyes of rabbits.1 Some studies indicated that Benzophenones-1, -2, and -4 were slightly to
moderately irritating at 100% concentration; however, Benzophenones-1 and -2 were nonirritating when tested at
16% in dimethyl phthalate (DMP) or petrolatum. Although Benzophenone-4 was irritating at concentrations of 8%
and 16% in DMP or petrolatum, it was nonirritating when tested as a 5% solution in water. Whereas one study
indicated that Benzophenone-11 (5% in DMP) was slightly irritating, another revealed that 16% Benzophenone-11
in DMP was nonirritating.
Benzophenone-3
The ocular irritation potential of a sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was
studied using 3 adult New Zealand albino rabbits.68 The formulation (100 mg) was instilled into the conjunctival sac
of the right eye of each animal. After instillation, eyes were examined macroscopically at intervals of 24 h, 48 h, and
72 h, and daily from 4 to 10 d, in accordance with the Draize scale. There were no signs of gross toxicity or adverse
effects. Corneal opacity and iritis were not observed during the study. At 1 h post-instillation, conjunctival
discharge was observed in 1 out of 3 eyes, but subsided within 96 h. The highest maximum mean total score
(MMTS) value (0.67) for ocular irritation was observed within 1 h after instillation, classifying the sunscreen
formulation as practically non-irritating to the eye.
Benzophenone-12
Benzophenone-12 was evaluated for ocular irritation potential using 6 New Zealand white rabbits (3 males, 3
females), in accordance with OECD TG 405.6 The undiluted test substance (0.1 g) was instilled once, and ocular
irritation was evaluated on days 1, 2, 3, 4, and 7. There was no evidence of ocular irritation (PII = 0).

CLINICAL STUDIES
Retrospective and Multicenter Studies
Benzophenone-3
Over 3400 patients (age range: 3 to 96 years) with suspected allergic contact dermatitis were evaluated and
then patch tested by 12 North American Contact Dermatitis Group dermatologists with a screening series of 50
allergens.115 The patients were patch tested (July 1, 1996 to June 30, 1998) using Finn chambers on Scanpor tape.
The patches remained in place for 48 hours, and sites were evaluated initially at 48 to 72 h, and, again, between 72
and 168 h after initial placement. A positive allergic patch test result was generally interpreted as a 1+, 2+, or 3+
reaction manifested by erythematous papules, vesicles, or a spreading reaction with crust and ulceration. The
relevance of the patch test reactions was determined in combination with the patient’s history and skin examination
findings, and were integrated to determine the diagnostic group. Of the 4094 patients patch tested with 3%
Benzophenone-3, 0.5% had allergic reactions (73.7% relevant reactions, i.e., definite, probable, or possible
relevance to patient’s present dermatitis).
A North American Contact Dermatitis Group (NACDG) study that was performed involved 5800 patients who
were patch tested with Benzophenone-3 (3% in petrolatum).116 Patch testing was performed from July of 1998 to
December of 2000. The patches remained in place for 48 h. Test sites were evaluated twice, initially at 48 h to 70 h
and, again, at between 72 h and 178 h after initial placement. A positive allergic patch test result was interpreted to
be a +, ++, or +++ reaction. Reactions of these types were manifested by erythematous papules, vesicles, or a
spreading reaction with crust and ulceration. The incidence of positive reactions was 0.6%. The relevance of this
incidence of positive reactions was classified as follows: 20.6% (definite relevance), 50% (possible relevance) and
2.9% (past relevance).
Data from 64 allergenicity studies (between 1992 and 2006) were aggregated and analyzed.117 This was done
in order to evaluate the irritation and sensitization potential of sunscreen products containing Benzophenone-3 at
concentrations between 1% and 6%. Forty-eight of 19,570 possible dermal responses were considered suggestive of
irritation or sensitization. The mean rate of responses across all formulations was 0.26%. Sensitization rates did not
correlate with Benzophenone-3 concentration. The available re-challenge data indicated that only 8 of these
responses were contact allergies due to Benzophenone-3. The mean rate of contact allergy to Benzophenone-3 was
0.07%. The authors concluded that these data indicate that that sunscreen products formulated with 1 to 6%
Benzophenone-3 do not possess a significant sensitization or irritation potential for the general public.
A cross-sectional analysis of patients patch tested by the NACDG between 2001 and 2010 was performed.118
Of the 23,908 patients who were patch tested, 219 (0.9%) had sunscreen coded as an allergen source. A frequent
allergen in sunscreens was Benzophenone-3, whereby 70.2% of the patients (26 of 37 patients patch tested) had an
allergic reaction to 10% Benzophenone-3 (in petrolatum) and 64.4% of the patients (56 of 87 patients patch tested)
had an allergic reaction to 3% Benzophenone-3 (in petrolatum). Values for the clinical relevance of allergic
reactions to 10% Benzophenone-3 (in petrolatum) in 26 of 37 patients were: definite relevance (5 of 26 patients
(19.2%)), probable relevance (11 of 26 patients (42.3%)), possible relevance (9 of 26 patients (34.6%)), and past
relevance (1 of 26 patients (3.8%). The clinical relevance values reported for 3% Benzophenone-3 (in petrolatum)
positive reactions in 56 of 87 patients were: definite relevance (13 of 56 patients (23.2%)), probable relevance (27
of 56 patients (48.2%)), possible relevance (15 of 56 patients (26.8%)), and past relevance (1 of 56 patients (1.8%).
NACDG patch testing results from January of 2007 to December of 2008 were reported.119 Standardized patch
testing was used at 13 centers in North America. A total of 5085 patients was tested. Five-hundred ninety-eight
patients (11.8%) had an occupationally-related skin condition and 3319 (65.3%) had at least 1 allergic patch test
reaction. Patches (Finn chambers, secured with tape) remained in place for 48 h. Reactions were scored at 48 h and
72 h to 168 h. At the end of testing, clinical relevance of positive patch test reactions was determined by
consideration of the patient’s history and clinical findings. Relevance of a positive allergen was categorized into
present, past, or unknown. Present relevance of a positive allergen was categorized as follows: definite (use test
with suspected item resulted in positive reaction, or a patch test to the object or product was positive); probable
(allergen could be verified as present in known skin contactants, and clinical presentation was consistent); and
possible (allergic reactions to Benzophenone-3 (3% in petrolatum) were observed in 22.7% (definite relevance) of
the patients. Other values relating to clinical relevance were: probable relevance (36.4%), possible relevance
(22.7%), and past relevance (6.8%).
A 3% aqueous solution of Benzophenone-3 was applied, in duplicate, to the midback of 4 patients, using Finn
chambers.120 At 48 h post-application, the test substance was removed, and sites evaluated for reactions. Reactions
were graded on a scale of +1 to +3. The other site containing the test substance was irradiated with UVA (8 J/cm2),
and then covered with light-opaque material. All sites were evaluated for reactions at 48 h post-irradiation. Contact
allergy was diagnosed as an equally positive reaction at nonirradiated and irradiated sites. Photoallergy was
diagnosed as a positive irradiated site with a negative unirradiated site. Allergy and photoallergy were diagnosed
when both sites were positive, but with the irradiated site having a greater reaction than the unirradiated site. The
results for a 3% aqueous solution of Benzophenone-3 are as follows: +2 reaction (without UVA) and +3 reaction
(with UVA) - Patient 1; +2 reaction (without UVA) and +3 reaction (with UVA) - Patient 2; +2 reaction (with UVA)
- Patient 3; and +2 reaction (without UVA) and +3 reaction (with UVA). All four patients were photoallergic to
Benzophenone-3. –
Over a 6-year period, 187 patients (76 males, 111 females) with a history of photosensitivity were photopatch
tested using standard techniques. Two-thirds of the patients were between the ages of 31 and 60 years.121 Phototest
allergens (Benzophenone-3, at 2% in petrolatum) were applied in duplicate to the patient’s midback, one set on
either side of the midline. Test material containing antigens was applied with aluminum disks (Finn Chambers) and
paper (Scanpor) tape. For the first two testing periods (January 1985 through February 1987 and March 1987
through August 1989), the antigens were left in place for 48 h. In the 3rd period (September 1989 through December
1990), the antigens were removed after 24 h. One set of antigens was then irradiated with UVA, 8 J/cm2 (January
1985 through August 1989) or 10 J/cm2 (September 1989 through December 1990). Both sets of antigens were then
covered with light opaque material (gauze pads and aluminum foil held in place with paper (Scanpor) tape). All
sites were evaluated for reactions at 48 hours post-irradiation. Second readings at day 7 post-irradiation were done
in the third test period (September 1989 through December 1990). Second readings were not done during the first
two test periods. Reactions were graded on a scale of ± to 3+. Testing revealed a total of 63 positive reactions,
classified as follows: 14 plain contact, 41 photocontact, and eight combined contact and photocontact in 37 (20%)
patients. Careful history taking resulted in a diagnosis of clinically relevant photoallergic contact dermatitis in 54%
of the 37 patients or 11% (20) of the total tested. Nine of the relevant responses were due to Benzophenone-3 (2%
in petrolatum).
Patients with positive photopatch tests to sunscreen agents were retrospectively selected from the database of
the contact dermatitis clinic at the Skin and Cancer Foundation in Australia.122 Benzophenone-3 (10% in white
petrolatum) was applied, in duplicate, using Finn chambers on Scanpor tape. Sites were covered with opaque
material. After 24 h, the test sites were examined and results were recorded. One site was irradiated with 5 J/cm2.
Reactions were scored on day 5 according to the standards of the ICDRG, and a final reading was performed on day
7. Nine patients had a positive photopatch test reaction to Benzophenone-3. Two patients had positive reactions at
non-irradiated sites.
 A study was performed to determine the proportion of photosensitive patients with photoallergic contact
dermatitis to Benzophenone-3.123 The study was a descriptive cross-sectional study involving 35 patients (11 men,
24 women) in Argentina with confirmed photosensitivity reactions. These patients had experienced at least 1
episode of photosensitivity reactions. Two sets of patches containing Benzophenone-3 (10% in petroleum jelly)
were applied to the back, 1 on the right and 1 on the left. At 48 h after patch application, 1 of the patches was
irradiated with a cumulative UVA dose (5 J/cm2; peak wavelengths of 350 nm, 365 nm, and 370 nm) over an 18-min
period. Reactions were scored at 30 min post-irradiation and at 96 h after patch application. A late reading was also
taken after 1 week. Photoallergic contact dermatitis was identified in 6 patients (17.14%). Five of these patients
(14.28%) had at least one positive reaction to Benzophenone-3 in the photocontact test. Four patients had a reaction
at the irradiated sites only, and 1 patient had a reaction at both irradiated and nonirradiated sites. The authors
concluded that photoallergic contact dermatitis to sunscreens containing Benzophenone-3 is common and is
probably underdiagnosed, due to a lack of confirmation by photopatch tests or other diagnostic tools.
Since 1990, seven sunscreen allergens have been included in the standard photopatch protocol at two Swedish
dermatology clinics.124 This study is based on 7 years of testing. Three-hundred fifty-five consecutive patients with
suspected photosensitivity were tested, and, in 28 of these (7.9%), a total of 42 allergic reactions was found. Eighty
percent of the reactions was of photocontact origin. The most common allergen was Benzophenone-3 (2% in
petrolatum), with 15 photocontact allergic reactions and 1 contact allergic reaction.
Benzophenone-4
In another study by the NACDG, 4857 patients were patch tested, and the positive reaction rate for
Benzophenone-4 (10% in petrolatum) was 2.1% (100 allergic reactions).125 Patch testing was performed using Finn
chambers. The values for clinical relevance of allergic reactions were: definite relevance (0), probable relevance
(20 of 100 patients (20%)), possible relevance (53 of 100 patients (53%)), and past relevance (9 of 100 patients
(9%)).
The phototoxicity of Benzophenone-4 was studied using 80 subjects.113 Benzophenone-4 was tested at
concentrations of 2%, 5%, and 10% in petrolatum. Each test concentration of Benzophenone-4 (20 µl) was applied
to an 8-mm diameter Finn chamber, secured with adhesive tape. Patches were applied (in duplicate) for 2 d to the
upper back, i.e., on non-paravertebral skin to the left and right of the upper back. At the time of patch removal, one
side of the back was covered with UV-opaque material, while the other side was irradiated with UV light (5 J/cm2;
99.2% UVA and 0.8% UVB). Reactions were scored according to the ICDRG grading scale. One subject had a
weak positive reaction (+ reaction), with no concomitant erythema score, to Benzophenone-4 (10% in petrolatum) at
the irradiated site.
Benzophenone-2, Benzophenone-3, and Benzophenone-4
Twenty-seven patients reported reactions due to sunscreen allergy (itchy bumps and burning).126 Of these, 11
(10 women, 1 man) patients agreed to photopatch testing. Finn chambers (8 mm, secured with tape) containing filter
paper wetted with the test substance (Benzophenone-2 or Benzophenone-3) were applied to the back. The chambers
were applied in duplicate for patch and photopatch testing. After 24 h, photopatches were removed and one set was
irradiated with UVA (10 J/cm2). Immediately after UVA exposure, photopatch tests were read to determine
immediate-type sensitivity reactions. Patch areas were then covered with opaque tape material. Another reading
was made 24 h later (day 3), and the final reading was made at 5 to 7 d. At the reading immediately after UVA
exposure, all reactions were negative, indicating the absence of contact urticaria. One patient had a delayed-type
hypersensitivity photopatch test reaction to Benzophenone-2 (1% in petrolatum), and another patient had a
photopatch test reaction to Benzophenone-3 (10% in petrolatum).
A retrospective analysis was performed, and involved the reviewing of 1527 charts in the University of British
Columbia Contact Dermatitis Clinic patch test database from January of 2009 to July of 2012.127 Twenty-three of
the patients were tested with the sunscreen series at the clinic. Also, as part of the regular screening at the clinic, all
1527 patients were patch tested with 70 allergens on the NACDG screening series. Benzophenone-3 and
Benzophenone-4 were tested at a concentration of 10% in petrolatum. Patch test chambers containing the test
substance were applied to the upper back and secured with tape for 48 h. Reactions were scored (using the ICDRG
grading scale) at the time of patch removal and at 96 h to 120 h. Of the 23 patients tested, 2 had positive reactions
(allergic contact dermatitis) to Benzophenone-3 and 1 had a positive reaction to Benzophenone-4. Additionally, of
the 1527 patients screened (no specific history of sunscreen allergy), 8 patients reacted to Benzophenone-3 in the
NACDG series. This number does not include the 2 patients who tested positive to Benzophenone-3, i.e., those who
presented with a positive history and were additionally tested with the sunscreen series.
 A total of 5592 patients was patch tested with Benzophenone-4 (10% in petrolatum, Finn chambers) in an
NADCG study.128 Of the patients patch tested, 93 had a positive (allergic) reaction. Values for the clinical
relevance of allergic reactions to 10% Benzophenone-4 (in petrolatum) were: definite relevance (3 of 93 patients
(3.2%)), probable relevance (12 of 93 patients (12.9%)), possible relevance (45 of 93 patients (48.4%)), and past
relevance (8 of 93 patients (8.6%)). In the same report, 5595 patients were patch tested with Benzophenone-3 (10%
in petrolatum, Finn chambers). Of the patients patch tested, 24 had an allergic reaction. Values for the clinical
relevance of allergic reactions to 10% Benzophenone-3 (in petrolatum) were: definite relevance (4 of 24 patients
(16.7%)), probable relevance (5 of 24 patients (20.8%)), possible relevance (11 of 24 patients (45.8%)), and past
relevance (1 of 24 patients (4.2%)).
The British Society for Cutaneous Allergy (BSCA) retrospectively reviewed the results from their facial patch
test series.129 This review involved 12 centers in the United Kingdom and Ireland for a 2-year period (January of
2016 to December of 2017). Of the 1390 patients patch tested with Benzophenone-4 (2% in petrolatum), 0.79%
(confidence interval (CI): 0.44% to 1.41%) had allergic reactions. Of 4224 patients patch tested with
Benzophenone-3 (10% in petrolatum), 0.17% (CI: 0.08% to 0.35%) had allergic reactions.
Fifteen patients (4 males, 11 females; mean age = 47.7 years) reacted to sunscreens.130 Eight patients had used
sunscreens before occasional sun exposure, and 6 had used them regularly for chronic lupus erythematosus,
melasma, vitiligo, rosacea, drug photosensitivity, and atopic dermatitis. One patient reacted to her daily cream
containing Benzophenone-3. Positive patch test (procedure not stated) results were as follows: 4 allergic contact
dermatitis reactions to Benzophenone-4, and 2 allergic contact dermatitis and 5 photoallergic contact dermatitis
reactions to Benzophenone-3.
Four-hundred-two patients (ages not stated) with suspected clinical photosensitivity were patch and photopatch
tested with UV absorbers, commercial sunscreens, facial cosmetics, fragrance materials, preservatives, and
emollients.131 Patch tests were performed according to ICDRG guidelines. A UVA dose of 5 or 10 J/cm2 was used
for photopatch testing. Benzophenone-3 and Benzophenone-4 were tested at a concentration of 10% in petrolatum.
Of the 402 patients, 80 patients (20%) presented with relevant allergic or photoallergic contact dermatitis to UV
absorbers. There were three allergic and nine photoallergic reactions to Benzophenone-3 and no photoallergic or
allergic reactions to Benzophenone-4.
Twelve patients with a history of acute eruption on photoexposed areas, induced by ketoprofen or tiaprofenic
acid, were patch tested.132 At least one month after the acute episode of contact dermatitis, the patients were patch
tested using the Finn Chamber technique. Finn chambers were mounted on Scanpor tape, and patches were removed
after 2 d. For UV irradiation, two sources of light were used (UVA alone and UVA + UVB). Photopatch test
results were positive for Benzophenone-3 (reactions in 3 of 12 patients) and negative for Benzophenone-4.
Photopatch testing (over 2-year period) of Benzophenone-3 and Benzophenone-4 was performed using 1155
patients from 17 centers across the United Kingdom, Ireland, and the Netherlands.133 Benzophenone-3 was tested at
a concentration of 10% in white paraffin. Benzophenone-4 was tested at concentrations of 5% and 10% in white
paraffin. Photopatch testing involved application of the test substance (on aluminum Finn chamber) to skin of the
mid-upper back (paravertebral area avoided) for 24 h or 48 h (depending on the center). The contact dermatitis units
traditionally applied allergens for 48 h, and photobiology units traditionally applied allergens for 24 h. Following
patch removal, one set (dark control) was covered with light-impermeable occlusive dressing, and the other was
irradiated with fluorescent UVA (5 J/cm2). Reactions were scored at 48 h post-irradiation, and, if possible, at 24 h
and 72 h. The ICDRG visual scoring system was used. Benzophenone-3 (10% in white paraffin) caused
photoallergic contact reactions in 27 patients. Benzophenone-4 (5% in white paraffin) and Benzophenone-4 (10% in
white paraffin) caused photoallergic contact reactions in 2 and 5 patients, respectively. The following allergic
reactions were also reported: 5% Benzophenone-4 (2 patients), 10% Benzophenone-3 (9 patients), and 10%
Benzophenone-4 (9 patients). Photoaugmentation and photoinhibition of contact allergy was observed in 1 patient
tested with 10% Benzophenone-3 and in 1 patient tested with 10% Benzophenone-4. The irritation reactions
observed included: 5% Benzophenone-4 (2 patients), 10% Benzophenone-3 (2 patients), and 10% Benzophenone-4
(4 patients).
A study was performed to identify the photoallergens that caused photoallergic contact dermatitis in the
population attending an outpatient clinic in Columbia.134 The study involved 82 patients with a clinical diagnosis of
photoallergic contact dermatitis. Photopatch tests were performed. The test substances (allergens, concentrations
not stated) were applied, in duplicate, to skin on the back. The test sites were covered with opaque tape for 24 h.
The panel on the right was irradiated with UVA (dose = 5 J/cm2; irradiance = 10.4 mW/cm2). Reactions were
scored 24 h after application of the allergen and at 24 h and 72 h post-irradiation. Both Benzophenone-3 and
Benzophenone-4 (concentrations not stated) induced a positive photopatch reaction. Benzophenone-3 was
photoallergenic in 22 of 82 patients (26.8%), and Benzophenone-4 was photoallergenic in 2 of 82 patients (2.4%).
An investigation of photoallergic contact dermatitis frequency was performed using 347 patients from centers
across 12 European countries.135 Benzophenone-3 (10% in petrolatum) or Benzophenone-4 (2% in petrolatum) was
applied to skin of the back, and removed at 48 h. One site was irradiated with UVA (5 J/cm2), and the other site was
covered with a UV-impermeable material. Reactions were scored at 48 h. Benzophenone-3 (10% in petrolatum)
elicited photoallergic contact dermatitis in 37 patients: + reaction (14 patients), ++ reaction (18 patients), and +++
reaction (5 patients). Benzophenone-4 (2% in petrolatum) elicited photoallergic contact dermatitis in 3 patients: +
reaction (1 patient) and ++ reaction (2 patients). Allergic contact dermatitis reactions (+ reactions) to
Benzophenone-3 (10% in petrolatum) were observed in 6 patients.
In a retrospective chart review, 160 patients (37 male, 123 female) underwent photopatch testing in Canada
between January of 2001 and December of 2010.136 Photoallergic, allergic, and irritant reactions were recorded for
26 common allergens. Duplicate sets of allergens (test concentration not stated) were applied to the patient’s back.
At 24 h, 1 set of allergens was uncovered and exposed to UVA at a dose of 5 J/cm2. The other set of allergens was
shielded from UVA exposure. Twenty-four-h reactions to the non-irradiated compounds were assessed at 15 to 20
min later. On the following day, the irradiated patches were read at 24 h post-irradiation. Reactions at non-
irradiated patch test sites were read 48 h after application. Benzophenone-3 induced photoallergic reactions in 12
patients, allergic reactions in 17 patients, and both allergic and photoallergic reactions in 6 patients. Benzophenone-
4 caused allergic contact dermatitis in 3 patients, but did not cause photoallergic reactions.
A prospective study was performed to evaluate the frequency and causes of photoallergic contact dermatitis
among dermatology outpatients.137 The study involved 1000 consecutive dermatology outpatients in Poland. All
patients with a history of dermatitis, induced or aggravated by exposure to light, were qualified by photopatch
testing. In the study group, 36 (3.6%; 95% CI: 2.4 - 4.8%) individuals required photopatch testing based on their
clinical symptoms. Because the total number of patients requiring patch tests of any kind amounted to 205, the
percentage of photopatch tested patients among all patch-tested patients was 17.5% (95% CI: 12.2 - 22.8%). Patch
tests (2 identical sets) were mounted on the back and remained under occlusion for 48 h. Some sites were irradiated
with UVA (5 J/cm2) and some were non-irradiated. Skin reactions were scored 24 h and 48 h after irradiation. The
presence of an inflammatory reaction at the irradiated sites and no reaction to the same hapten at non-irradiated sites
was interpreted as confirmation of photoallergy. In case of positive reactions to a hapten, both on irradiated and
non-irradiated sites, the classical contact allergy was recognized. Photoallergic contact dermatitis was ultimately
confirmed in 15 (1.5%; 95% CI: 0.7 to 2.3%) persons: 7 females and 8 males. Of these, 2 patients had a positive
reaction to Benzophenone-3 (10% in petrolatum). One patient had a positive reaction to Benzophenone-4 (2% in
petrolatum).
The photopatch testing of sunscreens was performed in a study involving 157 children (69 male, 88 female).138
Tests were performed in a single photo-investigation center during years 2000 to 2011. A duplicate series of UV
filters and the children’s own sunscreen products was applied to the back (ingredient test concentration not stated).
Reactions were scored at the time of sample removal and at 24 h and 48 h after exposure to UVA (5 J/cm2). Ten
children (5 to 7%) had positive photopatch reactions to UV filters and/or their sunscreen products (4 to 5% to UV
filters; 5 to 7% to their sunscreen products). Benzophenone-3 induced photoallergy (2+ reaction) in 33% of the
children (n = 3). A single case of a photoaugmentation reaction to Benzophenone-4 was reported. This patient had
a + reaction in the control panel, but had a ++ reaction in the irradiated panel.
Benzophenone-3, Benzophenone-4, and Benzophenone-10
From 1989 to 1991, 214 patients were patch tested to a sunscreen series containing 9 constituents.139 Standard
closed patch testing was employed, using Finn Chambers applied to the upper back skin. The patches were removed
at 2 d, and readings were made at the time of patch removal and at 3 or 4 d. Forty-five patients had photosensitivity
dermatitis/actinic reticuloid syndrome and 54 had polymorphic light eruption. Of the 214 patients, 16 reacted to one
or more sunscreens. The Benzophenones were the most frequent sensitizers. Benzophenone-3 and Benzophenone-
10 accounted for 27 and 8 positive patch tests, respectively.
Over a period of 3 years, 553 patients were patch tested (Finn chambers) with 10% Benzophenone-3, 10%
Benzophenone-4, and 10% Benzophenone-10.140 The dose per area was not stated. Results were recorded at 48 h
(day 2) and 96 h (day 4). Positive reactions (+ to +++) were graded according to International recommendations.
Thirteen patients (8 females, 5 males) and 1 patient had positive reactions to 10% Benzophenone-3 and 10%
Benzophenone-10, respectively. Thirteen patients had positive reactions to 10% Benzophenone-4. One patient had
a positive reaction to both Benzophenone-3 and Benzophenone-4.
A retrospective analysis of positive photopatch test episodes was undertaken using results retrieved from the
environmental dermatology database, and further verified with the original archived patch test documentation for
each individual patient.141 In 111 patients with positive reactions (4.1%), there were 155 allergic contact or
photoallergic reactions to allergens in the photopatch series. On day 0, the standard photoallergens were applied
(test concentration not stated) to the patient’s back in duplicate. On day 2, the patches were removed, and one series
irradiated with 5 J/cm2 of broadband UVA (2.5 J/cm2 used if history indicated clear episodes of severe
photosensitivity or patient suspected of having chronic actinic dermatitis). Eighty photoallergic reactions were
observed in 62 (2.3%) patients (32 men and 30 women), with UV filters accounting for 52 positive reactions. It
should be noted that 34 of the 62 patients (55%) had a preceding underlying photodermatosis. The most common
UV filter photoallergen was Benzophenone-3 (14 positive results), followed by Benzophenone-10 (n = 9). Forty-
nine patients (1.8%) had a total of 75 allergic contact reactions, 51 due to UV filters. Benzophenone-10 accounted
for 13 allergic contact reactions, and Benzophenone-3 accounted for eight allergic contact reactions.
A study was conducted to determine the threshold UVA elicitation dose in photopatch testing. Twenty-three
patients with a variety of photosensitive disorders were patch and photopatch tested.142 Benzophenone-3 and
Benzophenone-10 produced positive responses at 0.7 and 1.07 J/cm2, respectively. Isopropyl dibenzoyl
dibenzoylmethane produced a positive response at 1.0 J/cm2. These results demonstrate that high doses of UVA
(e.g., 10 to 15 J/cm2) are unnecessary, and that 5 J/cm2 should become the current standard.
Seven patients with ketoprofen-induced photodermatitis were patch tested and photopatch tested with
Benzophenone-3, Benzophenone-4, and Benzophenone-10 (test concentrations not stated).143 The aim of the study
was to evaluate the possibility of cross-reactivity between ketoprofen and benzophenones and other chemicals
because of their structural similarities. Patch tests (uninvolved skin of back) were performed using Finn chambers.
At 24 h post-application, a separate series of patch tests was exposed to suberythemal doses of UVB and UVA.
Irradiated and non-irradiated sites were evaluated at 72 h post-application. All non-irradiated patch test results for
the three benzophenones were negative. Four and 2 patients had positive UVA photopatch tests to Benzophenone-3
and Benzophenone-10, respectively. Photopatch test results for Benzophenone-4 were negative.
The photoallergenicity of Benzophenone-4 (10% in petrolatum) and Benzophenone-10 (10% in petrolatum)
was evaluated using 15 eczematous dermatitis patients.144 Testing was performed at least 3 months after complete
disappearance of the dermatitis. In photopatch tests, the test substance was applied to the back, under occlusion,
over a 2-d period. At 24 h, the occlusive patch was removed, and the site was exposed to UVA (5 J/cm2). Reactions
were scored at 48 h and 96 h (day 2 and day 4). There were no positive reactions to Benzophenone-4 (10% in
petrolatum). Three subjects had positive reactions to Benzophenone-10 (10% in petrolatum).
From February 1985 to March 1987, 280 patients with photosensitivity and other patients (ages not stated)
suspected of sunscreen dermatitis were patch and photopatch tested with a series of contact allergens and
photoallergens (test concentration = 2% in petrolatum).145 All tests were read at 2 d, and, at this time, the duplicate
light series was exposed to UVA (1 J/cm2). The second and final reading of all tests was carried out at 4 d. Fifteen
patients had positive patch and/or photopatch tests. Three were allergic to more than one UV absorber. During the
first 16 months of the study period (February 1985 to May 1986), there were two patients who were allergic to
Benzophenone-10. In the remaining 10 months, 4 patients were allergic to Benzophenone-10. Photopatch results
for Benzophenone-10 were negative.
Case Reports
Benzophenone-2
Epicutaneous tests were performed on two patients (both with itching erythema) who had been using nail
varnish and nail varnish remover and one patient who had artificial nails (itching erythema at perionychium of
several fingers; also marked erythema and edema).146 The three patients had sensitization reactions to important
allergens in nail varnish (tolunenesulfonamide-formaldehyde resin), nail varnish remover (Benzophenone-2), and
artificial nails (ethyl acrylate), respectively. Symptoms and skin changes disappeared when these three items were
no longer used.
A male patient presented with subacute chest and arm eczema after use of a toilet water product.147 A repeated
open application test with his product elicited a positive reaction after only two applications. Patch testing with an
ingredient of the product, Benzophenone-2 (2% in petrolatum), yielded a positive reaction (++). No reactions were
observed in 15 control subjects.
Severe dermatitis was observed in a female patient.148 The dermatitis worsened after sun exposure, and was
accompanied by severe itching. Cosmetic contact dermatitis was suspected and patch tests (protocol not stated)
were performed. Patch test results for Benzophenone-2 (1% in petrolatum) were positive. A reaction classified as
+++ (strong reaction: erythema, papules, and vesicles) was observed on days 2, 4, and 7.
Benzophenone-3
Erythema and blistering (at application) were observed after a female patient applied ketoprofen gel topically
to the right popliteal fossa and right shoulder. 149 After intermittent exposure to sunlight (over 24-h period), the
eruption extended to involve the legs, neck, hands, and other parts of the body. The patient was patch tested with
ketoprofen and its components using Finn Chambers on Scanpor tape. Photopatch testing (irradiation with 6 J/cm2
UVA) was also performed. Positive patch and photopatch test reactions to ketoprofen (up to 2% in petrolatum) were
reported. Patch test results for Benzophenone-3 were negative; however, a positive photopatch test reaction to
Benzophenone-3 (+++) was reported on day 4. The authors noted that, when irradiated with sunlight, ketoprofen is
broken down into various benzophenones that are structurally related to Benzophenone-3.
A female patient who applied a sunscreen experienced itching and a burning sensation of the nose, cheeks, and
dorsa of the hands after 3 h of direct sun exposure.150 Open photopatch testing of the sunscreen on a 2 cm2 area of
forearm skin produced an erythematous, papular response 24 h after a single exposure to UVA (25 J/cm2),
suggesting photoallergy. Patch testing with an ingredient of the sunscreen, Benzophenone-3 (2% in petrolatum),
yielded a +++ reaction. Histology of a biopsy from the Benzophenone-3 photopatch-test reaction showed a striking
epidermal spongiotic response and vesicle formation, with absence of vacuolation and sunburn cells. A prominent
mononuclear inflammatory cell filtrate was observed in the dermis.
Anaphylaxis (with generalized cutaneous wheal and flare reaction) was observed in a female patient after
widespread application of a sunscreen to the skin.151 The patient had a history of atopic dermatitis and allergic
rhinoconjunctivitis. A few days before testing, she experienced contact urticaria after coming in contact with
someone who had applied the same sunscreen to the face. Patch testing with the sunscreen (applied to normal skin)
yielded a 2-cm diameter wheal and flare reaction within 5 min. Blinded patch testing with Benzophenone-3
(sunscreen ingredient, concentration not stated) induced a wheal (16 mm) and flare reaction after 15 min. Non-
blinded patch tests for Benzophenone-3 in 2 control subjects yielded negative results. In prick tests, results for the
sunscreen and Benzophenone-3 were positive (wheal, 6 x 7 mm).
An acute, itchy rash was observed on a female patient (face, trunk, and limbs) after application of a sunscreen
to her daughter’s skin.152 Additionally, the patient subsequently applied a ‘false tan’ product to her skin and
developed a violent reaction, described as a severe cutaneous and systemic anaphylactic reaction. The patient was
patch tested with Benzophenone-3 (concentration not stated), and reactions were scored 20 min after patch
application. An acute urticarial wheal and flare (50 mm) reaction was observed at 20 min, and the reaction settled
within 1 h after oral drug treatment. The patch testing of 5 control subjects with Benzophenone-3 did not reveal any
reactivity at 20 min, 48 h, or 96 h.
In another case report, a male patient presented with the following history: intensely pruritic bilateral lip;
perioral, cheek, ear, hand, and forearm dermatitis; and painful ulcerations of the oral mucosa.153 On examination, 1
to 4 mm papules and papulovesicles (coalescing into edematous plaques) were present on the dorsal hands, fingers,
volar wrists, dorsal forearms, and upper arms. Patch testing was performed according to NACDG methods, using
Finn chambers secured with tape. The patient had a strong reaction to Benzophenone-3 (3% in petrolatum) at 48 h
(+++ reaction) and 96 h (+++ reaction).
A female patient experienced an anaphylactic reaction 15 min after applying a sunscreen all over her body.154
Generalized wheals were observed. The patient previously had pruritus and erythema within 30 min of putting her
bathing suit on, which she had worn earlier during sunscreen application. Patch testing with Benzophenone-3 (10%
in petrolatum) resulted in an urticarial reaction at the test site within 20 min. Patch testing did not elicit anaphylaxis
in this patient. Photopatch tests (without Benzophenone-3) were negative. No specific immunoglobulin E (IgE)
antibodies were found against inhalation allergens or latex. An assay for the detection of IgE to Benzophenone-3
was performed by incubating Benzophenone-3 with human serum albumin. No specific IgE to Benzophenone-3
was detected.
Benzophenone-3 and Benzophenone-4
Persisting erythema on light-exposed skin was reported in the history of a male patient who had applied
sunscreen on several occasions.155 Patch testing with ingredients of the sunscreen was performed. The patient was
patch tested using Finn chambers applied to the back, followed by UVA irradiation (dose = 10 J/cm2) at 24 h.
Reactions were scored at 20 min, and 24 h, 48 h, and 72 h post-irradiation. Photopatch test results were negative for
Benzophenone-4. Photopatch test results for Benzophenone-3 were as follows: + (at 24 hours), ++ (at 48 hours),
and +++ (at 72 hours).
Hand dermatitis was observed in a female hairdresser over a 2-year period.156 Patch testing with and
Benzophenone-4 yielded a positive (++) reaction. When she stopped using hair-care products with sun protection,
the dermatitis began to improve. Further patch testing with Benzophenone-4 (10% in petrolatum) yielded a positive
reaction, but patch test results for Benzophenone-3 were negative.
A female patient presented with a 2- to 3-year history of intermittent burning and pruritic facial eczema.157
Erythema of the cheeks bilaterally and on the neck, and minimal scale (but no vesicles) were observed. She had
used a facial moisturizer and a shampoo, both of which contained Benzophenone-3, for 2 years. Patch testing and
photopatch testing were performed using the Finn chamber technique. Results were scored on days 3 and 7.
Results were significant for a 2+ photocontact reaction to Benzophenone-3. A questionable photocontact reaction to
Benzophenone-4 was reported. There was no reaction to Benzophenone-3 when the site was not irradiated.
Immediately after irradiation (10 J of UVA), urticaria at the Benzophenone-3 photopatch test site was observed.
This reaction was consistent with photoallergic contact urticaria. The authors noted that the patient’s burning,
itching, and erythema resolved when she avoided contact with benzophenones in her personal care products.
Another case involved a female patient with a 1-year history of perioral itching and erythema, and a 3-d history
of erythematous swelling over her face and front of her neck.157 The patient had been sitting in the sun for a few
hours, several days before the swelling began. She had also used a lip balm and shampoo, both of which contained
Benzophenone-3. The patient had a 1+ reaction to Benzophenone-3 at both patch and photopatch test sites. The
perioral itching resolved within several days after discontinuing the lip balm. Additionally, the facial erythema
improved greatly after the shampoo was replaced with another that did not contain benzophenone.
A case of acute facial swelling in a diver has been reported.158 The diver’s face (left side) became swollen
after ascending to the surface of the water. An in vitro ImmunoCap IgE assay was positive to latex. Subsequent
patch testing (standard test) for contact dermatitis yielded a positive reaction to Benzophenone-4. The patch testing
protocol and test concentration were not stated.
Benzophenone-3, Benzophenone-4, and Benzophenone-10
A female patient presented with eyelid dermatitis for 1 year and facial dermatitis for two months.159 Patch tests
(procedure not stated) results for benzophenones were as follows: Benzophenone-3 (++), Benzophenone-4 (+), and
Benzophenone-10 (negative results).
Benzophenone-3, Benzophenone-8, and Benzophenone-10
Face eczema developed in a female patient after use of a cosmetic cream.160 Patch tests involving
Benzophenone-3 and Benzophenone-10 were performed using a polyethylene chamber secured with tape. Reactions
were scored on days 2 and 4 according to ICDRG methodology. Photopatch testing was also performed, whereby
the 2 benzophenones were applied in duplicate. The test substances were removed after 24 h, and the sites were
irradiated with UVA (5 J/cm2). Reactions were scored at day 1 and day 3 post-irradiation. For Benzophenone-3,
positive patch test (++ reaction) and photopatch test (+++ reaction) reactions were reported. For Benzophenone-10,
patch test results were negative, but photopatch test results were positive (+++ reaction).
A female patient was referred for phototesting and patch testing after recurrent episodes of dermatitis and systemic
symptoms.161 The first episode occurred 24 h after application of a sunscreen, and was described as follows:
edematous, painful pruritic eruption on the arms and neck; voice changes; and tachycardia. UVA phototesting at 10,
5 and 2.5 J/cm2 yielded normal results. Additionally, NACDG patch and photopatch test panels were applied. At 2
h after patch application and 1 h later, the patient experienced the following: raspy voice, dry mouth, difficulty with
swallowing, and tachycardia. On the next day, 24-h patch test reactions were as follows: fragrance mix (1+), 2(2-
hydroxy-5-methylphenyl)benzotriazole (++), and triclosan (+). Urticarial reactions to Benzophenone-3,
Benzophenone-8, and Benzophenone-10 at test sites were also observed. Because of the severe reactions, UVA
irradiation was not completed. The authors noted the occurrence of symptoms 2 h after application and severe
associated urticarial and systemic symptoms. The authors also stated that immediate reactions to and systemic
symptoms caused by these benzophenones are rare.
Other Clinical Reports
Benzophenone-3
Benzophenone-3 (2% to 10%), Benzophenone-4 (1 % to l0%), Benzophenone-8 (2% to 10%), and
Benzophenone-10 (0.5% to 10%) have been tested for sunscreen efficacy in large populations of human subjects,
and under various sources of UV radiation.1 In all tests combined, there were no reports of irritancy or phototoxic
reaction to these ingredients.
Benzophenone-3
A study was performed to identify association between exposure to potentially endocrine-activating chemicals
and the age of menarche in adolescent girls.162 Data from 1598 participants who had completed the reproductive
health questionnaire and laboratory examination for the Centers for Disease Control and Prevention’s National
Health and Nutrition Examination Survey (NHANES) for years 2003 to 2008 were used. Exposures were assessed
based on creatinine-corrected natural log urine concentrations of selected environmental chemicals and metabolites
found in at least 75% of samples in this study sample. The weighted mean age of menarche was 12 years of age.
Results for Benzophenone-3 included in this study indicated that exposure to this chemical was not significantly
associated with the age of menarche.
The association of Benzophenone-3 with serum total testosterone levels was examined using child and
adolescent participants in NHANES (2011–2012).163 Multivariable linear regression was performed to estimate
associations between natural log–transformed serum testosterone and quartiles of urinary Benzophenone-3 in male
and female children and adolescents. Serum testosterone was analyzed by isotope dilution LC-MS/MS, and was
natural log–transformed for analyses because the distribution of this variable was skewed left. Spot urine samples
were collected from study participants, and Benzophenone-3 was measured by solid phase extraction, coupled on-
line to HPLC/MS/MS. Statistical tests for linear trends were conducted by modeling quartiles as an ordinal variable
using integer values.
Male adolescents in the 3rd and 4th quartiles of Benzophenone-3 had statistically significantly lower
testosterone than males in the lowest quartile. Although the association was strongest for the 3rd quartile, the
overall trend was statistically significant (p-trend = 0.01). In female adolescents, testosterone was statistically
significantly higher for girls in the second versus first quartile of Benzophenone-3 exposure, but positive
associations were closer to the null and nonsignificant for the 3rd and 4th quartiles of exposure (p-trend = 0.14).
There were no significant associations between testosterone and Benzophenone-3 in male or female children, and no
evidence of consistent trends with increasing quartiles of exposure. Thus, Benzophenone-3 was associated with
statistically significantly lower testosterone in adolescent boys only. The authors concluded that urinary levels of
Benzophenone-3 were associated with lower levels of serum testosterone in male adolescents.
The influence of Benzophenone-3 and other chemicals on the age of menarche in 200 girls was studied.164 A
log w/v increase in childhood (pre-pubertal) urinary levels of Benzophenone-3 was associated with decreased time
to menarche. Benzophenone-3 urinary concentrations values were not reported.
The association between maternal urinary phenol concentrations during pregnancy and fetal growth was
studied in a population of 476 mothers wo had participated in a birth cohort between 2006 and 2008.165 An
association between urinary Benzophenone-3 and lower abdominal circumference in males was made. However,
the authors noted that this association should be verified in larger study populations with planned repeated
ultrasound measures during pregnancy.
A study was performed to study the association between prenatal exposure to Benzophenone-3 and gestation
age and birth weight.166 Specifically, relationships between birth outcomes and urinary concentrations of
Benzophenone-3 were evaluated. The study involved a cohort of 922 pregnant women. Urinary Benzophenone-3
was measured at 3 time points in pregnancy (visit 1: 16 - 20 wk; visit 2: 20 - 24 wk; visit 3: 24 - 28 wk). Multiple
linear regression (MLR) models were performed to regress gestational age and birthweight z-scores against each
woman’s log average concentrations of exposure biomarkers. Logistic regression models were performed to
calculate odds of preterm birth, small or large for gestational age (SGA and LGA), in association with each of the
exposure biomarkers. Results were transformed into the change in the birth outcome for an inter-quartile-range
difference in biomarker concentration (∆). Average Benzophenone-3 urinary concentrations were associated with
an increase in gestational age.
A study for determining an association between urinary phthalates, parabens, and phenols found in personal
care products with pubertal timing in girls and boys was performed.167 The study was a longitudinal cohort study
involving 338 children. No such association relating to urinary Benzophenone-3 was found.
Placental weights and birth weights were available for 473 mother-son pairs in a cohort for whom
Benzophenone-3 was measured in spot urine samples. Urine was collected between weeks 23 and 29 of gestation.168
A positive association between Benzophenone-3 and both placental weight and child birth weight was observed.
A study was performed to examine whether maternal and paternal preconception urinary concentrations of
Benzophenone-3 (e.g., from dietary and personal care product exposure) and other chemicals were associated with
the risk of preterm birth among couples attending fertility care.169 This study included 417 female and 229 male
participants of EARTH study who gave birth to 418 singleton infants between 2005 and 2018. Mothers and fathers
provided an average of 4 and 3 urine samples during the preconception period, respectively. The geometric mean of
Benzophenone-3 was calculated in order to estimate the preconception exposure of each participant. Risk ratios
(RRs) of preterm birth (live birth before 37 completed weeks of gestation) were estimated using modified Poisson
regression models adjusted for covariates. The mean gestational age among singletons was 39.3 (1.7) weeks and 8%
born preterm. No consistent pattern of association was observed for Benzophenone-3 in either parent.
Benzophenone-4 and Benzophenone-10
The allergenicity of Benzophenone-4 and Benzophenone-10 was evaluated using 15 eczematous dermatitis
patients (6 women, 9 men).144 The patients were patch tested (protocol not stated) at least 3 months after complete
disappearance of the dermatitis. None of the subjects had positive reactions to 10% Benzophenone-4 in petrolatum.
Two subjects had positive reactions to 10% Benzophenone-10 in petrolatum.

EPIDEMIOLOGICAL STUDIES
Benzophenone-3
A case-control study on idiopathic male infertility and exposure to phenols in the environment was
performed.170 The study involved 877 idiopathic infertile men and 713 fertile controls. Urinary concentrations and
semen parameters (semen volume, sperm concentration, and sperm number per ejaculate) were measured. There
was no evidence for an association between exposure to Benzophenone-3 and idiopathic male infertility.
Hirschsprung’s disease is a neonatal intestinal abnormality that is derived from a failure of enteric neural crest
cells migration during embryogenesis from 5 to 12 weeks.52 Urinary levels of Benzophenone-3 and the incidence of
Hirschsprung’s disease were investigated using a total of 423 patients in China.171 The patients were tested for
Benzophenone-3 in the urine via a spot test, and then divided into groups based on the presence of Hirschsprung’s
disease. Group 1 comprised 101 neonates with Hirschsprung’s disease who presented with intestinal obstruction
and chronic constipation, and were treated with surgery. Group 2 comprised 103 surgical control infants without
Hirschsprung’s disease. A third group (Group 3, non-surgical control) consisted of 219 neonates without
Hirschsprung’s disease. Results indicated a positive association between women identified with medium to high
levels of Benzophenone-3 (maximum detection level = 22,800 ppb) in the urine and the incidence of Hirschsprung’s
disease. A calculation relating to the concentration of Benzophenone-3 in the blood after a 4-h application of a
sunscreen product containing 6% Benzophenone-3 is presented at the end of the section on Absorption, Distribution,
Metabolism, and Excretion – Human.52 In this publication, the authors noted that since the embryonic period of
neural crest cell migration associated with Hirschsprung’s disease does not occur until weeks 5-12 of pregnancy,
women can unintentionally expose their fetus to extremely high levels of Benzophenone-3 over time. They noted
that the analysis of human exposure levels to Benzophenone-3 from sunscreen use, under normal conditions,
demonstrates that enough Benzophenone-3 can cross into the mother’s blood, making it available to the fetus at high
enough levels that can inhibit migration of neural crest cells during critical embryonic development.
Benzophenone-1, Benzophenone-2,
Benzophenone-3, and Benzophenone-8
A total of 413 men provided urine and semen samples (years 2005 to 2009), and the relationship between
urinary concentrations of benzophenones and semen quality was studied.172 Linear mixed models with fixed and
random effects were used to assess changes in semen endpoints associated with the following benzophenones that
were quantified in the urine: Benzophenone-1, Benzophenone-2, Benzophenone-3, and Benzophenone-8. The
investigators estimated the change (β-coefficients and accompanying 95% CI) in semen endpoints (e.g., sperm
concentration, total sperm count, and sperm motility) for men above the 75th percentile for each benzophenone
concentration relative to men below this percentile. Initially, regression models were run, including only the
benzophenone and creatinine concentrations. The rationale for modeling creatinine continuously was to account for
the interindividual variation in concentration, to more closely reflect men’s urinary dilution while preserving
statistical power. Benzophenone-2 was associated with findings such as diminished sperm concentration, more
immature sperm, and a decreased percentage of other tail abnormalities. Benzophenone-8 was associated with
decreased hypoosmotic swelling and higher acrosome area. No associations were observed for Benzophenone-1 or
Benzophenone-3. Overall, the authors noted that Benzophenone-2 and Benzophenone-8 were associated with
changes in semen endpoints, including sperm concentration, sperm viability, motility, sperm head, and morphology.
The authors noted that whether such changes are sufficient to affect couple fecundity, as measured by the time
needed to achieve pregnancy, or other couple-dependent fertility outcomes, remains to be established.
A study was performed to examine associations between urinary concentrations of benzophenone-type UV
filters and semen quality and reproductive hormone levels.173 The study was described as a cross-sectional study
involving 215 university students. All men provided urine, blood, and semen samples on a single day. Urinary
concentrations of the following benzophenones were measured: Benzophenone-1, Benzophenone-2,
Benzophenone-3, Benzophenone-8, and 4-hydroxybenzophenone. In the same subjects, semen quality was
evaluated by measuring volume, sperm counts, motility, and morphology. Serum samples were analyzed for the
following reproductive hormones: FSH, LH, testosterone (T), inhibin B, and E2. Associations between urinary
benzophenone concentrations, semen quality parameters, and reproductive hormone levels were examined using
linear regression, adjusting for potential cofounders.
Of the men tested, 97% had detectable urinary concentrations of at least 1 of the 5 benzophenone filters
quantified. After adjusting for important covariates (i.e., body mass index, smoking status, and time of blood
sample collection), the following results were: statistically significant positive association between urinary
Benzophenone-1 and Benzophenone-3 concentrations and serum FSH levels; urinary Benzophenone-1 concentration
statistically significantly positively associated with T/E2; and urinary Benzophenone-1 concentration negatively
associated with inhibin B/FSH ratio. No statistically significant associations between the following were found: the
other benzophenones and other reproductive hormone levels or between any semen parameters and any of the
urinary benzophenones. The authors concluded that, in young men, urinary benzophenone-type UV filters may be
associated with a modest alteration of some reproductive hormones, but the reported effects on reproductive
function are likely to be small, and of unclear clinical significance.
Benzophenone-1, Benzophenone-3
The presence of UV filters in semen, serum, and the urine was studied using 300 men.174 Samples were
collected during February to December of 2013, and only 6 of the men had used sunscreen during the 48 h preceding
sample collection. Benzophenone-1 and Benzophenone-3 were detected in 19% and 27% of the seminal fluid
samples, respectively, albeit at levels of 1 to 2 orders of magnitude lower than were detected in urine. For
Benzophenone-1 and Benzophenone-3, levels in the urine and seminal fluid were significantly correlated. The
authors concluded that chemical UV filters are present in men’s seminal fluid, some of which can activate the
human sperm-specific CatSper Ca2+ channel (calcium cation channel of sperm) and thereby potentially interfere with
the fertilization process.
Risk Assessment
Dermal
Benzophenone-3
Results from a risk assessment on Benzophenone-3 exposure indicated margin of safety (MOS) values of 42
for whole body sunscreen treatment twice per day over 6 h, and 1307 for face sunscreen treatment twice per day
over 6 h.25 The authors noted that a MOS of >100 is considered acceptable. Regarding the lower MOS value, the
authors noted that if personal care products containing Benzophenone-3 at the maximum concentration authorized in
the European Union and Australia (10%) would be applied on the total area of the human body (0.5 mg/cm2 twice
daily for 6 h), the MOS value of 42 indicates a possible health risk.
The daily systemic exposure dose and MOS for UV filters was estimated by in vitro permeation studies for the
6-h skin exposure of the face or the whole body in humans to a sunscreen, defined as a silicone-based oil-in-water
emulsion containing 10% Benzophenone-3 and 5% ethylhexyl triazone 175 Three in vitro experiments were
performed using a full-thickness porcine-ear skin mimicking in-use conditions. Ear skin was obtained from pigs that
were approximately 6 months old, and the skin disc was mounted in the diffusion cell. In the first experiment, the
sunscreen was spread uniformly onto the diffusion area (2 cm2), and the exact sunscreen dose was 1 mg/cm2. This
yielded a Benzophenone-3 dose of 100 µg/cm2 during the 6-h exposure. The receptor chamber was filled with
phosphate buffered saline. The second experiment involved a 3-h reapplication (100 µg/cm2 Benzophenone-3) of
the sunscreen to intact skin containing the 100 µg/cm2 Benzophenone-3 dose (total dose = 200 µg/cm2
Benzophenone-3). The procedure for the third experiment was the same as in first, except that freshly shaved skin
was exposed. The estimated systemic exposure dose of Benzophenone-3 after sunscreen application (at 1 mg/cm2)
for 6 h to the face and whole-body skin was estimated to be 136 mg/cm2 and 30 mg/cm2, respectively. Skin shaving
increased Benzophenone-3 bioavailability by 1.38-fold. MOS values were estimated according to guidelines
applicable for the European Union. For 3 realistic exposure scenarios, MOS values of 48, 34, and 34 for
Benzophenone-3 in sunscreen applied to the whole-body indicated some concerns regarding safety for consumers
(MOS < 100).
The following safety evaluation (including calculation of the MOS) of Benzophenone-3 was performed by the
Scientific Committee on Consumer Products (SCCP).176
Benzophenone-3 as a UV-filter in sunscreens up to 6%

Dermal absorption (6% formulation): 9.9% [mean (3.1%) + 2 SD (2 x 3.4%)]

Applied dose (sunscreen): 18 g/d
Typical human body weight: 60 kg
No observed effect level NOAEL (oral teratogenicity-rat): 200 mg/kg body weight/d

Systemic exposure dose (SED) = 18.103 mg/d x 6/100 x 9.9/100)/60 kg

= 1.78 mg/kg body weight/d

MoS = NOAEL/(SED) = 112

Benzophenone-3 as a UV-filter in cosmetics at 0.5% to protect formulations against sunlight

Dermal absorption (2% formulation): 8.0% [mean (4.0%) + 2 SD (2 x 2.0%)]

Applied dose (all cosmetic products): 17.79 g/d
Typical human body weight: 60 kg
No observed effect level NOAEL (teratogenicity-rat): 200 mg/kg body weight/d

Systemic exposure dose (SED) = (17.79.103 mg/d x 0.5/100 x 8.0/100)/60 kg

= 0.119 mg/kg body weight/d

MoS = NOAEL/SED = 1686

SCCP’s opinion on the safety of Benzophenone-3 is stated as follows: SCCP is of the opinion that the use of
Benzophenone-3 as a UV-filter up to 6% in cosmetic sunscreen products and up to 0.5% in all types of cosmetic
products to protect the formulation does not pose a risk to the health of the consumer, apart from its contact
allergenic and photoallergenic potential.

SUMMARY
Benzophenones-1 to -12 are substituted derivatives of a 2-hydroxybenzophenone. Most benzophenones are
soluble in inorganic solvents, but insoluble in water.
In the 1983 original report and in 2020, Benzophenone-2 (299 uses) and Benzophenone-4 (2259 uses) had the
highest reported use frequency, respectively. The use frequency of Benzophenone-2 (299 uses) in the 1983 original
report decreased to a value of 103 in 2020. The use frequency of Benzophenone-4 (240 uses) in the 1983 original
report increased substantially to a value of 2259 in 2020. Of the ingredients reviewed in the1983 report,
Benzophenone-4 had the highest use concentration (≤ 10% in suntan gels, creams and liquids (leave-on products)).
In 2020, Benzophenone-4 is the benzophenone with the highest reported use concentration, and is being used at
substantially lower concentrations of up to 1.6% in other non-coloring hair preparations (leave-on products).
According to the FDA, Benzophenones-3, -4, and -8 are active ingredients that are allowed in sunscreens. In
2019, FDA determined that there are insufficient data for determining that these 3 ingredients are GRASE in OTC
sunscreen drug products. In an in vitro skin penetration study using excised human epidermis, Benzophenone-3
passed through the skin in significant amounts (0.08 g/m2 or 10% of applied dose). Results from another in vitro
study (human skin) indicated that Benzophenone-3 penetrated very quickly in less than 30 min, and that there was
no difference in the mean quantity in the stratum corneum at 30 min versus 16 h. For Benzophenone-4, the quantity
in the stratum corneum at 30 min was statistically significantly lower at 30 min than at 16 h.
In rats, Benzophenone-2 was detected in the blood, liver, adipose tissue, and in the brain after application to
the skin. Metabolism to its sulfate and glucuronide forms was also reported. Results from another rat study indicate
that Benzophenone-3 was also detected in the plasma, liver, and brain after application to the skin, and that
Benzophenone-1 was the main metabolite.
After application of Benzophenone-3 (in solution/cream) to the skin of human subjects, it was detected in the
stratum corneum and was excreted in the urine. After dermal application of a sunscreen lotion containing
Benzophenone-3 to human subjects, Benzophenone-3 was detected in the stratum corneum, but not in the plasma or
urine. In another study, a sunscreen containing Benzophenone-3 was applied to human subjects. Some sites were
irradiated, whereas others were not. Benzophenone-3 was absorbed and excreted in the urine. Sunscreen
application has also resulted in the presence of Benzophenone-3 and the following metabolites in the urine:
Benzophenone-1, 2,3,4-trinydroxybenzophenone, and 2,2’-dihydroxymethoxybenzophenone. Other studies have
also supported the absorption and excretion of Benzophenone-3 after dermal application. Benzophenone-4 was also
detected in the stratum corneum of human subjects after dermal application.
Results from a SCCP risk assessment using data from a skin penetration study involving full-thickness pig ear
skin were used to arrive at a conclusion relating to the safety of Benzophenone-3. A MOS of 1686 was calculated,
and the SCCP concluded that the use of Benzophenone-3 as a UV-filter up to 6% in cosmetic sunscreen products
and up to 0.5% in all types of cosmetic products to protect the formulation does not pose a risk to the health of the
consumer, apart from its contact allergenic and photoallergenic potential.
In vitro toxicokinetic studies were performed using human and in vitro (whole zebrafish embryos) cell models.
Benzophenone-2 was metabolized into a variety of gluco- and sulfo-conjugated metabolites. When Benzophenone-
3 was incubated with rat liver microsomes in the presence of NADPH, the metabolites formed were 2,5-dihydroxy-
4-methoxybenzophenone and Benzophenone-1. In a similar experiment, the following Benzophenone-3 metabolites
were formed: Benzophenone-1; 2,4,5-trihydroxybenzophenone; 3-hydroxylated benzophenone-3; 5-hydroxylated
benzophenone-3; and 2,3,4-trihydroxybenzophenone. In the presence of human liver microsomes and NADPH,
Benzophenone-3 was metabolized to Benzophenone-1 and 5-hydroxylated benzophenone-3.
In a dermal metabolism and disposition study on [14C]Benzophenone-3 involving rats, the absorbed dose was
excreted mainly in the urine and feces, with ~3% to 10% of the absorbed dose remaining in the tissues.
When administered orally (gavage) to rats, Benzophenone-2 was metabolized to glucuronide- and sulfate-
conjugates. It was suggested that this biotransformation occurs in a first-pass effect in the gut wall or the liver.
Following the oral dosing (in corn oil) of rats with Benzophenone-3, it was converted to Benzophenone-1, which
was converted to 2,3,4-trihydroxybenzophenone. Benzophenone-3 was also metabolized to 2,2' dihydroxy-4-
methoxybenzophenone. In an oral (gavage) metabolism and disposition study involving rats and mice, overall,
[14C]Benzophenone-3 was well-absorbed and excreted mainly in the urine. The distribution of Benzophenone-3 in
tissues was minimal in rats and mice, and urinary metabolites included: benzophenone-3 glucuronide,
Benzophenone-1, benzophenone-1-glucuronide, and benzophenone-1 sulfates. Novel minor dihydroxy metabolites,
including 2,5-dihydroxy-4-methoxybenzophenone, were also detected. Results from an oral dosing (dietary) study
on Benzophenone-12 involving rats indicated metabolism to its glucuronide conjugate, and that Benzophenone-12
had no bioaccumulation potential.
Benzophenones-1, -2, and -3 have been detected in the urine of human subjects who had not been dosed with
either benzophenone. The same is true for Benzophenones-4, -6, and -8. Furthermore, Benzophenone-3 has been
detected in human brain white matter and in human breast tissue.
 In an acute dermal toxicity study (rats) on a sunscreen formulation containing 0.6% to 0.9% Benzophenone-3,
and LD50 of > 2000 mg/kg was reported. In a similar study on Benzophenone-12 involving rabbits, the LD50 was >
10,000 mg/kg.
After oral dosing (method not stated), Benzophenone-1 was classified as practically non-toxic (LD50 = 8600
mg/kg) in rats. The acute oral (gavage) LD50 (rats) for a sunscreen formulation containing Benzophenone-3 (0.6%
to 0.9%) was > 2000 mg/kg. An acute oral (gavage) LD50 of 3530 mg/kg for Benzophenone-4 was reported in a
study involving rats. Acute oral (gavage) dosing of rats with Benzophenone-8 resulted in an LD50 of > 2000 mg/kg.
An LD50 of > 10,000 mg/kg was reported for rats dosed orally (in water) with Benzophenone-12.
In a short-term (2 wk) oral (diet) toxicity study involving B6C3F1 mice, the NOAEL for microscopic lesions
was 6250 ppm. The same NOAEL for microscopic lesions was reported in a short-term (2 wk) oral toxicity study
involving groups of 10 F344/N rats. In a short-term oral (gavage) toxicity study, groups of 26 Wistar rats were
dosed orally with Benzophenone-4 at 2 wk prior to mating and 48 d thereafter. Female rats were dosed orally for a
total of 66 d. A NOAEL of 1250 mg/kg/d was reported for males and females. Groups of 6 male rats of the
Carworth Farms Elias strain were fed Benzophenone-12 in the diet for 35 d. No significant gross lesions were
observed. Repeated oral (gavage) dosing of groups of 24 Wistar rats with Benzophenone-12 (0.5%
carboxymethylcellulose suspension in drinking water) during a pre-mating period (10 wk for males; 2 wk for
females), a 2-wk mating period, and up to 30 d of lactation, a NOAEL of 1000 mg/kg/d for general systemic toxicity
was determined.
In a 2-wk dermal toxicity study involving groups of 10 B6C3F1 mice, dosed topically with Benzophenone-3
(0.5 to 8 mg in alcohol or lotion vehicle), minimal effects (variable increases in liver weight) were reported. In
another 2-wk study, groups of 10 F344/N rats received topical applications of Benzophenone-3 (1.25 to 20 mg in
alcohol or lotion vehicle). Minimal effects (small and variable increases in liver and kidney weights) were observed.
The findings reached statistical significance in the higher dose groups.
Benzophenone-3 (in ointment base, 100 mg/kg) was non-toxic when applied to the skin of groups of 4 to 6
male Sprague-Dawley rats twice daily for 4 wk. In another study, mated female Sprague-Dawley rats received
dermal applications of Benzophenone-3 (10% in cream; dose = 100 mg/kg) during the prenatal period and
adulthood. Their male offspring subsequently received dermal applications from 43 to 56 d of age. No adverse
effects on pregnant females or on the offspring were noted.
In a 90-d oral study (dosing method not stated) involving rats (number and strain not stated), a NOAEL of 236
mg/kg/d was reported. In a 13-wk oral toxicity study involving groups of 20 B6C3F1 mice, a NOAEL of 6250 ppm
was reported. When groups of 20 F344/N rats were fed Benzophenone-3 in the diet in this study, the same NOAEL
was reported. In another study, groups of 20 Sprague-Dawley rats received 10,000 ppm Benzophenone-3 in the diet
for 14 wk. In males, the absolute and relative liver and kidney weights were increased relative to the control group.
In females, the absolute kidney weight was significantly decreased, but the relative liver weight was significantly
increased relative to the control group.
The embryotoxicity of Benzophenone-3 was evaluated in an in vitro test involving zebrafish embryos.
Malformation of the somites was observed at concentrations of 0.0562 and 0.0789. The number of hatched embryos
at 96 h post-fertilization was also decreased.
Pregnant mice (number not stated in abstract) were exposed dermally to Benzophenone-3 (50 mg/kg/d) from
GD 0 to 6. Dermal exposure resulted in an intrauterine growth restriction (IUGR) phenotype, disturbed sex ratio,
and alterations in the growth curve of the offspring. In a 13-wk dermal dosing study involving groups of 20 B6C3F1
mice, it was not possible to establish a NOAEL for decreased epidermal sperm density due to this effect at doses up
to the highest dose of 364 mg/kg.
In a developmental toxicity study involving groups of 5 pregnant C57BL/6NCr mice, oral dosing with
Benzophenone-2 (6.25 mg) on GD 12 through 17, eight of 57 male fetuses had hypospadias (p = 0.0064). In a
continuous breeding study involving Swiss CD-1 mice, the animals were fed Benzophenone-3 at concentrations up
to 5% during a 7-d precohabitation period and a 98-d cohabitation period. Minimal effects on fertility and
reproduction were observed. From pregnancy (day 0) to the day before weaning (lactational day 21), mated
BALB/c female mice were dosed orally with Benzophenone-3 (in tocopherol-stripped corn oil) at doses of 30, 212,
and 3000 µg/kg/d. The offspring (no less than 9 litters per dose) were exposed in utero and during the first 21 d of
postnatal life. Study results suggested that even low doses of Benzophenone-3 can disrupt hormone sensitive organs
during critical windows of development.
 In an oral dosing study, the reproductive toxicity of Benzophenone-1 was evaluated using female rats (number
and strain not stated). After 3 d of dosing, a NOAEL of 10 mg/kg/d was reported. The oral dosing of groups of 5
Sprague-Dawley rats with Benzophenone-2 for 5 d caused a statistically significant increase in mean uterine weight
at the 2 highest doses of 33 mg/kg and 1000 mg/kg. The same effect for Benzophenone-2 was observed in a study
(same protocol) involving groups of ovariectomized rats of the same strain. In groups of 25 mated Wistar rats of the
Crl:WI (Han) strain, Benzophenone-3 (in corn oil) was orally at doses of 40, 200, and 1000 mg/kg/d on days 6
through 19 post-coitum. The NOAEL for Benzophenone-3 was 200 mg/kg/d. Groups of 25 pregnant Sprague-
Dawley rats were fed low-phytoestrogen chow containing 3000 or 30,000 ppm Benzophenone-3 from GD 6 until
postnatal day 21. The higher dose caused statistically significantly lower weights of the paired-testis, paired-
epididymis, and prostate. There were no changes in the relative weights of the paired epididymis and prostate in
either exposure group. There also were no differences in seminal vesicle weight. Groups (7 to 8 animals per group)
of mated female Sprague-Dawley rats were fed dietary concentrations up to 50,000 ppm Benzophenone-3 (in low-
phytoestrogen chow) from GD 6 until weaning on postnatal day 23. There were no statistically significant
differences in the following: mean number of implantation sites/litter, mean resorptions per litter, % litters with
resorptions, number and weights of live fetuses, or sex ratios between the control and Benzophenone-3 dose groups.
On GD 6, groups of 42, 35, 35, and 43 F0 time-mated female rats were fed diets containing 0, 1,000, 3,000, and
10,000 ppm Benzophenone-3, respectively, for 39 d. Groups of 50 (1,000 and 3,000 ppm) or 60 (0 and 10,000 ppm)
F1 rats per sex continued on study after weaning, and were fed diets containing the same exposure concentrations for
105 wk. Benzophenone-3 had no effects on the percentage of mated females producing pups, litter size, pup sex
distribution, or numbers of male or female pups. In a 13-wk oral dosing study, F344/N rats (10 males and 10
females per group) received diets containing 0, 3125, 6250, 12500, 25000, or 50000 ppm Benzophenone-3. At
50,000 ppm Benzophenone-3, markedly lower epididymal sperm density and an increase in the length of the estrous
cycle were observed.
In a study involving groups of 26 Wistar rats (13 males, 13 females/group), Benzophenone-4 was administered
orally (in corn oil, by gavage) at doses of 750, 1000, and 1250 mg/kg/d. The NOAEL (reproductive toxicity) for
Benzophenone-4 was 1250 mg/kg/d.
Benzophenone-12 (in 0.5% carboxymethylcellulose suspension in drinking water + 5 mg/100 ml Tween 80)
was administered orally to groups of Wistar rats (F0 animals: 12 males, 12 females/group) at doses of 100, 300, and
1000 mg/kg/d. The duration of treatment was described as follows: 10-wk premating period (males), 2-wk
premating period (females), 2-wk mating period (both sexes), ~2 d post-mating (males), entire gestation period, up
to 30 d of lactation (corresponding to 21 d of lactation and up to 9 d post-weaning), and 35 d post-mating (for
sperm-negative females). The NOAEL for reproductive performance and fertility of the F0 parental rats and
developmental toxicity in the offspring was 1000 mg/kg/d. The same NOAEL was reported in another study in
which Benzophenone-12 (in 0.5% carboxymethyl-cellulose suspension in drinking water + 5 mg/100 ml Tween 80)
was administered orally at doses of 100, 300, and 1000 mg/kg/d using groups of 50 Wistar rats (25 males, 25
females). The groups were dosed daily, from implantation to one day prior to the expected day of parturition (GD 6
to 19).
Six pregnant albino Swiss mice were injected s.c. with Benzophenone-3 (in peanut oil, 50 mg/kg) once daily
for 10 d (from the 7th to 16th day of gestation). Dosing resulted in severe apoptosis and neurotoxicity in neocortical
neurons. Thus, Benzophenone-3 can pass through the placenta and blood-brain barriers, and, therefore, can affect
infant neurodevelopment.
In a case-control study on idiopathic male infertility and environmental exposure to phenols, there was no
evidence for an association between exposure to Benzophenone-3 and idiopathic male infertility.
Urinary levels of Benzophenone-3 and the incidence of Hirschsprung’s disease were investigated using a total
of 423 patients. Results indicated a positive association between women identified with medium to high levels of
Benzophenone-3 (maximum detection level = 22,800 ppb) in the urine and the incidence of Hirschsprung’s disease.
The relationship between urinary concentrations of benzophenones and semen quality was studied using 413 men.
Benzophenone-2 was associated with findings such as diminished sperm concentration, more immature sperm, and a
decreased percentage of other tail abnormalities. Benzophenone-8 in the urine was associated with decreased
hypoosmotic swelling and higher acrosome area. No associations were observed for Benzophenone-1 or
Benzophenone-3. The clinical significance of these findings was not established.
In an in vitro genotoxicity test, micronuclei formation was detected in human keratinocytes treated with
Benzophenone-1 (10 µg/ml) in the presence of UVB. In a photogenotoxicity test involving human keratinocytes,
Benzophenone-1 photosensitized and generated reactive oxygen species in the presence of sunlight/UV radiation.
The in vitro luminescent umu test was used to evaluated the gentoxicity of Benzophenone-1, -3, -6, and -8 (doses up
to 10 µg/well) in Salmonella typhimurium strain TL210. Positive results were reported for Benzophenone-3.
The genotoxicity of Benzophenone-1 (doses up to 600 µg/plate), Benzophenone-3 (up to 200 µg/plate),
Benzophenone-6 (up to 2000 µg/plate), and Benzophenone-8 (up to 300 µg/plate) was evaluated in the Ames test
using Salmonella typhimurium strains TA98 and TA100 (with and without metabolic activation). Results were
negative for each benzophenone tested. The genotoxicity of Benzophenone-3 and Benzophenone-8 (each in
seawater, 1:10 or 1:1000) was evaluated at doses of 4 to 10 µl per plate using Salmonella typhimurium strain TA98
(without metabolic activation). Only Benzophenone-8 (1:10) had clear genotoxic activity that was dose-related
(doses of 4, 6, 8, and 10 µl). In another Ames test, a sunscreen formulation containing Benzophenone-3 (0.6% to
0.9%) was not genotoxic in the following Salmonella typhimurium strains at a dose of 5000 µg/plate: TA 98,
TA100, TA1535 and TA1538. Benzophenone-3 was also evaluated for genotoxicity at doses up to 6000 µg/plate
(with and without metabolic activation) using Salmonella typhimurium strains TA98 and TA100, and Escherichia
coli strain uvrA pKM101. Results were negative with and without metabolic activation.
The cytogenetic effect of Benzophenone-3 on human peripheral lymphocytes was evaluated using in vitro
chromosomal aberrations and micronuclei assays. Lymphocyte cultures were exposed to concentrations up to 0.2
µg/ml. A concentration-related, statistically significant increase in chromosomal aberrations and aberrant cell
frequencies was observed at all test concentrations. Micronuclei assay results were the same. The effect of
Benzophenone-3 on DNA damage was studied using human breast epithelial cells. Concentrations of 1 µM and 5
µM Benzophenone-3 increased DNA damage.
Benzophenone-8 (in ethanol) was evaluated at doses of 0.008 to 700 µg/plate in the Salmonella/mammalian
microsome mutagenicity assay using the following Salmonella typhimurium tester strains: TA98, TA100, TA1535,
TA1537, and TA1538. With metabolic activation, Benzophenone-8 caused a weak, but reproducibly significant
dose-dependent increase in the number of TA1537 revertants per plate. Benzophenone-8 (in ethanol) was tested in
the L5178Y TK+/- mouse lymphoma mutagenesis assay (with and without metabolic activation) at concentrations
ranging from 13 to 56 µg/ml. With metabolic activation, a significant dose-related increase in the mutant
frequencies was observed. A bacterial reverse mutation assay on Benzophenone-8 (in DMSO) was performed using
Salmonella typhimurium strain TA100 (doses up to 1500 µg/plate) and E. coli strain WP2vurA (doses up to 5000
µg/plate), with and without metabolic activation. Results were negative.
The genotoxicity of Benzophenone-12 (in DMSO) in the mammalian cell gene mutation assay using mouse
lymphoma L5178Y cells. doses up to 50 µg/ml and 52 µg/ml were tested with and without metabolic activation,
respectively. Results were negative without metabolic activation and ambiguous with metabolic activation.
In the in vivo micronucleus assay using mouse erythrocytes, results for Benzophenone-1 (doses not stated)
was were classified as inconclusive.
The genotoxicity of Benzophenone-3 was evaluated using the Drosophila somatic mutation and recombination
test (SMART). In the SMART assay, larva from a mating of “multiple wing hair” (mwh) females with
heterozygous “flare” (flr) males were exposed to 0, 3000, or 3500 ppm Benzophenone-3. None of the
Benzophenone-3-treated larva produced flies with significantly more single or multiple wing spots than controls. In
the same study, an in vivo cytogenetic assay on Benzophenone-3 using rat bone marrow cells was performed.
Sprague-Dawley rats were treated orally with doses of 0.5, 1.67, or 5 g/kg Benzophenone-3, or a single dose of 5
g/kg/d Benzophenone-3 for five consecutive days. Benzophenone-3 did not cause a significant increase in
chromosomal aberrations in this assay.
In the mammalian erythrocyte micronucleus test, the genotoxicity of a sunscreen formulation containing
Benzophenone-3 (0.6% to 0.9%) was evaluated using groups of 10 Wistar albino rats. Doses up to 2000 mg/kg
were administered dermally for 2 consecutive days, and Benzophenone-3 was classified as non-genotoxic. The
same sunscreen formulation (0.6% to 0.9% Benzophenone-3) was evaluated for genotoxicity in the mammalian
bone marrow chromosome aberration test using groups of 10 Wistar albino rats. Identical doses administered
according to the same procedure, and results were negative. Twelve ovariectomized Balb/c female mice were dosed
orally with Benzophenone-3 (3000 µg/kg/d) daily for 4 d. DNA damage was detected in mammary epithelial cells.
Effects of Benzophenone-1 on the proliferation and metastasis of MCF-7 human breast cancer cells expressing
estrogen receptors were studied. It was concluded that Benzophenone-1 may accelerate the growth of MCF-7 breast
cancer cells by regulating cell cycle-related genes and promote cancer metastasis through amplification of cathepsin
D. In a wound healing assay, Benzophenone-1 (10-6 M) statistically significantly enhanced the migration capability
of BG-1 ovarian cells by reducing the wounded area in the cell monolayer. It was noted that the results of this study
indicate that Benzophenone-1 may have the ability to induce ovarian cancer metastasis. The effect of
Benzophenone-3 (concentrations up to 150 µg/l) on cancer cell growth was studied using NCI-H460 lung cancer
cells. Results indicated that Benzophenone-3 had a cancer potentiating effect by enhancing anchorage-independent
survival and growth of lung cancer cells.
The oral carcinogenicity of Benzophenone-3 was evaluated in a National Toxicology Program (NTP) study
using male and female Sprague-Dawley rats and male and female B6C3F1/N mice. On D 6, groups of 42, 35, 35,
and 43 F0 time-mated female rats were fed diets containing 0, 1000, 3000, and 10,000 ppm Benzophenonoe-3,
respectively, for 39 d. Groups of 50 (1,000 and 3,000 ppm) or 60 (0 and 10,000 ppm) F1 rats per sex continued on
study after weaning and were fed diets containing the same exposure concentrations for 105 wk. There was
equivocal evidence of carcinogenic activity of Benzophenone-3 exposure in male Hsd:Sprague Dawley® SD® rats,
based on the occurrence of malignant meningiomas in the brain. There was equivocal evidence of carcinogenic
activity in female Hsd:Sprague Dawley® SD® rats, based on the increased incidence of thyroid C-cell adenomas
and the increased incidence of uterine stromal polyps. Groups of 50 male and 50 female mice were fed diets
containing 0, 1000, 3000, or 10,000 ppm Benzophenone-3 in the diet (equivalent to average daily doses of
approximately 113, 339, and 1207 mg Benzophenone-3/kg body weight for male mice and 109, 320, and 1278
mg/kg for female mice) for 104 (female mice) or 105 (male mice) wk. There was no evidence of carcinogenic
activity in male or female B6C3F1/N mice at exposure concentrations of 1000, 3000, and 10,000 ppm.
The xenoestrogenic effect of Benzophenone-1 on BG-1 human ovarian cancer cells expressing estrogen
receptors and relevant xenografted animal models, when compared to E2, was evaluated. In the in vitro cell
viability assay, Benzophenone-1 (10-8 to 10-5 M) statistically significantly increased BG-1 cell growth, as did E2. In
a second experiment, BG-1 cells (5 x 106) were injected s.c. into the backs of groups of 6 female mice of the
BALB/c nu/nu strain. Study results suggested that Benzophenone-1 is an endocrine disrupting chemical that exerts
xenoestrogenic effects by stimulating the proliferation of BG-1 ovarian cancer via the estrogen receptor signaling
pathway associated with the cell cycle.
A study was performed to evaluate the effects of Benzophenone-1 on prostate cancer progression.
Benzophenone-1 increased the viability of LNCaP prostate cancer cells at concentrations of 10-6 M and 10-7 M. In
the MTT assay, when the cells were co-treated with Benzophenone-1 (10-6 M) and biclutamide (10-9), the cell
viability that was increased by Benzopheonone-1 alone was statistically significantly reduced. These results suggest
that the proliferative effects of Benzophenone-1 on LNCaP cells was mediated by the androgen receptor signaling
pathway.
Benzophenones-1, -3, -6, and -8 were evaluated in the Bhas promotion assay at concentrations ranging from 2
to 100 µg/ml. Bhas 42 cells established from BALB/3T3 cells were used. Results indicated that none of the test
substances caused a statistically significant increase in the number of transformation foci (relative to the solvent
controls) over the range of concentrations. Thus, promotion activity was classified as negative.
The in vivo antitumor activity of Benzophenone-8 and Benzophenonone-12 was evaluated using a two-stage
mouse skin carcinogenesis model. Groups of 15 pathogen-free, female hairless mice of the HOS:HR-1 strain were
used, and skin tumors were induced by a single dose of NOR-1 (390 nmol). Each test substance was administered at
a concentration of 0.0025% to mice through drinking water, beginning at 1 week prior to tumor initiation and ending
at 1 week after tumor initiation. Benzophenone-8 was a more potent inhibitor of skin tumors than Benzophenone-
12.
Benzophenone-2 was applied (10 mg/kg) to the skin of 10 male Wistar rats for 4 wk. Benzophenone-2 did not
exacerbate oxidative stress and apoptosis markers in the hippocampus and frontal cortex; however, it did lower
oxidative stress in the frontal cortex.
In the neuroblastoma (SH-SY5Y) cell line, Benzophenone-2 and Benzophenone-3 adversely affected the
viability of nerve cells, most likely by enhancing the process of apoptosis. Both test substances produced a
statistically significant cytotoxic effect at concentrations of 10-5 M and 10-4 M. In another study (dermal exposure
to male offspring of Sprague-Dawley rats), it was noted that exposure to Benzophenone-3 induces the mitochondrial
apoptosis pathway in the rat frontal cortex. A 36% decrease in neuron viability was observed when cultures of rat
fetal primary cortical neurons were exposed to Benzophenone-3 (10 µg/ml) for 7 d. The authors noted that the
results of this study indicate that exposure to Benzophenone-3 induces the mitochondrial apoptosis pathway in the
rat frontal cortex. A continuous 24-h exposure of neocortical and hippocampal cultures (from Swiss mouse
embryos) to Benzophenone-3 (25 to 100 µM) induced apoptosis in mouse neuronal cells. Hippocampal cells
exhibited weaker vulnerability.
The neurotoxicity of Benzophenone-3 and its metabolite (Benzophenone-1) was studied using female Sprague-
Dawley rats and their offspring. Benzophenone-3 (10% in cream; dose = 100 mg/kg) was administered dermally
(shaved skin on back) twice daily to adult female rats (number not stated) during the prenatal period and adulthood.
Results indicated that dermal Benzophenone-3 exposure may cause damage to neurons that might be associated
with the increase in the level of extracellular glutamate.
Benzophenone-2 (at 250 and 500 µM) accelerated the conversion of dopachrome (intermediate in melanin
biosynthesis) to melanin.
Benzophenone-3 (in ethanol) was applied (volume = 100 µl; dose = 5 mg/kg [312.5 µg/cm2]) topically to a 4
cm2 area on the back (10 rats), daily for 30 d. Various behavioral testing protocols were used to assess the arousal
(open field tests), locomotion (open field and ladder test), habituation (open field test), and motor coordination (open
field and ladder test) of the animals over the study duration. No significant adverse behavioral effects were
observed.
Splenocytes were cultured in the presence of different concentrations of Benzophenone-2 (10-8 to 10-5 M).
Benzophenone-2 (10-5 M) shifted the Th1/Th2 balance toward a Th2 response (lower IFN-ℽ production and higher
IL-10). It was noted that these results show that Benzophenone-2 at high doses may possess immunomodulatory
effects. The dosing of 10 male Wistar rats with Benzophenone-3 (100 mg/kg) dermally for 4 wk did not have a
toxic effect on splenocytes and thymocytes, but increased the activity and function of these cells.
The immunosuppressive activity of Benzophenone-4 (0.01%) was evaluated using human dendritic cells (e.g.,
CD14+ human monocytes). Treatment with Benzophenonoe-4 did not impair the proliferation of lymphocytes.
In the zebrafish embryo assay on Benzophenone-1, Benzophenone-3, and Benzophenone-8, significant
decreases in whole-body T4 and T3 levels were observed at day 6 post-fertilization.
Groups of 11 ovariectomized adult Sprague-Dawley rats were dosed orally (by gavage) with 250 mg/kg and
1000 mg/kg Benzophenone-2 (1 ml) daily for 5 d. A dose-dependent suppression of T4 concentration by
Benzophenone-2 was observed. T3 levels were also reduced.
The estrogenic activity of Benzophenone-3 was evaluated (in a reporter gene assay) using the human cervical
epithelioid HeLa cell line as the host cell line for the generation of stable reporter cells for screening substances that
act via human estrogen receptor alpha (hERα) and β (hERβ). Assays were performed at concentrations between 10-7
and 10-5 M. Benzophenone-3 activated ERα moderately and had almost no effect on ERβ. Benzophenone-3 was not
considered estrogenic at 10-5 M. Exposure to Benzophenone-3 (10-10 M) for 24 h increased basal corticosterone
secretion from cultured adrenocortical cells.
Benzophenone-2 was applied to the skin of 10 male Wistar rats at a dose of 100 mg/kg for 4 wk. HPT activity
was increased, i.e., the level of TSH was reduced and the free fraction of T3 and T4 in the blood was increased.
Benzophenone-2 interference with thyroid function was evaluated in another study. Groups of 12 ovariectomized,
female Sprague-Dawley rats received oral doses ranging from 10 to 1000 mg/kg for up to 5 d. A dose-dependent
decrease in total serum T4 levels was observed, with statistically significant alterations at doses of 333 mg/kg and
1000 mg/kg. The small decrease in total T3 was not statistically significant.
The dosing of 10 male Wistar rats with Benzophenone-3 (100 mg/kg) dermally for 4 wk had no effect on the
following hematological parameters: leukocyte count, erythrocyte count, platelet count, erythrocyte morphology,
and erythrocyte hemoglobin content.
In Saccharomyces cerevisiae cultures, Benzophenone-2 was cytotoxic at concentrations > 2.5 x 10-3 M.
Benzophenone-3 was also cytotoxic to the Saccharomyces cerevisiae (IC50 = 0.0467 mM) and Aliivibrio fischeri
(IC50 = 0.0240 nM). The cytotoxicity of a sunscreen formulation composed of polymeric nanocapsules loading
Benzophenone-3 was evaluated using the L929 fibroblast cell line. The nanocapsules were seeded at a
concentration of 30 µg/ml, and the sunscreen formulation was found to be non-cytotoxic. In rat thymocytes, cell
mortality increased significantly after 3 h of exposure to 300 µM Benzophenone-3.
In MCF-7 breast cancer cells incubated for 6 d, Benzophenone-3 increased cell proliferation, with an EC50
between 1.56 and 3.73 µM. An increase in uterine weight (weak effect, active at dose of 1525 mg/kg/d) was
reported in a uterotrophic assay, whereby immature Long-Evans rats were fed Benzophenone-3 in the diet for 4 d.
The hormonal activity of Benzophenone-3 was evaluated using Saccharomyces cerevisiae strains BLYES and
BLYAS. In the estrogen assay, an EC50 value of 0.00644 mM (estrogenic activity) was reported for Benzophenone-
3. In the androgen assays, the androgenicity of Benzophenone-3 was not proven. However, Benzophenone-3 was
found to be antiandrogenic (EC50 = 0.0102 mM).
Benzophenone-8 (10 µM) upregulated PDE4B expression in normal human keratinocytes. Also,
Benzophenone-3 and UVB co-stimulation induced PDE4B upregulation. It was concluded that PDE4B has a role in
the mechanism of Benzophenone-3-induced phototoxicity.
A sunscreen formulation composed of polymeric nanocapsules loading Benzophenone-3 (0.005 wt%) was
classified as a non-irritant in the HET-CAM. Benzophenone-4 (25 mg) was considered corrosive to the skin when
evaluated using a three-dimensional human epidermis model.
There were no signs of erythema or edema in a group of 24 Wistar albino rats after a 24-h patch application of
a sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%). The same was true in 18 male New Zealand
rabbits after a 72-h patch application of the same formulation. When Benzophenone-3 (in isopropyl myristate and
SD alcohol vehicle) was applied to the skin of 30 female, Hartley albino guinea pigs, (followed by irradiation with
UVA), concentrations of 0.1% and 0.3% produced erythema grades greater than 1+. Solutions containing 3% and
6% Benzophenone-3 produced erythema grades of less than 1+ when applied to the skin of guinea pigs. The authors
noted that the erythema grade decreased with increasing concentration because the photoprotection afforded by
Benzophenone-3 was concentration-dependent. Benzophenone-8 (0.5 g in water) was evaluated for skin irritation
potential using 3 New Zealand white rabbits in a 4-h patch test. Skin irritation was not observed. Benzophenone-12
(0.5 g) was also classified as non-irritating to the skin of rabbits in a 4-h patch test.
In a 48-h patch test involving 80 subjects, Benzophenone-4 (5% in petrolatum) induced skin irritation in 4
subjects. Benzophenone-4 (10% in petrolatum) induced skin irritation in 6 subjects.
Benzophenone-8 was classified as a sensitizer in the in vitro KeratinoSens assay (HaCaT cell line) when tested
at concentrations up to 200 mM.
The skin sensitization potential of a sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was
evaluated in a study involving 30 adult male guinea pigs (3 groups of 10), and results were negative. The local
lymph node assay was also used to evaluate the sensitization potential of Benzophenone-3 (12.5%, 25%, and 50%),
and results were negative.
The maximization test was used to assess the cutaneous allergenic potential of Benzophenone-12, using 10
albino guinea pigs challenged with 40% Benzophenone-12 in PEG 300. Positive reactions were observed in 7
animals. Benzophenone-12 was evaluated for skin sensitization potential in another maximization test using 20
guinea pigs of the Pirbright white (Tif:DHP) strain. Sixty-five percent and 60% of the animals were sensitized to
Benzophenone-12 at 24 h and 48 h after challenge, respectively.
Of the 4094 patients (with suspected allergic contact dermatitis) patch tested with 3% Benzophenone-3, 0.5%
had allergic reactions. When 5,800 patients were patch tested with Benzopheonone-3 (3% in petrolatum), the
incidence of positive reactions was 0.6%. Data from 64 allergenicity studies were aggregated and analyzed, in order
to evaluate the irritation and sensitization potential of sunscreen products containing Benzophenone-3 (between 1%
and 6%). Forty-eight of 19,570 possible dermal responses were considered suggestive of irritation or sensitization.
The mean rate of contact allergy to Benzophenone-3 was 0.07%. Of 23,908 patients patch tested, 219 (0.9%) had
sunscreen coded as an allergen source. A frequent allergen in sunscreens was Benzophenone-3, whereby 70.2% of
the patients (26 of 37 patients patch tested) had an allergic reaction to 10% Benzophenone-3 (in petrolatum) and
64.4% of the patients (56 of 87 patients patch tested) had an allergic reaction to 3% Benzophenone-3 (in
petrolatum). In another study, 5085 patients were patch tested and allergic reactions to Benzophenone-3 (3% in
petrolatum) were observed in 22.7% of the patients.
When Benzophenone-4 (10% in petrolatum) was patch tested in a study involving 4857 patients, the positive
reaction rate was 2.1% (100 allergic reactions).
Of 214 patients patch tested, Benzophenone-10 and Benzophenone-3 accounted for 8 and 27 positive reactions,
respectively. Twenty-three patients with a variety of photosensitive disorders were photopatch tested.
Benzophenone-10 and Benzophenone-3 produced positive responses at 1.0 and 0.7 J/cm2, respectively. In a
retrospective review, 160 patients underwent photopatch testing. Benzophenone-3 caused both an allergic and
photoallergic reaction in 6 patients. Benzophenone-4 caused allergic contact dermatitis in 3 patients, but did not
cause photoallergic reactions. Another retrospective analysis involved the reviewing of 1527 charts in a patch test
database. Twenty-three of the patients were tested with the sunscreen series. Of the 23, two had positive reactions
(allergic contact dermatitis) to Benzophenone-3 (10% in petrolatum) and 1 had a positive reaction to Benzophenone-
4 (10% in petrolatum). Of the 1527 patients screened (no specific history of sunscreen allergy), 8 patients reacted to
Benzophenone-3.
A total of 5592 patients was patch tested with Benzophenone-4 and Benzopenone-3 (both at 10% in
petrolatum). Values for the clinical relevance of allergic reactions were: Benzophenone-4 (definite relevance: 3 of
93 patients (3.2%)) and Benzophenone-3 (definite relevance: 4 of 24 patients (16.7%)). Results from a facial patch
test series were reviewed retrospectively. Of the 1390 patients patch tested with Benzophenone-4 (2% in
petrolatum), 0.79% had allergic reactions. Of the 4224 patients patch tested with Benzophenone-3 (10% in
petrolatum), 0.17% had allergic reactions.
The allergenicity of Benzophenone-4 and Benzophenone-10 was evaluated using 15 eczematous dermatitis
patients. Patch test reactions to 10% Benzophenone-4 in petrolatum were negative. Two subjects had positive
reactions to 10% Benzophenone-10 in petrolatum. Five hundred fifty-three patients were patch tested with 10%
Benzophenone-3, 10% Benzophenone-4, and 10% Benzophenone-10. Thirteen patients and 1 patient had positive
reactions to 10% Benzophenone-3 and 10% Benzophenone-10, respectively. Thirteen patients also had positive
reactions to 10% Benzophenone-4.
Four patients were patch tested with 3% aqueous Benzophenone-3, with and without UVA (8 J/cm2). Each
patient was photoallergic to Benzophenone-3. Positive reactions were observed with and without UVA. Patients
(187 total) with a history of photosensitivity were photopatch tested with Benzophenone-3 (2% in petrolatum].
Reactions were positive in 9 patients. Nineteen patients with positive photopatch tests to sunscreen agents were
retrospectively selected from the database of a contact dermatitis clinic. Of the 19 patients, 9 had a positive
photopatch test reaction to 10% Benzophenone-3 in petrolatum. A study was performed, using 35 patients with
confirmed photosensitivity reactions, to determine the proportion of photosensitive patients with photoallergic
contact dermatitis to Benzophenone-3. Five of these patients (14.28%) had at least one positive reaction to
Benzophenone-3 in the photocontact test. Four patients had a reaction at the irradiated sites only, and 1 patient had
a reaction at both irradiated and nonirradiated sites.
Fifteen patients (4 males, 11 females; mean age = 47.7 years) with reactions to sunscreens were tested with
sunscreen ingredients. There were 4 allergic contact dermatitis reactions to Benzophenone-4, and 2 allergic contact
dermatitis and 5 photoallergic contact dermatitis reactions to Benzophenone-3. Four-hundred-two patients (ages not
stated) with suspected clinical photosensitivity were patch and photopatch tested with UV absorbers and commercial
sunscreens. There were 3 allergic and 9 photoallergic reactions to Benzophenone-3 (10% in petrolatum), and no
photoallergic or allergic reactions to Benzophenone-4 (10% in petrolatum). Twelve patients with a history of acute
eruption on photoexposed areas were photopatch tested. Results were positive for 3 patients tested with
Benzophenone-3, and negative for Benzophenone-4. In a population of 355 consecutive patients with suspected
photosensitivity, the most common allergen was Benzophenone-3 (2% in petrolatum), with 15 photocontact allergic
reactions and 1 contact allergic reaction.
Photopatch tests on Benzophenone-3 (10% in white paraffin) and Benzophenone-4 (5% and 10% in white
paraffin) were performed using 1155 patients. Benzophenone-3 (10% in white paraffin) caused photoallergic
contact reactions in 27 patients. Benzophenone-4 (10% in white paraffin) and Benzophenone-4 (5% in white
paraffin) caused photoallergic contact reactions in 5 and 2 patients, respectively. The following allergic reactions
were also reported: 10% Benzophenone-3 (9 patients), 10% Benzophenone-4 (9 patients), and 5% Benzophenone-4
(2 patients).
Eighty-two outpatients with photoallergic contact dermatitis were photopatch tested with Benzopheone-3 and
Benzopenone-4 (concentrations not stated). Benzophenone-3 was photoallergenic in 22 of 82 patients (26.8%), and
Benzophenone-4 was photoallergenic in 2 of 82 patients (2.4%). An investigation of photoallergic contact
dermatitis frequency was performed using 347 patients. Benzophenone-3 (10% in petrolatum) Benzophenone-4
(2% in petrolatum) elicited photoallergic contact dermatitis in 37 patients. Benzophenone-4 (2% in petrolatum)
elicited photoallergic contact dermatitis in 3 patients. Allergic contact dermatitis reactions to Benzophenone-3 (10%
in petrolatum) were observed in 6 patients. In a retrospective chart review, 160 patients underwent photopatch
testing. Benzophenone-3 induced photoallergic reactions in 12 patients, allergic reactions in 17 patients, and both
allergic and photoallergic reactions in 6 patients. Benzophenone-4 caused allergic contact dermatitis in 3 patients,
but did not cause photoallergic reactions.
A prospective study was performed using 1000 consecutive dermatology outpatients. Photoallergic contact
dermatitis was confirmed in 15 patients. Two of the 15 photopatch tested had a positive reaction to Benzophenone-3
(10% in petrolatum). One patient had a positive reaction to Benzophenone-4 (2% in petrolatum). The photopatch
testing of sunscreens was performed in a study involving 157 children. Benzophenone-3 induced photoallergy in 3
children, and a single case of a photoaugmentation reaction to Benzophenone-4 was reported. The phototoxicity of
Benzophenone-4 at concentrations of 2%, 5%, and 10% in petrolatum was studied using 80 subjects. One subject
had a weak positive reaction (+ reaction), with no concomitant erythema score, to Benzophenone-4 (10% in
petrolatum) at the irradiated site.
A retrospective analysis of positive photopatch test episodes was performed using results retrieved from a
dermatology database (111 patients positive). The most common UV filter photoallergen was Benzophenone-3 (14
positive results), followed by Benzophenone-10 (9 positive results). Benzophenone-10 accounted for 13 allergic
contact reactions, and Benzophenone-3 accounted for eight allergic contact reactions. Seven photodermatitis
patients were patch tested and photopatch tested with Benzophenone-3, Benzophenone-4, and Benzophenone-10
(test concentrations not stated). Four and 2 patients had positive photopatch tests to Benzophenone-3 and
Benzophenone-10, respectively. Photopatch test results for Benzophenone-4 were negative. The photoallergenicity
of Benzophenone-4 (10% in petrolatum) and Benzophenone-10 (10% in petrolatum) was evaluated using 15
eczematous dermatitis patients. There were no positive reactions to Benzophenone-4 (10% in petrolatum). Three
subjects had positive reactions to Benzophenone-10 (10% in petrolatum). Patients (280) patients with
photosensitivity were patch and photopatch tested with a series of contact allergens and photoallergens (test
concentration = 2% in petrolatum). Six patients were found to be allergic to Benzophenone-10, but photopatch test
results were negative.
A sunscreen formulation containing Benzophenone-3 (0.6% to 0.9%) was classified as practically non-
irritating to the eyes of 3 New Zealand albino rabbits. The ocular irritation potential of Benzophenone-4 (solid, 50
mg) was evaluated using the MatTek EpiOcular™ model. Test results classified Benzophenone-4 as irritating to the
human eye. Benzophenone-12 (0.1 g) was evaluated for ocular irritation potential using 6 New Zealand white
rabbits, and results were negative.
Benzophenone-3 had no effect on thyroid function when applied topically to 15 men (dose = 40 g) and 17
women (dose = 35 g) daily for 4 d. A study was performed to assess the relationship between exposure to
endocrine-disrupting chemicals and the age of menarche in adolescent girls. It was concluded that Benzophenone-3
exposure was not significantly associated with the age of menarche. The influence of Benzophenone-3 on the age of
menarche was also evaluated in a study involving 200 girls. A log ng/ml increase in childhood (pre-pubertal)
urinary levels of Benzophenone-3 was associated with decreased time to menarche. The association of
Benzophenone-3 with serum total testosterone levels was examined in a study involving child and adolescent
participants. Benzophenone-3 was associated with statistically significantly lower testosterone in adolescent boys
only.
The association between maternal urinary phenol concentrations during pregnancy and fetal growth was
studied in a population of 476 mothers wo had participated in a birth cohort. An association between urinary
Benzophenone and lower abdominal circumference in males was made. However, the authors noted that this
association should be verified in a larger population. A study was performed to examine paternal and maternal
preconception and maternal prenatal urinary phenol concentrations in relation to birth weight and head
circumference. Singletons (346) born to 346 mothers and 184 fathers (184 couples) from a prospective
preconception cohort of subfertile couples were evaluated. Benzophenone-3 concentration was associated with a
137 g increase in birth weight.
A study was performed to study the association between prenatal exposure to Benzophenone-3 and gestation
age and birth weight. Relationships between birth outcomes and urinary concentrations of Benzophenon-3 were
studied. Average Benzophenone-3 urinary concentrations were associated with an increase in gestational age.
Another study was designed to determine an association between urinary phthalates, parabens, and phenols found in
personal care products with pubertal timing in girls and boys (338 children total). No association relating to urinary
Benzophenone-3 was found. Placental weights and birth weights were available for 473 mother-son pairs in a
cohort whereby Benzophenone-3 was measured in spot urine samples. A positive association between
Benzophenone-3 and both placental weight and child birth weight was observed.
A study was performed to examine whether maternal and paternal preconception urinary concentrations of
Benzopheonone-3 (e.g., from dietary and personal care product exposure) and other chemicals were associated with
the risk of preterm birth among couples attending fertility care. This study involved 417 female and 229 male
participants and 418 singleton infant births. No consistent pattern of association was observed for Benzophenone-3
in either parent.
The following types of reactions were observed in case reports: sensitization reactions to Benzophenone-2 (at
1% and 2% in petrolatum); contact dermatitis and positive photopatch (2% in petrolatum) test reactions to
Benzophenone-3; photoallergic contact urticaria, contact urticaria (at 10% in petrolatum) and anaphylactic reactions
(wheal and flare) to Benzophenone-3; contact dermatitis (10% in petrolatum) and negative/questionable photopatch
reaction to Benzophenone-4; contact dermatitis and positive photopatch reactions to Benzophenone-10; and
anaphylactic reactions to Benzophenone-8 and Benzophenone-10.
DISCUSSION
The Panel published a safety assessment of benzophenones with the following conclusion in 1983: on the basis
of the available animal data and clinical human experience presented in this report, the Panel concludes that
Benzophenones-1, -3, -4, -5, -9, and -11 are safe for topical application to humans in the present practices of use and
concentration in cosmetics. During the same year, the Panel also published an addendum to this published safety
assessment, having concluded that Benzophenones-2, -6, and -8 are not mutagenic or genotoxic and that the
published conclusion on Benzophenones-1, -3, -4, -5, -9, and -11 is applicable to these 3 ingredients. In accordance
with Cosmetic Ingredient Review (CIR) Procedures & Support to the Expert Panel for Cosmetic Ingredient Safety,
the Panel evaluates the conclusions of previously-issued reports every 15 years. Thus, the Panel re-evaluated the
conclusion, and in 2005, published re-review summary that stated the Panel determined to not reopen the 1983
published safety assessment until results from National Toxicology Program (NTP) carcinogenicity studies on
benzophenones are available. An NTP oral carcinogenicity study on Benzophenone-3 was published in May 2020,
and results from this study have been reviewed by the Panel, along with other safety test data on Benzophenone-3,
as well as data on the other benzophenones that have been identified in the published literature since the original
safety assessment was published in 1983.

The Panel reviewed a number of systemic toxicity studies on benzophenones. However, the Panel noted that
these studies were performed at high concentrations that are not relevant to cosmetic exposure. The NTP oral
carcinogenicity study on Benzophenone-3 reviewed by the Panel involved rats and mice. Results indicated
equivocal evidence of carcinogenicity, i.e., male rats with benign thyroid tumors and malignant meningiomas in the
absence of a dose response, and no evidence of carcinogenicity in mice. Based on these results, the Panel did not
express any concern over the carcinogenic potential of benzophenones in cosmetic products.

The issue of incidental inhalation exposure from the use of Benzopheone-3 and Benzophenone-4 in cosmetic
products was discussed by the Panel. Benzophenone-3 is being used in aerosol hair spray (maximum concentration
of 0.014%), pump hair spray (maximum concentration of 0.05%), and in pump deodorant spray (at maximum
concentration of 0.08%). Benzophenone-4 is also being used in aerosol hair spray (maximum concentration of
0.015%) and pump hair spray (maximum concentrations of 0.001% to 0.1%). Relative to these uses, the Panel
stated that droplets/particles deposited in the nasopharyngeal or bronchial regions of the respiratory tract present no
toxicological concerns based on the chemical and biological properties of Benzophenone-3 or Benzophenone-4.
Benzophenone-3 is also being used in face powders (use concentrations unknown). The Panel noted that
conservative estimates of inhalation exposures to respirable particles during the use of loose powder cosmetic
products are 400-fold to 1000-fold less than protective regulatory and guidance limits for inert airborne respirable
particles in the workplace.

Finally, the Panel expressed concern about heavy metals that may be present in any of the benzophenones.
They stressed that the cosmetics industry should continue to use current good manufacturing practices (cGMPs) to
limit impurities.
CONCLUSION
The Expert Panel for Cosmetic Ingredient Safety concluded that the following benzophenone ingredients are
safe in cosmetics in the present practices of use and concentration described in this safety assessment.

Benzophenone-1 Benzophenone-5 Benzophenone-10*

Benzophenone-2 Benzophenone-6* Benzophenone-11*
Benzophenone-3 Benzophenone-8* Benzophenone-12*
Benzophenone-4 Benzophenone-9
 * Not reported to be in current use. Were ingredients in this group not in current use to be used in the future, the
expectation is that they would be used in product categories and at concentrations comparable to others in this group.
Table 1. Definitions, idealized structures, and reported functions of the ingredients in this safety assessment. (4,CIR Staff)
Ingredient /CAS No. Definition & Structures Function(s)
Benzophenone-1 Benzophenone-1 is a benzophenone derivative that conforms to the structure: Light Stabilizers
131-56-6

Benzophenone-2 Benzophenone-2 is a benzophenone derivative that conforms to the structure: Light Stabilizers
131-55-5

Benzophenone-3 Benzophenone-3 is a benzophenone derivative that conforms to the structure: Light Stabilizers; Sunscreen Agents
131-57-7

Benzophenone-4 Benzophenone-4 is a benzophenone derivative that conforms to the structure: Light Stabilizers; Sunscreen Agents
4065-45-6

Benzophenone-5 Benzophenone-5 is the sodium salt of Benzophenone-4 and conforms to the Light Stabilizers
6628-37-1 structure:
Table 1. Definitions, idealized structures, and reported functions of the ingredients in this safety assessment. (4,CIR Staff)
Ingredient /CAS No. Definition & Structures Function(s)
Benzophenone-6 Benzophenone-6 is a benzophenone derivative that conforms to the structure: Fragrance Ingredients; Light
131-54-4 Stabilizers

Benzophenone-8 Benzophenone-8 is a benzophenone derivative that conforms to the structure: Light Stabilizers; Sunscreen Agents
131-53-3

Benzophenone-9 Benzophenone-9 is a benzophenone derivative that conforms to the structure: Light Stabilizers
76656-36-5

Benzophenone-10 Benzophenone-10 is a benzophenone derivative that conforms to the structure: Light Stabilizers
1641-17-4

Benzophenone-11 Benzophenone-11 is a mixture of Benzophenone-6, Benzophenone-2, and other tetra- Light Stabilizers
1341-54-4 substituted benzophenone materials.
Benzophenone-12 Benzophenone-12 is a benzophenone derivative that conforms to the structure: Light Stabilizers
1843-05-6
Table 2. Chemical Properties
Property Value/Results Reference
Benzophenone-1
Form Light-yellow powder 1

Molecular weight (g/mol) 214.21 1

Specific gravity (g/ml) 1.27 1

Solubility Soluble in methanol, ethanol, ethyl acetate, methyl ethyl ketone, acetone, ether, 1

and acetic acid; slightly soluble in benzene; insoluble in water

Melting point (ºC) 144 1

log Kow 2.96 (estimated) 11

UV absorption λmax (nm) 290 1

Benzophenone-2
Form Yellow crystalline solid 1

Molecular weight (g/mol) 302.33 1

Solubility Soluble in methanol, ethanol, methyl ethyl ketone; slightly soluble in water 1

Melting point (ºC) 195 1

log Kow 2.78 (estimated) 11

UV absorption λmax (nm) 283 1

Benzophenone-3
Form Light, cream-colored powder 1

Molecular weight (g/mol) 228.26 1

Solubility Soluble in most organic solvents; insoluble in water 1

Melting point (ºC) 66 1

log Kow 3.79 (estimated) 11

UV absorption λmax (nm) 289 1

Benzophenone-4
Form Pale, ivory-colored powder 1

Molecular weight (g/mol) 318.39 1

Solubility Soluble in water, methanol, and ethanol 1

Melting point (ºC) 147 1

log Kow 0.37 (estimated) 11

UV absorption λmax (nm) 288 1

Benzophenone-5
Formula weight (g/mol) 330.29 (sodium cation is 22.99) 1

log Kow -1.42 (estimated) 11

Benzophenone-6
Form Light yellow solid 1

Molecular weight (g/mol) 274.26 1

Specific gravity (g/ml) 1.34 1

Solubility Soluble in methanol, ethanol, ethyl acetate, methyl ethyl ketone, and toluene; 1

insoluble in water
Melting point (ºC) 124 1

log Kow 3.90 (estimated) 11

UV absorption λmax (nm) 281 1

1
11

Benzophenone-8
Form Yellow crystalline solid 1

Molecular weight (g/mol) 244.24 1

Solubility Soluble in methanol, ethanol, ethyl acetate, isopropanol, ether, and acetone; 1

slightly soluble in water

Boiling point (ºC @ 1 mm Hg) 164-166 1

Melting point (ºC) 73.5-74.5 1

log Kow 3.82 (estimated) 11

UV absorption λmax (nm) 285 1

Benzophenone-9
Form Light yellow powder 1

Formula weight (g/mol) 478.35 (2 sodium cations are 45.97) 1

Solubility Soluble in methanol and ethanol; insoluble in ethyl acetate and benzene 1

Melting point (ºC) 350 1

log Kow -2.78 (estimated) 11

UV absorption λmax (nm) 284 1

Benzophenone-10
Molecular weight (g/mol) 242.27 1

log Kow 4.07 (estimated) 11

UV absorption λmax (nm) 300 1

 Property Value/Results Reference
Benzophenone-11
Form Yellow or tan powder 1

Specific gravity (g/ml) 1.38 1

Solubility Soluble in methanol, ethanol, ethyl acetate, and methyl ethyl ketone; insoluble 1

in water
Melting range (ºC) 85-105 1

UV absorption λmax (nm) 285 1

Benzophenone-12
Molecular weight (g/mol) 326.44 1

log Kow 6.96 (estimated) 11

 Table 3. Current and historical frequency and concentration of use of benzophenones according to duration and exposure
# of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%)
Benzophenone-1 Benzophenone-2
202012 19831 202013 19831 202012 19831 202013 19831
Totals* 595 142 0.009-1.1 0.1-1 103 299 NR 0.1-5
Duration of Use
Leave-On 566 128 0.05-1.1 0.1-1 95 254 NR 0.1-5
Rinse-Off 28 11 0.009-0.15 0.1 7 31 NR 0.1-1
Diluted for (Bath) Use 1 3 NR 0.1 1 14 NR 0.1-1

Eye Area NR NR NR NR NR NR NR NR
Incidental Ingestion NR 7 0.05 0.1-1 NR NR NR NR
Incidental Inhalation-Spray 42;2c 8;5a;2c NR 0.1;0.1-1a;0.1c 81;4a;2c 157;39a;8c NR 0.1-5;0.1a;0.1c
Incidental Inhalation-Powder 2c 2c NR 0.1c 2c 8c NR 0.1c
Dermal Contact 55 25 0.15-0.5 0.1-1 101 274 NR 0.1-5
Deodorant (underarm) NR NR NR NR 1a NR NR NR
Hair - Non-Coloring NR 14 NR 0.1-1 2 25 NR 0.1-1
Hair-Coloring NR NR NR NR NR NR NR NR
Nail 540 96 0.009-1.1 0.1-1 NR NR NR NR
Mucous Membrane 1 10 0.05 0.1-1 5 15 NR 0.1-1
Baby Products 2 NR NR NR NR NR NR NR
Benzophenone-3 Benzophenone-4
202012 19831 202013 19831 202012 19831 202013 19831
Totals* 989 47 0.001-0.5 0.1-1 2259 240 0.000035-1.6 0.1-10
Duration of Use
Leave-On 853 43 0.014-0.5 0.1-1 625 102 0.0001-1.6 0.1-10
Rinse-Off 100 3 0.001-0.5 0.1 1562 121 0.000035-0.5 0.1-5
Diluted for (Bath) Use 36 1 NR 0.1 72 17 0.15 0.1
Exposure Type
Eye Area 5 NR NR NR 10 1 0.2 0.1-1
Incidental Ingestion 101 NR 0.5 NR 10 NR NR NR
Incidental Inhalation-Spray 390;110a;78b 2;1a 0.014-0.05;0.1- 0.1-1;0.1a 91;290a;87c 20;35a;9c 0.001- 0.1;0.1-
0.5b 0.1;0.0001-0.5a 10a;0.1c
Incidental Inhalation-Powder 4;78b NR 0.3-0.35b NR 87c 9c 0.1-0.2b 0.1c
Dermal Contact 752 10 0.0092-0.5 0.1-1 1694 104 0.005-0.5 0.1-10
Deodorant (underarm) 4a NR 0.08 (spray) NR NR NR NR NR
Hair - Non-Coloring 71 1 0.014-0.5 0.1 503 133 0.000035-1.6 0.1-5
Hair-Coloring 14 NR 0.15 NR 39 1 0.05-0.1 0.1-1
Nail 51 36 0.001-0.4 0.1-1 6 2 0.2 0.1
Mucous Membrane 157 1 0.05-0.5 0.1 1196 19 0.15-0.2 0.1-1
Baby Products 1 NR 0.05-0.25 NR 2 2 NR 0.1
Benzophenone-5 Benzophenone-6
202012 19831 202013 19831 202012 19831 202013 19831
Totals* 14 10 0.06 0.1 NR 90 NR 0.1-1
Duration of Use
Leave-On 12 10 NR 0.1 NR 84 NR 0.1-1
Rinse-Off 2 NR 0.06 NR NR 4 NR 0.1
Diluted for (Bath) Use NR NR NR NR NR 2 NR 0.1
Exposure Type
Eye Area 2 NR 0.06 NR NR NR NR NR
Incidental Ingestion NR NR NR NR NR NR NR NR
Incidental Inhalation-Spray 2a;7c NR;3a;7c NR NR;0.1a;0.1c NR 3 NR 0.1-1
Incidental Inhalation-Powder 7c 7c NR 0.1c NR NR NR NR
Dermal Contact 12 10 0.06 0.1 NR 8 NR 0.1-1
Deodorant (underarm) NR NR NR NR NR NR NR NR
Hair - Non-Coloring 1 NR NR NR NR 4 NR 0.1
Hair-Coloring NR NR NR NR NR NR NR NR
Nail NR NR NR NR NR 78 NR 0.1-1
Mucous Membrane NR NR NR NR NR 2 NR 0.1
Baby Products NR NR NR NR NR NR NR NR
*Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses.
a
It is possible these products are sprays, but it is not specified whether the reported uses are sprays.
b
It is possible these products are powders, but it is not specified whether the reported uses are powders.
c
Not specified whether a spray or a powder, but it is possible the use can be as a spray or a powder, therefore the information is captured in both categories
NR – no reported use
 Table 3. Current and historical frequency and concentration of use of benzophenones according to duration and exposure
# of Uses Max Conc of Use (%) # of Uses Max Conc of Use (%)
Benzophenone-8 Benzophenone-9
202012 19831 202013 19831 202012 19831 202013 19831
Totals* NR 4 NR 0.1-1 71 123 NR 0.1-1
Duration of Use
Leave-On NR 1 NR 0.1 41 41 NR 0.1-1
Rinse-Off NR 2 NR 0.1-1 29 27 NR 0.1-1
Diluted for (Bath) Use NR 1 NR 0.1-1 1 55 NR 0.1
Exposure Type
Eye Area NR NR NR NR NR NR NR NR
Incidental Ingestion NR NR NR NR 2 NR NR NR
Incidental Inhalation-Spray NR 1a NR 0.1a 13;5a;15c 4;13a;14c NR 0.1-1;0.1-
1a;0.1-1c
Incidental Inhalation-Powder NR NR NR NR 15c 14c NR 0.1-1c
Dermal Contact NR 2 NR 0.1-1 68 96 NR 0.1-1
Deodorant (underarm) NR NR NR NR NR NR NR NR
Hair - Non-Coloring NR 2 NR 0.1-1 NR 23 NR 0.1-1
Hair-Coloring NR NR NR NR NR 1 NR 0.1
Nail NR NR NR NR 1 3 NR 0.1
Mucous Membrane NR 1 NR 0.1-1 28 55 NR 0.1
Baby Products NR NR NR NR NR NR NR NR
Benzophenone-11
202012 19831 202013 19831
Totals* NR 168 NR 0.1-5
Duration of Use
Leave-On NR 140 NR 0.1-5
Rinse-Off NR 19 NR 0.1
Diluted for (Bath) Use NR 9 NR 0.1-1
Exposure Type
Eye Area NR NR NR NR
Incidental Ingestion NR NR NR NR
Incidental Inhalation-Spray NR 85;25a;2c NR 0.1-5;0.1-
1a;0.1c
Incidental Inhalation-Powder NR 2c NR 0.1c
Dermal Contact NR 144 NR 0.1-1
Deodorant (underarm) NR NR NR NR
Hair - Non-Coloring NR 21 NR 0.1-5
Hair-Coloring NR NR NR NR
Nail NR 3 NR 0.1
Mucous Membrane NR 12 NR 0.1-1
Baby Products NR NR NR NR
*Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses.
a
It is possible these products are sprays, but it is not specified whether the reported uses are sprays.
b
It is possible these products are powders, but it is not specified whether the reported uses are powders.
c
Not specified whether a spray or a powder, but it is possible the use can be as a spray or a powder, therefore the information is captured in both categories
NR – no reported use
Table 4. Acute toxicity studies
Ingredient Animals No./Group Vehicle Concentration/Dose/Protocol LD50/Results Reference
DERMAL
Benzophenone-3 Wistar albino 12 males and 12 Sunscreen 0.6% to 0.9%. OECD TG 402. No statistically significant 68

rats females formulation Formulation (2000 mg/kg) applied to 2" changes in terminal body
x 2", 4-ply gauze pad, and patch placed weight between test and
(secured with surgical tape) on hairless, controls. Hematological
dorsal skin. Patch remained in place for and serum biochemistry
24 h. Animals observed for 14 d, after parameters normal. No
which animals killed. abnormalities at necropsy
or microscopic
examination. LD50 > 2000
mg/kg.

Benzophenone-3 New Zealand 6 males Sunscreen 0.6% to 0.9%. OECD TG 404. Systemic toxicity not 68

rabbits formulation Formulation applied to 25 cm2 area of observed.

dorsal skin, using 2" x 3", 4-ply gauze
pad (secured with surgical tape). 72-h
application period.
Benzophenone-12 Albino rabbits 5 rabbits Water 10,000 mg/kg. OECD TG 402. No deaths, and no clinical 6

Applied, under an occlusive or semi- signs or adverse findings.

occlusive patch, for 24 h to skin. Patch The LD50 > 10,000 mg/kg.
removal followed by 7-d observation
period.
ORAL
Benzophenone-1 Rats Number and Not stated Details not stated LD50 = 8600 mg/kg. 7

strain not stated Practically non-toxic.

Benzophenone-3 Female Wistar 10 rats Sunscreen 0.6% to 0.9%. OECD TG 423. All animals survived and 68

albino rats. formulation in Formulation (2000 mg/kg) administered gained normal body
0.5% by gavage to 1 fasted rat. Thereafter, weight; no clinical signs of
carboxymethyl each 48 h, the same dose administered toxicity observed. No
cellulose to 4 rats. 5 control rats. Dosing evidence of gross
followed by 14-d observation period, abnormalities, adverse
after which animals killed. Following pharmacological effects, or
organs examined macroscopically: abnormal behavior. LD50 >
heart, lungs, liver, kidneys, and spleen. 2000 mg/kg.
Benzophenone-8 Female Wistar 6 rats Propylene glycol Test substance (200 mg/ml) None of the animals died. 9

rats of the administered via gavage at dose of 2000 No treatment-related

CLR:(WI) mg/kg. Dosing followed by 14-d adverse effects. No
strain observation period. Animals killed; evidence of macroscopic
gross and microscopic examinations changes. The LD50 > 2000
performed. mg/kg.
Benzophenone-12 Male rats of 10 rats Water 20% suspension. Test substance None of the animals died. 6

the CF Nelson administered at dose of 10,000 mg/kg. No clinical signs and no

strain Dosing followed by a 7-d observation findings at necropsy. LD50
period. > 10,000 mg/kg.
Table 5. Repeated dose toxicity studies
Ingredient Animals/Group Study Vehicle Dose/Concentration Results
Reference
Duration
DERMAL
Benzophenone-3 B6C3F1 mice; 5 2 weeks Acetone or 0.5 to 8 mg applied topically. Minimal, variable increases in liver and kidney weights, primarily in the 69

males and 5 females lotion higher dose groups.

Benzophenone-3 F344/N rats; 5 males 2 weeks Acetone or 1.25 to 20 mg applied topically Small and variable increases in liver and kidney weights, reaching a 69

and 5 females lotion statistical significance primarily in the higher dose groups.
Benzophenone-3 Male Sprague- 4 weeks Ointment 100 mg applied topically twice Body weight, organ-to-body weight ratios, and hematological and 70

Dawley rats; groups base daily clinical chemistry parameters not affected. Pathological examinations
of 4 to 6 revealed no significant changes between control and treated animals. No
gross external abnormalities observed. Non-toxic to rats.
Benzophenone-3 Female Sprague- Adults: first Cream Adults received dermal Dosing of adult pregnant females did not significantly alter body weight 35

Dawley rats and their to last day of applications (10% in cream; dose or cause apparent adverse effects, when compared to controls. No
offspring pregnancy = 100 mg/kg) twice daily. At 21 d significant differences in body weight and sex-ratio observed in
(~22 to 23 after birth, offspring (male and offspring, when compared to controls.
days). female) divided into groups of 5
Offspring: males and groups of 5 females.
from 43 to From 43 to 56 d age, test
56 d age substance administered dermally
to male offspring.
ORAL
Benzophenone-3 B6C3F1 mice (5 2 weeks Feed 0, 3125, 6250, 12,500, 25,000, or Dose-related increase in liver weight, associated with hepatocyte 69

males and 5 females 50,000 ppm cytoplasmic vacuolization. NOAEL for microscopic lesions was 6250
per group) ppm.
Benzophenone-3 F344/N rats (5 males 2 weeks Feed 0, 3125, 6250, 12,500, 25,000, or One high-dose female rat died. Liver and kidney weights increased. 69

and 5 females per 50,000 ppm Enlarged livers associated with marked hepatocyte cytoplasmic
group) vacuolization at ≥ 6250 ppm. Renal lesions, consisting of dilated tubules
and regeneration of tubular epithelial cells, found primarily in high-dose
rats. NOAEL for microscopic lesions was 6250 ppm.
Benzophenone-4 Groups of 26 Wistar 48 days of Corn oil OECD TG 422. 0, 750, 1000, and No morbidity observed during dosing period. No test substance-related 8

rats (13 males, 13 dosing 1250 mg/kg/d (by gavage). Male mortalities. Clinical findings sporadic and of no biological significance.
females/group). (males); 66 rats were treated 2 wk before Body weight changes restricted to statistically significant decrease in %
days of mating and thereafter. Female rats body weight change in recovery group of male rats treated at 1250 mg/kg
dosing treated 2 wk before mating, and from day 1 – 22, as compared to the control group. This effect on body
(females) during mating, gestation, and weight considered incidental and not test substance-related. Food
lactation. Recovery groups of consumption unaffected by treatment. Observed changes in hematology
male and female rats (5/sex/dose) and clinical chemistry not of toxicological importance. Detailed clinical
treated at 0 or 1250 mg/kg body examinations and microscopic examination of eyes, with optic nerve (in
weight/d for 66 d total. Animals in 0 and 1250 mg/kg groups), did not reveal abnormalities. Hormonal data
recovery groups allowed to showed no significant effects on concentrations of T4 or TSH (male and
recover for 2 wk after final dose females), testosterone (males), or estradiol (females). No significant
given. effects on either the absolute or relative weight of brain, adrenals, heart,
liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or
epididymides. All adult animals normal externally. Visceral findings
included case of mild splenic enlargement at 1000 mg/kg and one case of
mild testicular shrinkage at 1250 mg/kg. Microscopic examination
revealed no treatment-related effects, that is, incidences and types of
lesions observed at 1250 mg/kg comparable to concurrent control groups.
NOAEL (systemic toxicity) established at 1250 mg/kg/d for male and
female rats.
Table 5. Repeated dose toxicity studies
Ingredient Animals/Group Study Vehicle Dose/Concentration Results
Reference
Duration
Benzophenone-12 Groups of 6 male rats 35 d Feed OECD TG 417. Concentrations of No significant gross lesions observed in rats killed on day 11, 22, or 35. 6

(Carworth Farms 1.25% and 5% daily No lesions of liver or kidneys at histological examination.
Elias strain)
Benzophenone-12 Groups of Wistar rats Premating to 0.5% OECD TG 416. Administered by No treatment-related gross pathological or histopathological findings; no 6

(F0 animals: 12 post-weaning carboxymet gavage during 10-wk premating deaths. No clinical signs or changes in general behavior observed in
males, 12 hylcellulose period (males), 2-wk premating male or female F0 parental animals of any dose group. No treatment-
females/group) at suspension period (females), 2-wk mating related body weight changes or effects on food consumption. No
doses of 100, 300, in drinking period (both sexes), ~2 d post- hematological findings or treatment-related clinical biochemical findings.
and 1000 mg/kg/d. water + 5 mating (males), entire gestation NOAEL of 1000 mg/kg/d for general, systemic toxicity.
mg/100 ml period, up to 30 d of lactation
Tween 80 (corresponding to 21 d of lactation
and up to 9 d post-weaning), and
35 d post-mating (for sperm-
negative females). Pups from the
F1 litter were selected (F1 rearing
animals) for specific post-weaning
examinations. The study was
terminated with the terminal
sacrifice of the F1 rearing animals.
All F0 parental animals were also
killed. Gross necropsy and
histopathological examination
performed on animals killed.
Benzophenone-1 Male and female rats 90 days Unknown Details relating to test protocol not NOAEL of 236 mg/kg body weight/d. Regarding organ toxicity 7

(number and strain stated endpoint, critical effects observed unspecified.

not stated)
Benzophenone-3 B6C3F1 mice (10 13 weeks Feed 0, 3125, 6250, 12,500, 25,000, or Decreased, body weight gains (dose-related). Mild increases in liver 69

males and 10 females 50,000 ppm weights observed in dosed mice of both sexes. Kidney weights increased
per group) variably in dosed females. Microscopic lesions noted only in kidneys of
males at 50,000 ppm: eosinophilic protein casts in dilated renal tubules
and mild inflammation associated with dilated tubules. NOAEL for
microscopic lesions of 6250 ppm.
Benzophenone-3 F344/N rats (10 13 weeks Feed 0, 3125, 6250, 12,500, 25,000, or Body weight gains of high-dose male and female rats reduced. Liver and 69

males and 10 females 50,000 ppm. kidney weights increased. Kidney lesions progressed to include
per group) papillary degeneration, or necrosis, and inflammation, while liver lesion
appeared to regress. Liver enzymes in serum remained elevated.
NOAEL for microscopic lesions of 6250 ppm.
Table 5. Repeated dose toxicity studies
Ingredient Animals/Group Study Vehicle Dose/Concentration Results
Reference
Duration
Benzophenone-3 Sprague-Dawley rats 14 weeks Feed 0 or 10,000 ppm Mean body weight of 10,000 ppm males not significantly different from 71

(10 male and 10 control males, but mean body weight of 10,000 ppm females
females per group) significantly decreased, and was approximately 87% of control value. In
males, absolute and relative liver and right kidney weights increased in
10,000 ppm group, compared to control group. In females, absolute
kidney weight significantly decreased, and relative liver weight
significantly increased, relative to control group. Incidence of mixed-cell
cellular infiltration in liver significantly increased in 10,000 ppm males,
relative to the control group. Cellular infiltrates composed of
mononuclear cells with scarce neutrophils, and had no specific
predisposition to specific area of liver lobule. Unlikely that cellular
infiltrates, all of minimal severity, would be responsible for changes in
liver weights observed in male rats at this time point. No other histologic
findings observed that would explain differences in organ weights, but in
females, body weight changes could have influenced absolute kidney
weight decrease and relative liver weight increase. However, increase in
relative liver weight in exposed females accompanied by nonsignificant
absolute liver weight increase. Unlikely that body weight was
responsible for liver weight change. Transcriptome analysis was
performed on RNA extracted from microarray study of male rat livers
from 10,000 ppm and control groups. Observed effects on transcription
consistent with mild induction of xenobiotic metabolism-related
processes, likely related to observed liver weight increases. Analysis of
subset of estrogen-responsive genes showed no change in response to
Benzophenone-3.
Table 6. Reproductive and developmental toxicity studies
Test Article Animals/Group Vehicle Dose/Concentration Procedure Results Reference
DERMAL
Benzophenone-3 B6C3F1 mice Acetone 22.75 to 364 mg/kg 13-wk dermal dosing study Epididymal sperm density decreased at all 3 dose levels 74

(10 males and evaluated (22.75, 91.0, and 364.0 mg/kg). Not possible to
10 females per establish NOAEL for decreased epididymal sperm density. In
group) female mice, no significant difference in estrous cycle length
between control group and each dose group.
Benzophenone-3 Pregnant mice Olive oil 50 mg/kg/d Exposed dermally from GD 0 to Dosing resulted in reduced fetal weight at GD 14 and feto- 75

(number not 6. High-frequency ultrasound placental index (first pregnancy), with 16.13% of fetuses under
stated) imaging was used to follow fetal 5th percentile; uterine artery parameters showed altered
and placental growth in vivo. pattern at GD 10. Benzophenone-3 detected in serum 4 h after
Blood flow parameters in uterine exposure on GD 6, and in amniotic fluid, on GD 14. Weight of
and umbilical arteries were offspring of first progeny lower in test group. Placental
analyzed by Doppler weights in test group decreased in second pregnancy. First and
measurements. Mice killed on second progenies of exposed mothers showed higher
GD 5, 10, and 14 (during first percentage of females (female sex ratio). Dermal exposure
pregnancy), and on GD 10 and during early pregnancy resulted in intrauterine growth
14 (during second pregnancy). restriction. (IUGR) phenotype, disturbed sex ratio, and
Benzophenone-3 levels analyzed alterations in the growth curve of the offspring in the mouse
in serum and amniotic fluid. model.
ORAL
Benzophenone-1 Female rats Unknown 10 mg/kg/d. Other Oral dosing for 3 d. NOAEL of 10 mg/kg/d. Any reproductive effects observed not 7

(number and administered doses specified.

strain not stated) not stated
Benzophenone-2 Groups of 5 10% ethanol/90% 6.25 mg/d Administered via gavage on GD In the test group, 8 of 57 male fetuses had hypospadias (p = 76

timed pregnant corn oil vehicle 12 through 17. Control pregnant 0.0064, when compared to controls). No changes in body
C57BL/6NCr mice dosed with vehicle only. mass-adjusted anogenital distance. Co-administration of
mice. Animals killed on GD 18. Benzophenone-2 with estrogen receptor antagonist yielded
Anogenital distance in male normal genital tubercles; i.e., no hypospadias in 26 of 26 mice.
fetuses measured and genital Hypospadias was not observed after dosing with the estrogen
tubercles examined receptor antagonist only or after dosing with vehicle only.
histologically. Quantitative Reverse transcriptase-polymerase chain reaction analysis
reverse transcriptase-polymerase showed that genital tubercles of treated male mice had higher
chain reaction analysis of genes levels of estrogen receptor-β, when compared to male controls
purportedly involved in genital (p = 0.04). Rersults indicated that Benzophenone-2 may cause
tubercle development also hypospadias via signaling through estrogen receptor.
performed. Also co-
administration of
Benzophenone-2 with estrogen
receptor antagonist (10 µg in
vehicle (s.c.) during gestation,
Table 6. Reproductive and developmental toxicity studies
Test Article Animals/Group Vehicle Dose/Concentration Procedure Results Reference
Benzophenone-3 Swiss CD-1 Feed 1.25%, 2.5%, and Continuous breeding protocol. Feed consumption in the 2.5% and 5.0% groups consistently 77

mice 5.0% (w/w) Male and female mice higher, but F0 body weights consistently lower. These findings
continuously exposed for a 7-d suggest that Benzophenone-3 may be adversely affecting
precohabitation and a 98-d metabolism or digestive process. In 2.5% and 5.0% dose
cohabitation period. F1 groups, number of live pups per litter significantly reduced.
generation from control, 2.5%, During lactation and nursing of F1 pups, pup survival
and 5.0% groups weaned for significantly below control value in 2.5% and 5.0% groups.
second generation studies. Minimal effects on fertility in F1 generation, but pup weights
significantly reduced. Epididymal sperm motility, sperm
count, and percentage of abnormal sperm not affected by
treatment. No apparent effects on estrual cyclicity or the
average estrous cycle length in treated females. Results
indicated that Benzophenone-3 caused systemic toxicity, but
had minimal effects on fertility and reproduction.
Benzophenone-3 B6C3F1 mice Feed 0, 3125, 6250, 13-wk oral dosing study Mice in the highest dose group (50,000 ppm in feed) exhibited 69

(10 males and 12,500, 25,000, or a decrease in epididymal sperm density and an increase in
10 females per 50,000 ppm length of the estrous cycle.
group)
Benzophenone-3 3 groups of Tocopherol- 30 µg/kg/d, 212 Oral dosing from pregnancy day Developmental exposures reduced size and growth of 78

mated BALB/c stripped corn oil µg/kg/d, and 3000 0 until the day before weaning mammary gland in males prior to (at postnatal day 21,
female mice µg/kg/d (lactational day 21). Sample statistically significant reduction) and during puberty
sizes for treatment groups were: (reduction not statistically significant). In females, reduced
30 µg/kg/d (10 litters), 212 mammary cell proliferation (statistically significant at 30
µg/kg/d (11 litters), and 3000 µg/kg/d), decreased number of cells expressing estrogen
µg/kg/d (9 litters). Sample size receptor α (statistically significant at 30 or 212 µg/kg/d), and
for controls was 11 litters. Pups altered mammary gland morphology (dose response) in
were weaned on postnatal day 21 adulthood. In males, anogenital index reduced after exposure
and co-housed with same-sex to 30 and 212 µg/kg/d at postnatal day 21 and in puberty. In
animals of the same treatment adult males, no differences in anogenital distance observed.
group for the remainder of the No effect on male body weight observed. In females,
experiment. anogenital index unaffected at postnatal day 21, but decreased
(at 212 µg/kg/d) when measured at puberty. No effects on
female anogenital index observed in adulthood.
Benzophenone-3 F344/N rats (10 Feed 0, 3125, 6250, 13-wk oral dosing study. Rats receiving diet with 50,000 ppm showed markedly lower 69

males and 10 12,500, 25,000, or epididymal sperm density and an increase in the length of the
females per 50,000 ppm estrous cycle at the end of the study.
group)
Table 6. Reproductive and developmental toxicity studies
Test Article Animals/Group Vehicle Dose/Concentration Procedure Results Reference
Benzophenone-3 Groups (7 to 8 low-phytoestrogen 1000; 3000; 10,000; Feeding from GD 6 until No exposure-related clinical signs were observed. On GD 10, 40

animals per chow 25,000; or 50,000 weaning on postnatal day 23. 15, and 20, the body weights of dams decreased in a dose-
group) of mated ppm. Control group fed low- dependent manner. Absolute and relative kidney weights in
female Sprague- phytoestrogen chow only. dams statistically significantly higher in 50,000 ppm exposure
Dawley rats group, when compared to control group. On GD 10, 15, and
20, body weights of dams decreased in dose-dependent
manner. Absolute and relative kidney weights in dams
statistically significantly higher in 50,000 ppm exposure
group, when compared to control group. Exposure associated
with reduced body and organ weights (kidney) in male and
female offspring. No statistically significant differences in
mean number of implantation sites/litter, mean resorptions per
litter, % litters with resorptions, number and weights of live
fetuses, or sex ratios between control and dose groups. One
fetus in 50,000 ppm group had hydrocephaly, but no other
malformations. Normalized anogenital distance in male pups
at postnatal day 23 decreased in 50,000 ppm exposure group.
Exposure to 50,000 ppm also caused impairment of
spermatocyte development in testes of male offspring. In
females, follicular development delayed in 50,000 ppm
exposure group. Authors concluded that few adverse effects in
dams and offspring dosed maternally and lactationally at
10,000 ppm or less. At higher concentrations, possible that
dosing produced delay in postnatal growth, which could have
adversely affected reproductive organ development; however,
this is not clear. Authors noted that further work needed to
clarify possible decreases in spermatogenesis and
folliculogenesis observed.
Benzophenone-3 Groups of 25 Chow and milk 3000 or 30,000 ppm Dosing from GD 6 until Daily observation of male offspring did not reveal any clinical 79

pregnant postnatal day 21. Male offspring observations related to perinatal exposure. At necropsy on
Sprague- weaned on postnatal day 28 and postnatal day 30, body weights 22% lower in 30,000 ppm
Dawley rats then dosed with same group when compared to control group. Rats exposed
concentrations. Animals killed perinatally to 30,000 ppm also had statistically significantly
on postnatal day 30. Controls lower weights of paired-testis, paired-epididymis, and prostate.
received diet without test These weights lower in males exposed to 30,000 ppm when
substance compared to controls (26%, 17.6%, and 18.5%, respectively).
Paired-testis weight to body weight ratio also statistically
significantly lower in 30,000 ppm group; however, no changes
in relative weights of paired epididymis and prostate in 30,000
ppm group. Rats exposed did not have differences in seminal
vesicle weight. Serum testosterone concentrations in rats
exposed perinatally to 3000 and 30,000 ppm Benzophenone-3
were 13.5% and 28.3% lower when compared to controls, with
statistical significance obtained in the 30,000 ppm
Benzophenone-3 exposure group. Also, liver and paired-
kidney weights lower in dose-dependent manner in 30,000
ppm group, attaining statistical significance. However,
relative liver and paired-kidney weights similar to controls.
Table 6. Reproductive and developmental toxicity studies
Test Article Animals/Group Vehicle Dose/Concentration Procedure Results Reference
Benzophenone-3 Groups of 42, Feed 0, 1000, 3000, and 39-day feeding period, beginning Gestation body weights of dams receiving 10,000 ppm slightly 71

35, 35, and 43 10,000 ppm on GD 6. Groups of 50 (1000 lower (~3%) than those of control group and showed
F0 time-mated and 3000 ppm) or 60 (0 and statistically significant differences. Dams receiving 3000 or
female Sprague- 10,000 ppm) F1 rats per sex 10,000 ppm displayed slight decreases in GD 6 - 21 body
Dawley rats continued on study after weight gain (~10%) relative to control group, which attained
weaning, and were fed diets statistical significance. Lower body weight gain over GD 6 - 9
containing same concentrations (10,000 ppm) and 18 - 21 (3000 and 10,000 ppm) intervals,
for 105 wk; 10 F1 rats per sex which was associated with slightly lower feed consumption
from 0 and 10,000 ppm groups over GD 18 - 21 interval. Authors noted that these collective
were evaluated at 14 wk. effects are minimal and would not be expected to affect
Dietary concentrations of 1000, normal development of offspring. Dosing had no effects on
3000, and 10,000 ppm percentage of mated females producing pups, litter size, pup
Benzophenone-3 resulted in sex distribution, or numbers of male or female pups. Authors
average daily doses of noted that decrease in percentage of females pregnant in
approximately 70, 206, and 660 10,000 ppm group can be attributed to 7 animals with no
mg Benzophenone-3/kg body evidence of pregnancy, as shown by absence of implantation
weight/d during gestation, and sites. Therefore, lower pregnancy rate not exposure-related,
157, 478, and 1609 mg/kg/d over given that exposure began after implantation. Dams dosed did
lactation days (LD) 1 - 14. not display any adverse clinical findings before or after
parturition. Litter size of 10,000 ppm group slightly lower on
postnatal days 7 and 10.

Benzophenone-3 Groups of 25 Corn oil 40, 200, and 1000 OECD TG 414. Dosing once All fetal pathological findings were indicative of a minor 10

mated Wistar mg/kg/d daily (by gavage) on days 6 disturbance and delay in ossification at the highest dose tested
rats of the through 19 post-coitum. Dose (1000 mg/kg/d). No test substance-induced effects on fetal
Crl:WI (Han) volume of 5 ml/kg. Animals morphology were observed at doses of 40 or 200 mg/kg/d. In
strain. killed on day 20. all dose groups, was scattered occurrence of few external, soft
tissue, and skeletal malformations without a consistent pattern.
Findings also occurred without clear dose-response
relationship and/or incidence, and not test substance-related.
External variations not observed in any fetuses. Authors
concluded that Benzophenone-3 did not possess any selective
teratogenic properties. NOAEL of 200 mg/kg/d.
Table 6. Reproductive and developmental toxicity studies
Test Article Animals/Group Vehicle Dose/Concentration Procedure Results Reference
Benzophenone-4 Groups of 26 Corn oil 750, 1000, and 1250 OECD TG 422. Male rats Pregnancy rates of 77, 62, 77, and 77% at 0, 750, 1000, and 8

Wistar rats (13 mg/kg/d treated 2 wk before mating and 1250 mg/kg, respectively. No significant effects observed on
males, 13 thereafter for total of 48 d of gestation length or litter size. Likewise, no significant effects
females/group) dosing (by gavage). Female rats were observed on the number of live births, pup survival, pup
treated 2 wk before mating, weight or sex ratio. Four pups in 750 mg/kg dose group
during mating, during gestation cannibalized. All other pups at 0, 750, 1000, and 1250 mg/kg
and during lactation, for total of normal externally. Internal examination of pups revealed no
~ 63 d of dosing. Control rats test substance-related abnormalities. Microscopic examination
dosed with corn oil only. of pups’ thyroid and parathyroid glands in 0 and 1250 mg/kg
Recovery groups of male and dose groups revealed no abnormalities. NOAEL (reproductive
female rats (5/sex/dose) treated toxicity) of 1250 mg/kg/d.
at 0 or 1250 mg/kg body
weight/d for 66 d total. Animals
in recovery groups allowed to
recover for 2 wk after final dose
given. No morbidity observed.
Estrous cyclicity unaffected by
treatment. All females showed
evidence of copulation after
cohabitation/mating period.
Benzophenone-12 Groups of 50 0.5% 100, 300, and 1000 Administered (gavage) to mated Neither clinical signs nor effects on body weight (or 6

Wistar rats (25 carboxymethyl- mg/kg/d. females from implantation to one organ/body weight ratios) were observed. No test substance-
males (for cellulose day prior to expected day of related necropsy findings were observed after dosing of dams.
mating), 25 suspension in parturition (GD 6 to 19). Female No evidence of dead/aborted fetuses or pre- and post-
females) drinking water + 5 rats killed on GD 20, and fetuses implantation loss. Test substance-related external, skeletal, or
mg/100 ml Tween removed from uterus. visceral malformations not observed. NOAEL (for maternal
80 and prenatal developmental toxicity) of 1000 mg/kg/d.

Benzophenone-12 Groups of 0.5% 100, 300, and 1000 Administered (gavage) as Clinical examinations of F0 parental animals did not reveal test 6

Wistar rats (F0 carboxymethylcell mg/kg/d. follows: 10-wk premating substance-related adverse findings, and no effects on
animals: 12 ulose suspension period (males), 2-wk premating reproductive performance. No test substance-related adverse
males, 12 in drinking water period (females), 2-wk mating findings at clinical or gross examination of F1 pups. For F1
females/group) + 5 mg/100 ml period (both sexes), ~2 d post- rearing animals, no test sub-stance-related findings during
Tween 80 mating (males), entire gestation clinical examinations and sexual maturation, and no gross
period, up to 30 d findings. NOAEL (for reproductive performance and fertility
(corresponding to 21 d of of F0 parental rats and develop-mental toxicity in offspring) of
lactation and up to 9 d post- 1000 mg/kg/d.
weaning), and 35 d post-mating
(for sperm-negative females).
Control group (12 males, 12
females) dosed with vehicle
only. Pups from F1 litter
selected (F1 rearing animals) for
specific post-weaning
examinations. Terminal
sacrifice of F1 rearing animals.
All F0 parental animals also
killed.
Table 6. Reproductive and developmental toxicity studies
Test Article Animals/Group Vehicle Dose/Concentration Procedure Results Reference
EMBRYO/OVARY CULTURES
Benzophenone-3 Zebrafish DMSO 0.116 mM, 0.0789 Modified OECD TG 236. Fish Cumulative mortality under 10% in negative and solvent 72

embryos (40 per mM, 0.0523 mM, embryotoxicity test (4 control groups at the end of the experiment. In positive
test 0.0307 mM, 0.0219 replicates). Dosing up to 120 h control group, cumulative mortality of 75%. %. In negative
concentration) mM, and 0.00535 post-fertilization, because this and solvent control groups, percentage of hatched embryos
mM period includes time points at was 95%. No hatched embryos observed in positive control
which different developmental group. Except for one in solvent control group (no swim
states can be observed. The bladder was observed), no malformations in negative and
positive control was 3,4- solvent control groups. LC50 values reported: 0.0766 mM (at
dichloroaniline (0.0247 mM), 72 h post-fertilization), 0.0698 mM (at 96 h), and 0.0573 mM
and water served as the negative (at 120 h). At 0.00438 mM, all embryos able to inflate tswim
control. DMSO served as the bladder. At higher concentrations, absence of swim bladder
solvent control. Endpoints inflation in concentration-dependent manner. EC50 value of
evaluated: mortality, 0.0295 mM after 120 h post-fertilization. At 72 h post-
malformations, hatching, and fertilization, deformation of tail observed (EC50 = 0.0419
inflation of the swim bladder. mM). Malformation of somites at 0.0526 and 0.0789 mM.
Decreased number of hatched embryos after 96 h post-
fertilization (EC50 = 0.0543 mM). Other malformations
observed, but frequency not concentration-dependent:
pericardial and yolk sac edema, deformed jaw and ventricle or
dilated gut, and jaw deformity. Benzophenone-3 caused
mortality, unsuccessful hatching, and different malformations
to zebrafish.
Benzophenone-3 Whole ovary DMSO 5.8 nM, 276 nM, Effect on follicular assembly Exposure to 5.8 mM decreased the population of total oocytes, 73

cultures 576 nM, and 876 nM. studied. Pups from the same number of nests per ovary, and number of early primary
collected from litters were randomly assigned to follicles. 5.8 mM stimulated process of germ cell nest
Wistar rats. different treatment groups so that breakdown and caused decrease in reserve of total oocytes.
Ovaries (n = each group contained ovaries of 276 mM increased population of total oocytes and number of
120) collected different pups from different nests per ovary, but decreased number of primary follicles. At
from rats at litters. ovary cultures were 576 nM and 876 nM, no changes observed in number of
birth (postnatal treated for 7 d. Vehicle control oocytes, germ cell nests per ovary, and assembled follicles in
day 0). cultures were treated with 0.01% ovaries.
DMSO. Positive control cultures
were treated with the estrogen
receptor β (ESR2) antagonist, 4-
(2-phenyl-5,7-
bis(trifluoromethyl)pyrazolo-1,5-
α-pyrimidin-3-yl) phenol
(PHTPP) in DMSO.
Table 7. Genotoxicity studies
Test Article Concentration/Dose Vehicle Test System Procedure Results Reference
IN VITRO
Benzophenone-1 1-25 µg/ml Culture medium Human keratinocytes (HaCaT Photo-genotoxicity of Benzophenone-1 and Benzophenone-1 photosensitized and 80

cells). apoptotic parameters assessed by western blot, generated intracellular reactive oxygen
immunocytochemistry, flow cytometry, the comet species (2.02 folds) under sunlight/UV
assay (for DNA damage), and transmission radiation. Decrease in cell viability was
electron microscopy (TEM) imaging. Apoptotic recorded as 80.06%, 60.98%, and
cells detected by annexin V/pro-propidium iodide 56.24% under sunlight, UVA, and
(PI) staining and sub G1 population of cell cycle. UVB, respectively. Benzophenone-1
Annexin V is a protein that is commonly used to enhanced lipid peroxidation, and
detect apoptotic cells. PI is a fluorescent agent leakage of lactate dehydrogenase
that is used to stain cells. (LDH) enzyme (61.7%).
Benzophenone-1 induced upregulation
of apoptotic proteins Bax.Bcl2 ratio,
Apaf-1, cytochrome c, Smac/DIABLO,
and cleaved caspase3 observed.
Benzophenone-1 5-25 µg/ml Culture medium HaCaT cells HaCaT cells treated with Benzophenone-1 in Immunostaining results showed 80

presence of UVB (1.08 J/cm2) or UVA (2.7 maximum CPD formation by

J/cm2). Genotoxicity potential of Benzophenone- Benzophenone-1 at a concentration of
1 confirmed through photo-micronuclei and 25 µg/ml (in presence of UVB). CPD
cyclobutene pyrimidine dimers (CPDs) formation formation not observed in control cells
(detected using immunostaining and fluorescence (exposed in dark or in light).
microscopy). Micronuclei formation detected in
HaCaT cells treated with 10 µg/ml in
presence of UVB . Simultaneously,
micronuclei not detected in control cells
exposed in dark or in light. Maximum
tail DNA (29.1%) recorded at 25 µg/ml,
compared to control value of 4.8%.
Cells exposed to different
concentrations in presence of UVA (2.7
J/cm2) exhibited statistically significant
(p > 0.01) DNA damage when
compared to control cells. Similarly,
highest olive tail moment (OTM) of
3.57 units recorded at concentration of
25 µg/ml (with UVA irradiation), when
compared to control cells (0.54 units).
Authors noted that study established
involvement of Benzophenone-1 in
photogenotoxicity and apoptosis via the
release of cytochrome c and
Smac/DIABLO.
Benzophenones -1, -3, Doses up to 10 µg/well DMSO or Salmonella typhimurium Luminescent umu-test Positive results for Benzophenone-3 81

-6, and -8 methanol strain TL210 and pseudo-positive results for

Benzophenone-1 and Benzophenone-8.

Benzophenones -1, -3, Benzophenone-1 (doses up DMSO S. typhimurium strains TA98 Ames test. Benzo[a]pyrene (BaP) was positive None of test substances produced clear 81

-6, and -8 to 600 µg/plate), and TA100 (with and without control (with activation), and 2-(20-furyl)-3-(5- positive results with or without
Benzophenone-3 (up to 200 metabolic activation). nitro-2-furyl) acrylamide (AF2) was positive metabolic activation. Results classified
µg/plate), Benzophenone-6 control (without activation). as negative.
(up to 2000 µg/plate), and
Table 7. Genotoxicity studies
Test Article Concentration/Dose Vehicle Test System Procedure Results Reference
Benzophenone-8 (up to 300
µg/plate)
Benzophenone-3 and 4 to 10 µl per plate Seawater S. typhimurium strain TA98 Ames test. Positive control was 2,4,7- Neither ingredient was genotoxic. 82

Benzophenone-8 (without metabolic trinitrofluorene Positive control was genotoxic.

activation).
Benzophenone-3 and Each ingredient (chlorinated, Chlorinated S. typhimurium strain TA98 Ames test. Positive control was 2,4,7- Only Benzophenone-8 (1:10) had clear 82

Benzophenone-8 doses up to 10 µl per plate) bromide-rich without metabolic activation. trinitrofluorene genotoxic activity that was dose-related
tested in seawater at ratios of water (artificial (doses of 4, 6, 8, and 10 µl). No
1:10 and 1:1000. seawater) genotoxic activity observed for either
ingredient at a ratio of 1:1000. Positive
control was genotoxic.
Sunscreen formulation 5000 µg/plate Sunscreen S. typhimurium strains: TA Ames test. Positive controls: sodium azide, 2- No observable increase in number of 68

containing 98, TA100, TA1535 and nitrofluorene, and 2-aminofluorene revertant colonies with or without
Benzophenone-3 TA1538 (with and without metabolic activation. Benzophenone-3
(0.6% to 0.9%) metabolic activation) was non-genotoxic. Positive controls
were genotoxic.
Benzophenone-3 0.20 µg/ml, 0.10 µg/ml, 0.05 DMSO Human peripheral Chromosomal aberrations (24-h exposure) assay. Benzophenone-3 induced following 7 83

µg/ml, 0.025 µg/ml, and lymphocytes Positive control (mitomycin C) types of structural chromosomal
0.0125 µg/ml aberrations in the chromosomal
aberrations assay: gaps, chromatid and
chromosome breaks, dicentric
chromosomes, rings, tri- or tetra-radials,
acentric fragments, and rearrangements.
Most frequent aberrations were acentric
fragments and chromatid aberrations;
numerical aberrations not found.
Statistically significant increase in
chromosomal aberrations and aberrant
cell frequencies at all test
concentrations, when compared to
solvent control. No statistically
significant differences between the
solvent and untreated control cultures
were observed. Positive control caused
statistically significant increase in
chromosomal aberrations and aberrant
cell frequencies (when dose-response
also observed), considering that
regression analysis revealed statistically
significant (p < 0.001) correlation
between Benzophenone-3
concentrations and level of genomic
damage, compared to all test
concentrations of Benzophenone-3 and
negative and untreated controls.
Benzophenone-3 0.20 µg/ml, 0.10 µg/ml, 0.05 DMSO Human peripheral Micronuclei (48-h exposure) assay. Positive Benzophenone-3 caused statistically 83

µg/ml, 0.025 µg/ml, and lymphocytes control (mitomycin C) significant increase in micronuclei
0.0125 µg/ml formation at all test concentrations. A
dose-response was also observed,
considering that a regression analysis
revealed a statistically significant
correlation (p < 0.001, compared to
Table 7. Genotoxicity studies
Test Article Concentration/Dose Vehicle Test System Procedure Results Reference
negative control) between
Benzophenone-3 concentrations and
frequencies of micronuclei and cells
with micronuclei. Results for vehicle,
untreated, and positive controls were
same as those reported in chromosome
aberrations assay above.
Benzophenone-3 1 µM and 5 µM Human breast epithelial cells. DNA damage assay. Immunostaining with Benzophenone-3 increased DNA 84

antibodies against markers of DNA damage, ℽ- damage in manner similar to E2, and in
H2AX (phosphorylated histone H2AX) and p53- an estrogen-receptor alpha (ERα)-
binding protein 1 (53BP1). dependent manner. However,
Benzophenone-3 had limited
transactivation of target genes at same 2
concentrations. Exposure caused R-
loop formation in normal human breast
epithelial cell line when ERα
introduced. Authors concluded that
Benzophenone-3 induces DNA damage,
mediated by formation of ERα-
dependent R-loops at concentrations
10-fold lower than those required for
transactivation.
Benzophenone-3 Doses up to 6000 µg/plate S. typhimurium strains TA98 Ames test (with and without metabolic activation) Non-genotoxic with and without 71

and TA100, and Escherichia metabolic activation.

coli strain uvrA pKM101
Benzophenone-8 Doses up to 1500 µg/plate DMSO S. typhimurium strain TA100 OECD TG 471. Bacterial reverse mutation assay No significant increases in frequency of 9

(strain TA100) and up to and E. coli (E. coli) strain (with and without metabolic activation). revertant colonies were noted for either
5000 µg/plate (strain WP2vurA Benzophenone-8 caused visible reduction in bacterial strain, at any dose level either
WP2vurA) growth of the bacterial background lawns of both with or without metabolic activation.
strains (with and without metabolic activation), Authors concluded that Benzophenone-
initially from 500 µg/plate. Therefore,test 8 was negative for genotoxicity.
substance evaluated up to either maximum
recommended dose of 5000 µg/plate or the toxic
limit (depending on the bacterial strain type).
Benzophenone-8 0.008 to 700 µg/plate Ethanol S. typhimurium tester strains: Salmonella/mammalian microsome mutagenicity Benzophenone-8 caused weak, but 85

TA98, TA100, TA1535, assay (with and without metabolic activation) reproducibly significant, increase in
TA1537, and TA1538. number of TA1537 revertants per plate.
Increase was dependent upon increasing
concentrations of the test substance, and
was totally dependent on the presence
of metabolic activation.
Benzophenone-8 13 to 56 µg/ml Ethanol L5178Y TK+/- mouse L5178Y TK+/- mouse lymphoma mutagenesis Cultures treated without metabolic 86

lymphoma cells assay (with and without metabolic activation) activation exhibited mutant frequencies
not significantly different from those of
solvent controls. Cultures treated with
metabolic activation exhibited
significant increase in mutant
frequencies, and dose response evident.
Two highest concentrations, 24 and 32
µg/ml, exhibited mutant frequencies
that were 3.8 and 2.0 times greater,
respectively, than average mutant
Table 7. Genotoxicity studies
Test Article Concentration/Dose Vehicle Test System Procedure Results Reference
frequency of solvent controls.
Benzophenone-8 was genotoxic.
Benzophenone-12 Doses up to 50 µg/ml (with DMSO Mouse lymphoma L5178Y OECD TG 476. Mammalian cell gene mutation Benzophenone-12 was non-genotoxic 6

metabolic activation) and up cells assay (with and without metabolic activation). without metabolic activation. Results
to 52 µg/ml (without Positive effect defined as doubling of mutant ambiguous with metabolic activation.
metabolic activation). frequency over concurrent solvent-treated control Relative to these results (with metabolic
value, together with evidence of dose-related activation), authors noted that a less
increase. than 3-fold increase in mutant
frequency occurred at highly toxic
concentrations.
IN VIVO
Benzophenone-3 0, 3000, or 3500 ppm Larva from a mating of Drosophila somatic mutation and recombination None of the Benzophenone-3-treated 87

Benzophenone-3 “multiple wing hair” (mwh) test (SMART). Test substance exposure for 72 h. larva produced flies with significantly
females with heterozygous Positive control: 25 ppm dimethylnitrosamine. A more single or multiple wing spots than
“flare” (flr) males recombination between the mwh and flr genes controls. In contrast, DMN-treated
(Drosophila melanogaster) produces twin wing spots, while events such as larva produced flies with significantly
deletions produce single spots. more single or multiple wing spots than
controls.

Benzophenone-3 0.0, 0.5, 1.67, or 5 g/kg Sprague-Dawley rat bone In vivo cytogenetics assay to evaluate Under either treatment protocol, none 87

marrow cells clastogenicity. Rats treated by oral gavage with of the Benzophenone-3 concentrations
single administration of each dose for 5 caused significant increase in
consecutive days. Cyclophosphamide (CP) was chromosomal aberrations.
positive control, administered at dose of 20
mg/kg. Colchicine growth-arrested bone marrow
cells collected 8 and 12 hours after single
treatment, and 12 hours after last daily treatment.
Benzophenone-3 0.6% to 0.9% Sunscreen Groups of 10 Wistar albino OECD TG 474. Mammalian erythrocyte Neither sunscreen formulation (all 68

formulation rats dosed prior to assay micronucleus test Doses of 0.5 g/kg, 1 g/kg, and 2 doses) nor placebo statistically
g/kg administered dermally (method not stated) significantly increased ratio of
for 2 consecutive days (at intervals of 24 h). micronucleus polychromatic
Positive control group dosed i.p. with erythrocyte (MNPCE)/polychromatic
cyclophosphamide (0.04 g/kg); negative control erythrocyte (PCE) and PCE/(PCE +
group dosed dermally with placebo formulation (2 normochromatic erythrocyte (NCE)).
g/kg). At 48 h after first dose, all rats killed and Positive control statistically
bone marrow extracted from the femur. 200 significantly increased these ratios.
erythrocytes in bone marrow cells of each animal Authors concluded that sunscreen (2
used to score total number of mature and g/kg) did not statistically significantly
immature erythrocytes. Number of micronuclei increase number of micronucleated
per 2000 immature erythrocytes recorded. immature erythrocytes or systemic
toxicity at 48 h, classifying sunscreen
formulation as non-genotoxic.

Benzophenone-3 0.6% to 0.9% Sunscreen Groups of 10 Wistar albino Mammalian bone marrow chromosome aberration No increment in the total number of 68

formulation rats dosed prior to assay test (modification of OECD TG 475). Doses of aberrant cells or in the chromosome
the sunscreen, 0.5 g/kg, 1 g/kg, and 2 g/kg, aberration percentage for the sunscreen
administered according to procedure stated formulation or placebo formulation
immediately above. Same is true for positive and observed. Positive control facilitated
negative controls (cyclophosphamide and placebo increase in number of aberrant cells.
formulation; same doses). Animals killed after Authors concluded that sunscreen
dosing, bone marrow extracted from femur, and formulation was non-genotoxic.
Table 7. Genotoxicity studies
Test Article Concentration/Dose Vehicle Test System Procedure Results Reference
slides prepared. Light microscopy used to
evaluate any evidence of chromosomal
abnormalities.
Benzophenone-3 3000 µg/kg/d Tocopherol- 12 ovariectomized mice DNA damage assay. Mice exposed to Results indicated that R-loops and DNA 84

stripped Corn oil (Balb/c female mice) dose Benzophenone-3 at 10 d after surgical procedure. damage detected in mammary epithelial
prior to assay 8 mice dosed orally with E2, and 12 mice dosed cells of mice treated with
orally with Benzophenone-3 daily for 4 d. Each Benzophenone-3. Authors concluded
mouse administered 1 µl of tocopherol-stripped that acute exposure to Benzophenone-3
corn oil per gram of body weight to deliver E2 in mice induces DNA damage,
(250 µg/kg/d) or Benzophenone-3 (3000 µg/kg/d). mediated by formation of ERα-
Immunostaining of mouse mammary epithelium dependent R-loops at concentrations
was performed to quantify R-loops and DNA 10-fold lower than those required for
damage in vivo. transactivation.
Table 8. Dermal irritation and sensitization studies
Test Article Concentration/Dose Test Population Procedure Results Reference
IN CHEMICO / IN VITRO STUDIES
Sunscreen formulation Benzophenone-3 (0.005 Hen’s egg The hen’s egg-chorioallantoic membrane test (HET-CAM). Formulation classified as non-irritant. 110

composed of polymeric wt %) (embryonated Nanocapsules contained poly(ℇ-caprolactone), carrot oil, a

nanocapsules loading membrane) non-ionic surfactant, and Benzophenone-3 (0.005 wt%). Eggs
Benzophenone-3. incubated for 10 d, after which membrane removed and CAM
was exposed. Formulation then added on embryonated hen’s
egg membrane; effects studied for 300 s. As positive control
(for vascular hemorrhage and lysis), 300 µl of sodium
hydroxide solution (0.1 M) applied. Sodium chloride solution
(0.9 wt%) applied as a negative control. Diluted (distilled
water) formulation (300 µl) applied to eggs also. Assay
monitored for any event (hemorrhage, lysis, and coagulation)
for 300 s.

Benzophenone-4 25 mg Three-dimensional Dermal corrosion potential studied according to OECD Test Benzophenone-4 considered corrosive to skin. 8

human epidermis TG 431. Before dosing, tissues moistened with sterile water
model (25 µl). Solid test article evenly applied to apical surface of
each tissue. Each treatment (test article or control) conducted
in duplicate. Exposure period for test article and control was 3
and 60 min. For 60-min exposure, dosed tissues placed in
incubator for remainder of 60-min exposure. MTT assay
performed using tissues transferred to 24-well plates. Mean
optical density for test chemical determined to be 2.098 and
0.315 for 3-min endpoint and 1-h endpoint, respectively. Mean
% tissue viability, compared to the negative control (n = 3),
determined to be 85.7 % and 13.4 % for 3-min endpoint and 1-
h endpoint, respectively.
Benzophenone-8 up to 200 mM (in KeratinoSens cell OECD TG 442D. In vitro antioxidant response element Benzophenone-8 classified as positive. Authors 9

DMSO) line (immortalized (ARE)-nuclear erythroid 2-related factor 2 (Nrf2) Luciferase stated that further testing is required, having noted
adherent human test method. Experiment involved 2 independent runs. that this test is part of tiered strategy for
keratinocyte cell line Maximal average fold induction of luciferase activity (Imax) evaluation of skin sensitization potential.
(HaCaT cell line), response for luciferase gene expression as well as sensitization
transfected with a potential determined. In both repetitions, induction of
selectable plasmid to luciferase above threshold of 1.5 noted. Imax was > 1.5-fold
quantify luciferase and statistically significantly different, as compared to
gene induction) negative control (DMSO).

ANIMAL
Sunscreen formulation 0.6% to 0.9% in Wistar albino rats OECD TG 402. Dose applied to 2" x 2", 4-ply gauze pad, and No signs of erythema or edema. 68

containing Benzophenone-3 formulation (formulation (12 males, 12 patch placed (secured with surgical tape) on hairless, dorsal
dose = 2000 mg/kg) females) skin. Patch remained in place for 24 h.
Table 8. Dermal irritation and sensitization studies
Test Article Concentration/Dose Test Population Procedure Results Reference
Sunscreen formulation 0.6% to 0.9% in 18 male New OECD TG 404. 3 groups: test, positive control (0.8% aqueous No evidence of erythema or edema in test or 68

containing Benzophenone-3 formulation Zealand rabbits (3 formaldehyde), and negative control (placebo sunscreen placebo groups (PII = 0). Positive control was
groups of 6) formulation), respectively. Each material applied to 25 cm2 severely irritating (PII = 10.43). No signs of
area of dorsal skin, using 2" x 3", 4-ply gauze pad (secured systemic toxicity.
with surgical tape). Application period of 72 h, after which
patches removed. Reactions scored for erythema and edema
at 24 h, 48h, and 72 h; primary irritation index (PII)
calculated.
Benzophenone-8 0.5 g in water (0.5 ml) 3 New Zealand white The test substance applied to skin for 4 h using semi-occlusive Skin irritation not observed in animals tested, and 9

rabbits patch. Reactions scored for up to 72 h post-application. Benzophenone-8 classified as a non-irritant.

Benzophenone-12 Fine powder (0.5 g) 3 male New Zealand OECD TG 404. The test substance applied (abraded and No evidence of erythema or edema during study 6

white rabbits intact skin of back) for 4 h under occlusive patch. Reactions (modified PII = 0); no clinical signs observed.
scored at 24 h, 48 h, and 72 h after patch removal using Draize Benzophenone-12 classified as non-irritating to
system. Modified PII calculated using 24-h and 72-h scores. skin of rabbits.
Sunscreen formulation 0.6% to 0.9% in 30 adult male guinea OECD TG 406. One group treated with the sunscreen None of the animals treated with sunscreen 68

containing Benzophenone-3 formulation pigs (3 groups of 10) formulation. The other 2 groups ere treated with 0.1% w/v 1- formulation or placebo had sensitization
chloro-2,4-dinitrobenzene (CDNB) in 10% propylene glycol reactions. Positive control induced skin
(positive control group) and a placebo formulation (cream sensitization. Authors classified sunscreen
base only, negative control group). Induction applications formulation as non-sensitizer.
(sunscreen formulation, positive control, or placebo) made to
the groups of animals. Inducing agents loaded on 2 cm x 4 cm
filter paper secured with occlusive dressing. Observations
relating to challenge reactions assessed after 24 h of induction,
and reactions scored.
Benzophenone-3 12.5%, 25%, and 50% Groups of 4 female Applications made to dorsum of each ear lobe on 3 At each concentration, stimulation index less than 10

mice of the CBA consecutive days. No local findings or clinical signs of limit criterion of 3. Benzophenone-3 classified as
strain toxicity, and no mortalities. At 5 d after topical application, non-sensitizer.
animals killed. Lymph nodes excised and single cell
suspensions prepared. Incorporation of [ H]methyl thymidine
3

measured.

Benzophenone-12 Intradernal induction at 10 test and 5 control Maximization test. Intradermal induction of sensitization in No toxic symptoms evident in test or control 114

15% (in PEG 300 and in female albino guinea test group performed in nuchal region. Epidermal induction of group. Seven of 9 surviving test animals had
emulsion of Freund’s pigs sensitization conducted for 48 h under occlusion 1 week after discrete/patchy to moderate/confluent erythema at
Complete Adjuvant intradermal induction, and following pretreatment of test areas the 24- and 48-h reading after challenge treatment
(FCA)/physiological with 10% sodium lauryl sulfate (SLS) 23 h prior to application with Benzophenone-12. No skin effect observed
saline); epidermal of test substance. Control animals intradermally induced with in control group. Benzophenone-12 classified as
induction and challenge PEG 300 and FCA/physiological saline, and epidermally skin sensitizer.
with 40% in PEG 300 induced with PEG 300 under occlusion following pretreatment
with 10% SLS. Two weeks after epidermal injection, control
and test animals challenged by epidermal application of test
substance and PEG 300 alone under occlusive dressing.
Cutaneous reactions evaluated at 24 h and 48 h after removal
of dressing .
Table 8. Dermal irritation and sensitization studies
Test Article Concentration/Dose Test Population Procedure Results Reference
Benzophenone-12 Intradermal injection at 20 guinea pigs (10 Maximization test. During first week of induction, intradermal Results indicated that 65% and 60% of animals 6

induction: males, 10 females) of injections (neck, 0.1 ml per injection; 3 pairs): adjuvant /saline sensitized to Benzophenone-12 under
Benzophenone-12 (5%) the Pirbright white mixture 1:1 (v/v), Benzophenone-12 (5%) in oleum arachidis experimental conditions employed at 24 h and 48
in oleum arachidis (w/v), (Tif:DHP) strain. (w/v), and Benzophenone-12 (5%) in the adjuvant/saline h after challenge, respectively. Authors classified
and Benzophenone-12 mixture (w/v). During the second week of induction (filter Benzophenone-12 as sensitizer.
(5%) in the paper patch application), Benzophenone-12 (30%) in
adjuvant/saline mixture petrolatum (w/w) applied to the neck for 48 h (2 cm x 4 cm
(w/v). Induction patch occlusive patch contained 0.4 g of paste). Control group of
application: 30% in 10 guinea pigs (5 males, 5 females) also treated during
petrolatum (w/w). induction. Challenge phase (week 5; i.e., 2 wk after
Challenge patch induction) consisted of single, 24-h application of
application: 20% in Benzophenone-12 (20% in petrolatum (w/w)). Test substance
petrolatum (w/w). (0.2 g paste) applied to flank using 2 cm x 2 cm occlusive
challenge patch. Reactions scored at 24 h and 48 h using
Draize scale. During challenge, control group treated with
vehicle and test substance to check for maximum sub-irritant
concentration of test substance in adjuvant-treated animals.
HUMAN
Benzophenone-4 2%, 5%, and 10% in 80 subjects Three concentrations tested on each subject. Dose applied to Benzophenone-4 (5% in petrolatum) induced skin 113

petrolatum (20 µl dose of 8-mm diameter Finn chamber, secured with adhesive tape. irritation in 4 subjects. Benzophenone-4 (10% in
each applied) Patches applied for 2 d to upper back. Reactions scored petrolatum) induced skin irritation in 6 subjects.
according to International Contact Dermatitis Research Group
(ICDRG) grading scale.
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