NTP TechNical RePoRT oN

The ToxiciTy STudy of

chiToSaN
(caSRN 9012-76-4)
admiNiSTeRed iN feed To
SPRague dawley
[cRl:cd(Sd)] R aTS
NTP TOX 93

DECEMBER 2017
 NTP Technical Report on the
Toxicity Study of Chitosan (CASRN 9012-76-4)
Administered in Feed to Sprague Dawley
[Crl:CD(SD)] Rats
Toxicity Report 93

December 2017

National Toxicology Program

Public Health Service
U.S. Department of Health and Human Services
ISSN: 2378-8992

Research Triangle Park, North Carolina, USA

 Chitosan, NTP TOX 93

Foreword
The National Toxicology Program (NTP) is an interagency program within the Public Health
Service (PHS) of the Department of Health and Human Services (HHS) and is headquartered at
the National Institute of Environmental Health Sciences of the National Institutes of Health
(NIEHS/NIH). Three agencies contribute resources to the program: NIEHS/NIH, the National
Institute for Occupational Safety and Health of the Centers for Disease Control and Prevention
(NIOSH/CDC), and the National Center for Toxicological Research of the Food and Drug
Administration (NCTR/FDA). Established in 1978, NTP is charged with coordinating
toxicological testing activities, strengthening the science base in toxicology, developing and
validating improved testing methods, and providing information about potentially toxic
substances to health regulatory and research agencies, scientific and medical communities, and
the public.
The Toxicity Study Report series began in 1991. The studies described in the Toxicity Study
Report series are designed and conducted to characterize and evaluate the toxicologic potential
of selected substances in laboratory animals (usually two species, rats and mice). Substances
selected for NTP toxicity studies are chosen primarily on the basis of human exposure, level of
production, and chemical structure. The interpretive conclusions presented in the Toxicity Study
Reports are based only on the results of these NTP studies. Extrapolation of these results to other
species, including characterization of hazards and risks to humans, requires analyses beyond the
intent of these reports. Selection per se is not an indicator of a substance’s toxic potential.
NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA
Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and
local health and safety regulations. Animal care and use are in accordance with the Public Health
Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective
quality assurance audits before being presented for public review.
NTP Toxicity Study Reports are indexed in the National Center for Biotechnology Information
(NCBI) Bookshelf and are available free of charge electronically on the NTP website
(http://ntp.niehs.nih.gov). Toxicity data are available through NTP’s Chemical Effects in
Biological Systems (CEBS) database:
https://www.niehs.nih.gov/research/resources/databases/index.cfm.

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Table of Contents
Foreword ......................................................................................................................................... ii
Tables ............................................................................................................................................. iv
Figures............................................................................................................................................ iv
About This Report............................................................................................................................v
Peer Review .................................................................................................................................. vii
Publication Details ....................................................................................................................... viii
Abstract .......................................................................................................................................... ix
Introduction ......................................................................................................................................1
Chemical and Physical Properties ...............................................................................................1
Production, Use, and Human Exposure ......................................................................................1
Absorption, Distribution, Metabolism, Excretion, and Toxicokinetics ......................................2
Toxicity .......................................................................................................................................3
Experimental Animals .........................................................................................................3
Humans ................................................................................................................................4
Carcinogenicity ...........................................................................................................................4
Developmental and Reproductive Toxicity ................................................................................4
Genetic Toxicity ..........................................................................................................................5
Study Rationale ...........................................................................................................................5
Materials and Methods .....................................................................................................................6
Procurement and Characterization of Chitosan...........................................................................6
Preparation and Analysis of Dose Formulations .........................................................................6
Animal Source.............................................................................................................................7
Animal Welfare ...........................................................................................................................7
Six-month Study .........................................................................................................................7
Statistical Methods ....................................................................................................................13
Calculation and Analysis of Lesion Incidences .................................................................13
Analysis of Continuous Variables .....................................................................................13
Quality Assurance Methods ......................................................................................................14
Results ............................................................................................................................................15
Six-month Study .......................................................................................................................15
Discussion ......................................................................................................................................28
References ......................................................................................................................................33
Appendix A. Summary of Lesions in Rats in the Six-month Feed Study of Chitosan ............... A-1
Appendix B. Clinical Pathology Results .....................................................................................B-1
Appendix C. Vitamin Concentration and Bone Parameter Results .............................................C-1
Appendix D. Organ Weights and Organ-Weight-to-Body-Weight Ratios ................................. D-1
Appendix E. Reproductive Tissue Evaluations ........................................................................... E-1

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Appendix F. Chemical Characterization and Dose Formulation Studies .................................... F-1

Appendix G. Feed and Compound Consumption in the Six-month Feed Study of
Chitosan ................................................................................................................ G-1
Appendix H. Ingredients and Nutrient Composition in AIN-93M Maintenance Purified
Diet ........................................................................................................................ H-1
Appendix I. Sentinel Animal Program.......................................................................................... I-1

Tables
Summary of Findings Considered to be Toxicologically Relevant in Sprague Dawley
Rats Exposed to Chitosan in Feed for Six Months ............................................................x
Table 1. Distribution of Evaluated Parameters ................................................................................8
Table 2. Experimental Design and Materials and Methods in the Six-month Feed Study of
Chitosan ...........................................................................................................................11
Table 3. Survival, Body Weights, and Feed Consumption of Group A Rats in the Six-
month Feed Study of Chitosan ........................................................................................15
Table 4. Selected Clinical Chemistry and Urinalysis Data for Group C Rats in the Six-
month Feed Study of Chitosan ........................................................................................18
Table 5. Serum and Hepatic Vitamin Concentration Data for Group B Rats in the Six-
month Feed Study of Chitosan ........................................................................................21
Table 6. Digestive Data for Group C Rats in the Six-month Feed Study of Chitosan ..................23
Table 7. Liver Parameter Data for Group A Rats in the Six-month Feed Study of Chitosan........25

Figures
Figure 1. Chitosan (CASRN 9012-76-4; Chemical Formula: [C6H11NO4]n) ..................................1
Figure 2. Growth Curves for Group A Rats Exposed to Chitosan in Feed for Six Months ..........16
Figure 3. Section of the Liver from a Control Male Sprague Dawley Rat from the Six-
month Feed Study of Chitosan with a Moderate Degree of Fatty Change (H&E) ........26
Figure 4. Higher Magnification of Figure 3 (H&E) ......................................................................26
Figure 5. Section of the Liver with a Lack of Fatty Change from a Male Sprague Dawley
Rat Exposed to 9% Chitosan in Feed for Six Months (H&E) ........................................27

This report has been reformatted to meet new NTP publishing requirements;
its content has not changed.

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About This Report

National Toxicology Program1
1
Division of the National Toxicology Program, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina, USA

Collaborators
K.A. Shipkowski, B.C. Sayers, S.A. Elmore, C.R. Blystone, M.C. Cora, L.M. Fomby, P.M.
Foster, M.R. Hejtmancik, M.J. Hooth, A.P. King-Herbert, G.E. Kissling, D.E. Malarkey, B.S.
McIntyre, J.T. Painter, T.A. Peace, K.R. Shockley, S.L. Smith-Roe, M.D. Stout, G.S. Travlos,
R.W. Tyl, M.K. Vallant, D.Y. Vasconcelos, S. Waidyanatha, N.J. Walker, K.L. Witt

Division of the National Toxicology Program, National Institute of Environmental Health

Sciences, Research Triangle Park, North Carolina, USA
Evaluated and interpreted results and reported findings
K.A. Shipkowski, Ph.D., Co-Study Scientist
B.C. Sayers, Ph.D., Co-Study Scientist
S.A. Elmore, D.V.M., Study Pathologist
C.R. Blystone, Ph.D.
M.C. Cora, D.V.M.
P.M. Foster, Ph.D.
M.J. Hooth, Ph.D.
A.P. King-Herbert, D.V.M.
G.E. Kissling, Ph.D.
D.E. Malarkey, D.V.M., Ph.D.
B.S. McIntyre, Ph.D.
K.R. Shockley, Ph.D.
S.L. Smith-Roe, Ph.D.
M.D. Stout, Ph.D.
G.S. Travlos, D.V.M.
M.K. Vallant, M.S., MT
S. Waidyanatha, Ph.D.
N.J. Walker, Ph.D.
K.L. Witt, M.S.

Battelle Columbus Operations, Columbus, Ohio, USA

Conducted study and evaluated pathology findings
M.R. Hejtmancik, Ph.D., Principal Investigator
L.M. Fomby, D.V.M., Ph.D.
T.A. Peace, D.V.M.
D.Y. Vasconcelos, D.V.M., Ph.D.

ILS, Inc., Research Triangle Park, North Carolina, USA

Coordinated NTP Pathology Peer Review (December 19, 2008)
J.T. Painter, D.V.M., Ph.D.

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 Chitosan, NTP TOX 93

RTI International, Research Triangle Park, North Carolina, USA

Provided sperm parameter data
R.W. Tyl, Ph.D., Principal Investigator

Contributors
NTP Pathology Peer Review, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, USA
Participated in NTP Pathology Peer Review (December 19, 2008)
S.A. Elmore, D.V.M., National Toxicology Program

Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina, USA
Supervised pathology review
M.H. Hamlin, II, D.V.M., Principal Investigator

RTI International, Research Triangle Park, North Carolina, USA

Supported sperm parameter data collection
K. Vick, B.S.

Dynamac Corporation, Research Triangle Park, North Carolina, USA

Prepared quality assessment audits
S. Brecher, Ph.D., Principal Investigator
S. Iyer, B.S.
V.S. Tharakan, D.V.M.

Social & Scientific Systems, Inc., Research Triangle Park, North Carolina, USA
Provided statistical analyses
M.V. Smith, Ph.D., Principal Investigator
L.J. Betz, M.S.
S.F. Harris, B.S.
J.D. Krause, Ph.D.
C.G. Leach, M.S.

Biotechnical Services, Inc., Little Rock, Arkansas, USA

Prepared Toxicity Study Report
S.R. Gunnels, M.A., Principal Investigator
L.M. Harper, B.S.
T.S. Kumpe, M.A.
E.S. Rathman, M.S.
D.C. Serbus, Ph.D.

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 Chitosan, NTP TOX 93

Peer Review
The draft NTP Technical Report on the Toxicity Study of Chitosan (CASRN 9012-76-4)
Administered in Feed to Sprague Dawley [Crl:CD(SD)] Rats was evaluated by the reviewers
listed below. These reviewers served as independent scientists, not as representatives of any
institution, company, or governmental agency. In this capacity, reviewers determined if the
design and conditions of this NTP study were appropriate and ensured that this Toxicity Study
Report presents the experimental results and conclusions fully and clearly.

Peer Reviewers
Diane Birt, Ph.D.
Iowa State University (Retired)
Ames, Iowa, USA
Melissa G. Rhodes, Ph.D.
Roivant Sciences, Inc.
Durham, North Carolina, USA

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 Chitosan, NTP TOX 93

Publication Details
Publisher: National Toxicology Program
Publishing Location: Research Triangle Park, NC
ISSN: 2378-8992
DOI: https://doi.org/10.22427/NTP-TOX-93
Report Series: NTP Toxicity Report Series
Report Series Number: 93
Official citation: National Toxicology Program (NTP). 2017. NTP technical report on
the toxicity study of chitosan (CASRN 9012-76-4) administered in feed to Sprague
Dawley [Crl:CD(SD)] rats. Research Triangle Park, NC: National Toxicology Program.
Toxicity Report 93.

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 Chitosan, NTP TOX 93

Abstract
Chitosan is a cationic carbohydrate polymer that is commercially derived from the deacetylation
of chitin obtained from seafood shells. The most widespread route of human exposure to
chitosan is as a dietary supplement for body weight reduction. Chitosan was nominated by the
National Cancer Institute for mechanistic studies designed to measure the potential for vitamin E
depletion and osteoporosis following ingestion. Male and female Sprague Dawley rats were
exposed to chitosan (86.5% deacetylated, with an average molecular weight of approximately
82 kilodaltons and estimated to be approximately 94% pure) in feed for 6 months.
In this 6-month study, groups of 10 male and 10 female core study rats (Group A) were fed
control diets (AIN-93M) or diets containing chitosan at concentrations of 1%, 3%, or 9%, for up
to 25 weeks. Two additional groups of 10 male and 10 female rats (Groups B and C) were given
the same dietary concentrations for up to 26 weeks. All male and female Group A rats survived
to the end of the study. Mean body weights and feed consumption of exposed Group A groups
were similar to those of the control groups. Dietary concentrations of 1%, 3%, and 9% resulted
in average daily doses of approximately 450, 1,500, and 5,200 mg chitosan/kg body weight per
day to males and 650, 1,800, and 6,000 mg/kg per day to females. There were no treatment-
related clinical findings in core study animals.
The 9% male and female rats had significantly decreased cholesterol values (26% to 48%),
compared to the controls, at all time points. Triglycerides were significantly decreased in 9%
male and female rats, but not at every time point. Phosphorus levels were significantly decreased
in 9% male rats at weeks 13, 19, and 25; a decrease also occurred in 3% males at week 13.
Phosphorus levels were significantly decreased in 3% and 9% females at weeks 13 and 25.
Compared to those of the controls, serum vitamin A concentrations were significantly decreased
(approximately 30%) at weeks 13, 19, and 26 in 9% males, at weeks 13 and 26 in 3% males
(approximately 15%), and at weeks 19 and 26 in 9% females (approximately 20%). Serum
vitamin E concentrations were significantly decreased at all time points in 3% (33% to 42%) and
9% (79% to 82%) males, in 1% (17%) males at week 13, and in 9% (62% to 65%) females at all
time points. Hepatic vitamin E concentrations were significantly decreased at week 26 in 3%
(48%) and 9% (87%) males and 9% (80%) females. Serum concentrations of 1,25 (OH)2
vitamin D were significantly increased in 9% (105% to 142%) males and (100% to 180%)
females at weeks 7, 19, and 26.
Compared to the control groups, percent fat digested was significantly decreased during week 6
in 9% males and females, during week 12 in 3% and 9% males, during week 18 in 9% males and
females, and during week 24 in all exposed groups of males and females. Calcium absorption
was significantly increased in 9% females during weeks 12 and 24. Fecal weight was
significantly increased in 3% and 9% males and females during each collection period, and in
1% females during weeks 12, 18, and 24. Fecal moisture was significantly increased in 9% males
(up to 170%) and 9% females at all time points, in 3% males during week 6, and in 3% females
during weeks 12 and 18.
Results of this study did not support chitosan as a cause of bone resorption. Significant elevation
of parathyroid hormone levels occurred occasionally and inconsistently, while calcium levels
remained relatively stable. Bone calcium, bone length, and the histology findings did not indicate
calcium loss from the bone following chitosan exposure.

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The absolute and relative liver weights of 9% males and females and the absolute and relative
thymus weights of 3% males and 9% males and females were significantly less than those of the
control groups.
There was a treatment-related decrease in the incidence of periportal fatty change in the liver of
9% females relative to the control group. A decreased incidence of periportal fatty change was
observed in the liver of 9% males relative to the control group as well, but this decrease was not
significant, and it was the same as that observed in 1% males. The appearance of periportal fatty
change was similar in both males and females and in both exposed and control groups.
Under the conditions of the 6-month feed study of chitosan, male and female rats fed 3% and 9%
chitosan in the diet had significantly decreased levels of serum vitamin A and serum and hepatic
vitamin E and increased levels of serum 1,25 (OH)2 vitamin D. Consumption of high levels of
chitosan decreased percentage fat digestion and increased fecal weight and moisture, as well as
reduced levels of phosphorous, cholesterol, and triglycerides. Female rats exposed to 9%
chitosan also had significant liver weight and histologic changes. Based on the above results, the
lowest-observed-effect level for chitosan exposure was 1% (approximately equivalent to
450 mg/kg) in male and 9% (approximately equivalent to 6,000 mg/kg) in female rats.
Synonyms: 2-Amino-2-deoxy-beta-D-glucosamine; deacetylated chitin; poliglusam; poly
(D-glucosamine)
Trade names: Celox, Chicol, Chitopearl, CTFA 04299, Flonac N, Kytex H, Sea Cure F

Summary of Findings Considered to be Toxicologically Relevant in Sprague Dawley Rats Exposed

to Chitosan in Feed for Six Months
Male Rats Female Rats
Concentrations in feed 0%, 1%, 3%, 9% 0%, 1%, 3%, 9%
Survival rates Group A: 10/10, 10/10, 10/10, 10/10 Group A: 10/10, 10/10, 10/10, 10/10
Group B: 9/10, 10/10, 10/10, 8/10 Group B: 10/10, 10/10, 9/10, 10/10
Group C: 10/10, 10/10, 10/10, 10/10 Group C: 10/10, 9/10, 10/10, 10/10
Body weights Exposed groups similar to the control group Exposed groups similar to the control group
Clinical findings None None
Clinical pathology ↓ Phosphorus ↓ Phosphorus
↓ Cholesterol ↓ Cholesterol
↓ Triglycerides ↓ Triglycerides
Vitamin ↓ Serum vitamin A ↓ Serum vitamin A
concentrations ↑ Serum 1,25 (OH)2 vitamin D ↑ Serum 1,25 (OH)2 vitamin D
↓ Serum vitamin E ↓ Serum vitamin E
↓ Hepatic vitamin E ↓ Hepatic vitamin E
Digestive parameters ↓ Percent fat digested ↓ Percent fat digested
↑ Fecal weight ↑ Fecal weight
↑ Fecal moisture ↑ Fecal moisture
↑ Calcium absorbed
Bone parameters None None
Reproductive toxicity None Not determined
Organ weights ↓ Absolute and relative liver weights ↓ Absolute and relative liver weights
↓ Absolute and relative thymus weights ↓ Absolute and relative thymus weights
Nonneoplastic effects None Liver: periportal, fatty change (7/10, 4/10,
4/10, 0/10)

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 Chitosan, NTP TOX 93

Introduction

Figure 1. Chitosan (CASRN 9012-76-4; Chemical Formula: [C6H11NO4]n)

Synonyms: 2-Amino-2-deoxy-beta-D-glucosamine; deacetylated chitin; poliglusam; poly. (D-glucosamine).

Trade names: Celox, Chicol, Chitopearl, CTFA 04299, Flonac N, Kytex H, Sea Cure F.

Chemical and Physical Properties

Chitosan is a cationic carbohydrate polymer that is commercially derived from the deacetylation
of chitin. The primary unit of the chitosan polymer is D-glucosamine. Chitosan exists in multiple
forms that can differ in molecular weight [3 to 3,600 kilodaltons (kDa)] and in the degree of
deacetylation (40% to 100%)1. Chitosan is defined as chitin that is sufficiently deacetylated to
form soluble amine salts. Solubility in aqueous, acidic media occurs when deacetylation of chitin
reaches approximately 50%2. In addition to the degree of deacetylation, chitosan solubility is
also dependent on the molecular weight and the distribution of the remaining acetyl groups on
the polymer3. Chitosan is insoluble in alkaline solutions at pH levels above 6.5. Chitosan
products are highly viscous, resembling natural gums4.

Production, Use, and Human Exposure

Chitin, from which chitosan is derived, is a naturally occurring carbohydrate polymer second
only to cellulose in abundance. Chitin is a structural component found in the exoskeleton of
arthropods and in the cell walls of fungi and yeast2. The primary unit of chitin, N-acetyl-D-
glucosamine, forms the polymeric structure via 1 → 4 glycosidic bonds. Discarded crab and
shrimp shells from the seafood industry are the primary source material of chitin for the
commercial production of chitosan5. For chitosan production, seafood shells are deproteinized by
treatment with an aqueous 3% to 5% sodium hydroxide (NaOH) solution. The resulting product
is neutralized and calcium is removed by treatment with an aqueous 3% to 5% hydrochloric acid
(HCl) solution at room temperature resulting in a white or slightly pink precipitate of chitin. The
N-deacetylation of chitin is done by treatment with an aqueous 40% to 45% NaOH solution, and
the precipitate is washed with water. The precipitate is then dissolved in aqueous 2% acetic acid
and the insoluble material is removed. The resulting clear supernatant solution is neutralized with
aqueous NaOH solution producing chitosan as a white precipitate.
Chitosan is used in a wide range of products including use as a flocculating agent for water and
waste treatment and as a chelating agent for removal of traces of heavy metals from aqueous

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 Chitosan, NTP TOX 93

solutions4. In agriculture, chitosan is used as a plant growth regulator through foliar application
and as an antimicrobial agent and a time-release reservoir for fertilizers in soil amendments.
Chitosan has several current or proposed biomedical applications. Chitosan is considered to be
hemostatic due to its cationic nature. As such, wound dressings manufactured from chitosan are
available for clinical use6. Several drug delivery systems based on chitosan nanoparticles are
currently being investigated. Chitosan nanoparticles are capable of permeating the blood brain
barrier, and the mucoadhesive properties of chitosan have been shown to enhance drug
absorption2; 7. Chitosan has also been evaluated for the manufacture of ocular bandage lenses and
biodegradable surgical and dental implants8.
In cosmetics, chitosan is used in a variety of hair and skin products, including hair and body
washes, coloring shampoos, and agents for skin cleaning and protection9. Chitosan has also been
evaluated for use as an additive to toothpaste for prevention of enamel erosion10.
As a dietary supplement, chitosan is marketed and sold in weight-loss products, but the
mechanism behind chitosan-induced inhibition of fat digestion is not well understood. It has been
proposed that chitosan acts as a weak anion exchanger and decreases intestinal cholesterol
absorption while also increasing the excretion of bile acids11-13. Another possible mechanism is
that chitosan traps fat in the intestines by increasing the viscosity of the intestinal contents and
preventing the hydrolysis of triglycerides13-15. The manufacturer-recommended consumption of
chitosan as a weight-loss product in humans typically averages 1,000 mg per day, or
approximately 14.3 mg/kg per day (based on a 70 kg adult)16; 17. There are no available dose or
prevalence data for human consumption of chitosan as a dietary supplement.

Absorption, Distribution, Metabolism, Excretion, and

Toxicokinetics
The systemic absorption and distribution of chitosan following oral exposure are likely
influenced by the molecular weight of the polymer. The effect of molecular weight on chitosan
absorption has been evaluated in male Sprague Dawley rats. Oral gavage administration of
chitosan with molecular weights of 3.8, 7.5, 13, 22, or 230 kDa resulted in maximum plasma
chitosan concentrations (Cmax) of 20.23, 9.30, 5.86, 4.32, or less than 0.5 μg/mL, respectively18.
The results of this study suggest that the absorption of chitosan from the gastrointestinal tract
following oral exposure is inversely related to chitosan molecular weight, as there is likely low
bioavalability associated with the higher molecular weight chitosan polymers.
The biodegradation of chitosan influences absorption and distribution because both are
dependent on molecular weight. The biodegradation of chitosan in vivo is dependent on the
degree of deacetylation19. Enzymatic degradation of chitosan depends on the ability to hydrolyze
glucosamine-glucosamine, glucosamine-N-acetyl-glucosamine and N-acetyl-glucosamine-N-
acetyl-glucosamine linkages1. Degradation of chitosan in vertebrates is thought to occur
predominantly by lysozymes and bacterial enzymes in the colon1. While eight human chitinases
have been identified with three showing enzymatic activity, their capacity to degrade chitosan
has not been investigated1; 20.

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Toxicity
Experimental Animals
The acute toxicities of chitosan and chitosan oligomers prepared by enzymatic depolymerization
of chitosan have been evaluated. Hirano5 reported the oral LD50 for chitosan as 16 g/kg body
weight in mice. No clinical signs of toxicity were observed following a single oral administration
of chitosan oligomers up to 10 g/kg in male and female Kunming strain mice21.
No significant differences in weight gain were observed between exposed male Charles River
albino rats and the controls in a 4-week study with 1% or 5% dietary chitosan22. In male Wistar
rats, no significant differences in growth, feed intake, liver weight, or dried fecal weight were
observed between control and chitosan-fed (2% or 5%) animals after 21 days23. In male Sprague
Dawley rats fed chitosan in the diet for 8 weeks, no toxicity was observed in animals at
concentrations up to 5%, progressive growth reductions and clinical pathology disturbances
occurred at 10% and 15%, and enlargement of the liver and kidneys was observed at 15%24.
In female BALB/c mice fed a 5% (4.4 ± 0.7 g/day per animal) chitosan diet for 4 weeks, body
weight reduction correlated with significantly decreased feed consumption and alterations in
normal gut flora25.
In a study to evaluate mineral and fat-soluble vitamin status in male Charles River Japan Sprague
Dawley rats, exposure to a diet containing 5% chitosan for 2 weeks caused a decrease in mineral
absorption and bone mineral content26. Decreased serum vitamin E was observed in rats fed 5%
chitosan with ascorbic acid supplementation in the diet. Serum vitamin E depletion was not
observed in rats given glucosamine instead of chitosan.
Depletion of fat-soluble vitamins has been associated with a variety of neurologic and metabolic
disorders. Male C57BL/6 mice fed a vitamin E-deficient diet showed signs of cognitive decline
after 3 months of exposure and had increased lipid peroxidation products in brain tissue after
6 months of exposure27. Male rats fed a vitamin A-deficient diet for 3 months had lower levels of
serum cholesterol, HDL-cholesterol, and triacylglycerol, as well as decreased synthesis of liver
fatty acids28.
The toxicity of glucosamine oligomers has been evaluated in male and female Charles River
Japan F344 rats fed 0%, 0.04%, 0.2%, or 1% oligoglucosamine in the diet for 90 days29.
Glucosamine oligomers are prepared by hydrolysis of chitosan and, similar to the chitosan
utilized in this 6-month study, are considered low molecular weight chitosan. In the 1%
(653.1 mg/kg per day in males, 719.8 mg/kg per day in females) group, erythema and edema in
the snout and on the forelimbs and loss of fur on the forelimbs were observed in both male and
female rats. Neutrophilic infiltration in the nasal cavity was also observed in both sexes in the
1% group. These findings were considered to be caused by topical exposure to glucosamine
oligomers during feeding and grooming. Decreased feed consumption and body weight gain
were also observed in animals in the 1% group in this study and were thought to be the result of
feeding difficulty due to the snout and forelimb lesions described above. Rats receiving
1% oligoglucosamine also displayed lower weights of the uterus, ovary, seminal vesicles, and
testes (with fewer germ cells).

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 Chitosan, NTP TOX 93

The intravenous administration of chitosan has been investigated due to the development of
chitosan formulations for drug delivery. No adverse effects were reported in rabbits up to
60 days following intravenous administration of chitosan oligosaccharides (prepared by
oxidative depolymerization of chitosan) at doses up to 8.6 mg/kg daily for 5 consecutive days30.
In this study, increased lysozyme activity was observed in rabbit serum collected the day after
the last intravenous injection. Chemical modifications and nanoparticle suspensions of chitosan
are currently being investigated for drug delivery1. As such, modifications made to chitosan
could alter the toxicity of the unmodified chitosan polymer.
No adverse effects of chitosan were reported in eye or skin irritation tests in rabbits or guinea
pigs, respectively31.

Humans
Studies designed to evaluate the effectiveness of chitosan as a weight-loss supplement suggest
that chitosan is well tolerated in humans. No adverse effects were reported in male (4.5 g
chitosan per day) or female (2.5 g per day) volunteers following oral chitosan administration for
12 days32; 33. Additionally, no adverse effects were reported following oral administration of
chitosan at up to 6.75 g per day for 8 weeks in male and female volunteers34.

Carcinogenicity
No 2-year carcinogenicity studies of chitosan were identified in the available literature.
Carcinogenicity and chronic toxicity have been evaluated for N-acetyl-D-glucosamine, a
monomeric constituent of chitosan. F344 rats administered N-acetyl-D-glucosamine at
concentrations up to 5% in the diet (1,935 mg/kg per day in males and 2,244 mg/kg per day in
females) for 104 weeks had no associated increases in tumor response35. In a second study in
F344 rats, administration of N-acetyl-D-glucosamine in feed at concentrations up to 5% in the
diet (2,323 mg/kg per day in males and 2,545 mg/kg per day in females) for 52 weeks did not
induce an increase in tumor response35.

Developmental and Reproductive Toxicity

A limited number of developmental and reproductive toxicity studies were identified in the
literature.
In a multigenerational prenatal and postnatal assessment of high molecular weight chitosan
(HMWCS), F0 time-mated ICR mice were administered 0, 125, 500, or 2,000 mg/kg HMWCS
via a single intraperitoneal injection on gestational day 6 (GD 6) and subjected to a laparotomy
or allowed to litter36. F1 offspring (1 mouse/sex per litter) from the same exposure group were
mated and females similarly subjected to either a laparotomy or allowed to litter to produce an F2
generation. F0 dams in the 2,000 mg/kg group exhibited signs of maternal toxicity (mortality and
diarrhea). F0 dams in the 500 and 2,000 mg/kg groups displayed dose-dependent increases in
vaginal bleeding, postimplantation loss, and lower spleen weights. Fetal weights for both
generations were lower in the 2,000 mg/kg group. There were no external, visceral, or skeletal
malformations attributed to chitosan administration. F0 dams allowed to litter displayed a dose-
related reduction in litter size. F1 mice exposed in utero to 2,000 mg/kg HMWCS and examined
on postnatal day 21 (PND 21) exhibited higher uterus, ovary, and thymus weights. Female F1

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 Chitosan, NTP TOX 93

mice exposed in utero to 2,000 mg/kg HMWCS displayed lower thymus weights on PND 56. F2
mice exposed in utero to 2,000 mg/kg HMWCS displayed lower testis and ovary weights on
PNDs 21 and 56.
Chitosan oligomers did not induce morphologic sperm abnormalities in male mice following oral
gavage daily for 5 days with up to 5,000 mg/kg21.
The effects of chitosan nanoparticles (spherical; 200 ± 6 nm or 340 ± 10 nm diameter) have been
examined in zebrafish (Danio rerio) embryos. Embryos exposed 4 to 5 hours after fertilization to
0, 5, 10, 20, 30, or 40 μg/mL (200 nm particles) or 0, 10, 20, or 40 μg/mL (340 nm particles)
displayed concentration-dependent decreases in hatching rates and increases in mortality
96 hours after exposure37. Increased rates of cell death and reactive oxygen species production
were observed in all exposure groups. Exposure to 200 nm, but not 340 nm, chitosan
nanoparticles induced developmental malformations in embryos, including bent spines,
pericardial edema, and opaque yolks.

Genetic Toxicity
No in vitro or in vivo studies evaluating chitosan for mutagenic effects were identified in the
available literature.
Chitosan oligomers were negative at concentrations up to 5,000 μg/plate in Salmonella
typhimurium strains TA97, TA98, TA100, and TA102 with and without rat liver S9 metabolic
activation enzymes, and they were negative for micronucleus induction in mouse bone marrow
following oral gavage for 2 days at up to 5,000 mg/kg21.

Study Rationale
Chitosan was nominated for study by the National Cancer Institute due to widespread human
exposure, especially through use as a dietary supplement for body weight reduction, and for
concerns regarding potential vitamin E and bone mineral depletion following ingestion. NTP
conducted a 6-month study evaluated the effects of dietary chitosan on the development of
osteopenia/osteoporosis, fat and calcium absorption, fat-soluble vitamin depletion, and general
toxicity effects in Charles River Sprague Dawley rats.

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Materials and Methods

Procurement and Characterization of Chitosan
Chitosan was obtained from Vanson HaloSource, Inc. (Redmond, WA), in one lot (02-ASSF-
0715), which was used in the 6-month study. Identity, purity, and stability analyses were
conducted by the analytical chemistry laboratory at Midwest Research Institute (MRI) (Kansas
City, MO) and by the study laboratory at Battelle Columbus Operations (Columbus, OH)
(Appendix F). Reports on analyses performed in support of the chitosan studies are on file at the
National Institute of Environmental Health Sciences.
The test article, an off-white powder, was identified using infrared and proton nuclear magnetic
resonance (NMR) spectroscopy. The percentage of deacetylation of the test article, determined
by proton NMR, ranged from 85.97% to 87.17%, with an average of 86.5%. All spectra were
consistent with the literature spectra38; 39, and with the Sadtler spectral database.
The moisture content for lot 02-ASSF-0715 was determined using weight loss on drying, the
inorganic content was determined on the dried test article by ashing, viscosity was determined
using a Brookfield viscometer, and the most abundant molecular weight was determined using
gel permeation chromatography (GPC) with refractive index (RI) detection.
Moisture content was 4.50% water, the average inorganic content was 2.13%, and viscosity was
81.3 centipoise. GPC/RI indicated one major peak and the determined molecular weight of the
bulk chemical ranged from 62,755 to 87,343 daltons (Da). This resulted in an average molecular
weight of 81,644 g/mol, or approximately 82 kDa, classifying the test article as a low molecular
weight chitosan (LMWCS). A sample of lot 02-ASSF-0715 was submitted to Covance
Laboratories, Inc. (Madison, WI), for nutritional and contaminant testing using standard
methods. Levels of organochlorine and organophosphorous pesticides, nitrosamines, and
aflatoxins were below the detection limits of the analytical methods. The purity of lot
02-ASSF-0715 was estimated to be approximately 94% based on the analysis of moisture and
inorganic content. Taken together, these data indicated that the test article was chitosan.
To ensure stability, the test article was stored in sealed amber glass vials at room temperature.
Reanalysis of the test article was performed during the study using GPC/RI and no degradation
of the test article was detected.

Preparation and Analysis of Dose Formulations

The dose formulations were prepared approximately monthly by mixing chitosan with feed.
Dose formulations were stored in lined plastic buckets sealed with lids and stored at −30°C to
−15°C for up to 42 days.
Homogeneity studies of approximately 0.5% and 9% formulations (5,046 and 90,049 µg/g,
respectively) and stability studies of an approximately 0.5% (5,046 µg/g) formulation were
performed by the analytical chemistry laboratory using GPC/RI. Two peaks were attributed to
chitosan with retention times of approximately 6.9 minutes and 12.1 minutes, respectively.
Chitosan quantitation was based on the larger polymeric components of the first peak only
because vehicle components co-eluted with the later oligomeric peak. Homogeneity studies of

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 Chitosan, NTP TOX 93

1% and 9% (10 and 90 mg/g in feed, respectively) dose formulations were performed by the
study laboratory using GPC/RI. Homogeneity was confirmed, and stability was confirmed for at
least 42 days for dose formulations stored in lined plastic buckets sealed with lids at
temperatures up to room temperature and for at least 7 days under simulated animal room
conditions.
Periodic analyses of the dose formulations of chitosan were performed by the study laboratory
using GPC/RI. Of the dose formulations analyzed, all nine were within 10% of the target
concentrations (Table F-3). Animal room samples were also analyzed; all three were within 10%
of the target concentrations.

Animal Source
Male and female Sprague Dawley [Crl:CD(SD)] rats were obtained from Charles River
Laboratories (Portage, MI) for use in the 6-month study.

Animal Welfare
Animal care and use are in accordance with the Public Health Service Policy on Humane Care
and Use of Animals. All animal studies were conducted in an animal facility accredited by the
Association for the Assessment and Accreditation of Laboratory Animal Care International.
Studies were approved by the Battelle Columbus Operations Animal Care and Use Committee
and conducted in accordance with all relevant NIH and NTP animal care and use policies and
applicable federal, state, and local regulations and guidelines.

Six-month Study
The rats were 5 to 6 weeks old upon receipt. Rats were quarantined for 12 to 15 days and were 7
to 9 weeks old on the first day of the study. Before the study began, five male and five female
rats were randomly selected for parasite evaluation and gross observation for evidence of
disease. The health of the animals was monitored during the study according to the protocols of
the NTP Sentinel Animal Program (Appendix I). A positive test result for parvovirus occurred in
one animal at the 4-week timepoint. Additional testing of serum from this animal and other
sentinel animals via other testing methodologies deemed the original positive result to be a false
positive. All other test results were negative for rodent pathogens.
The animals in this study were split into three groups, the core group, Group A, and two special
study groups, Groups B (vitamin and bone analysis) and C (fat digestion, hematology, clinical
chemistry, and urinalysis). Different parameters were evaluated in each group, which allowed for
the collection of extensive endpoints (Table 1). Groups of 10 male and 10 female rats were
examined per endpoint and there was no crossover of analyses between any of the groups.
Group A rats were fed diets containing 0%, 1%, 3%, or 9% chitosan for 25 weeks. Groups B and
C rats were fed diets containing the same concentrations for up to 26 weeks. Feed and water
were available ad libitum. The AIN-93M diet was used for this study instead of the NTP-2000
diet because of the high levels of fat-soluble vitamins and higher total fat content found in the
NTP-2000 diet. The NTP-2000 feed contains almost double the amount of required fat-soluble
vitamins and has a higher fat content (7% to 8%) than the AIN-93M feed (4%)40-42. One of the
primary rationales for this chitosan study was the potential for decreases in fat-soluble vitamin

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concentrations, and therefore, utilizing a diet with lower levels of preexisting vitamins and a
lower fat content was ideal to avoid confounding potential results. Rats were housed
individually. Feed consumption was recorded weekly for core study rats. Core study rats were
weighed and clinical findings were recorded initially, on day 8, weekly thereafter, and at the end
of the study. Details of the study design and animal maintenance are summarized in Table 2.

Table 1. Distribution of Evaluated Parameters

Group
Parameter A B C
Feed consumption X – –
Body weights X – –
Clinical findings X – –
SMVCE X – –
Bone histomorphometry X – –
Gross lesions and histopathology X – –
Vitamin A (serum and liver) – X –
Vitamin E (serum and liver) – X –
1,25 (OH)2 Vitamin D (serum) – X –
Bone calcium, ash, and moisture – X –
Hematology – – X
Clinical chemistry – – X
Vitamin K1 (plasma and liver) – – X
Feed and fecal analysis – – X
Urinalysis – – X

On the first day of weeks 7, 13, 19, and 26, blood was collected from all Group B rats via the
retroorbital plexus under CO2/O2 anesthesia for determination of vitamins A, E, and
1,25 (OH)2 vitamin D concentrations. Blood was collected into tubes, allowed to clot, and
centrifuged. Sera were stored at approximately −70°C until analysis. Blood samples for
vitamin K1 concentrations in Group C rats, collected into tubes containing EDTA at the same
time as hematology collections, were centrifuged; the plasma was harvested, snap frozen, and
stored at −70°C protected from light. At study termination (week 26), liver samples were
collected from surviving Group B and C rats, processed, and stored frozen for determination of
vitamins A and E (Group B) or vitamin K1 (Group C) concentrations. Blood and liver samples
were analyzed by high performance liquid chromatography for vitamins A and E (Covance
Laboratories, Inc.), by competitive enzyme immunoassay for 1,25 (OH)2 vitamin D (Antech
Diagnostics, Morrisville, NC), or by gas chromatography/mass spectrometry for vitamin K1
(Analytics, Inc., Gaithersburg, MD). Because most values for vitamin K1 were below the limit of
quantitation, the results are not presented in this Toxicity Study Report.

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For 8 days beginning during weeks 6, 12, 18, and 24, Group C rats were placed in metabolism
cages (Nalgene Company, Rochester, NY) for fecal and urine collection. During collection
periods, rats were allowed control or dosed feed and water ad libitum, and feed samples were
collected. Feces were collected for a period of 8 days, with each day’s collection being combined
with previous days’ collection and stored at approximately −20°C. Feces were stored at −70°C
after each collection period until shipping to Covance Laboratories, Inc., on dry ice for analyses
of calcium, fat, and moisture; the feed samples were also sent for analysis. Fat content in feed
and feces was determined gravimetrically by Soxhlet extraction. Feed consumption, fat intake
[(total feed consumed per interval) × (% fat in feed/100)], and fat excretion
[fecal weight × (% fecal fat/100)], were calculated to estimate fat digestion:
{[(fat intake − fat excreted in feces)/fat intake] × 100}. Calcium concentrations in feed and feces
were determined using inductively coupled plasma emission spectrometry. Moisture was
determined by weight loss upon drying. Urine was collected on ice for each Group C rat over a
24-hour period during the last day in the metabolism cage and coincided with the last day of
fecal collection. Total urine collected was transferred to centrifuge tubes and the volume was
recorded. Urine creatinine was measured using a Hitachi 911TM chemistry analyzer (Roche
Diagnostics, Indianapolis, IN), and deoxypyridinoline was measured using a Metra Total DPD
Enzyme Immunoassay Kit (Quidel, San Diego, CA).
On the last day in the metabolism cage, at the beginning of weeks 7, 13, 19, and 25, blood was
collected from all Group C rats via the retroorbital plexus under CO2/O2 anesthesia for
hematology (week 25 only) and clinical chemistry. Blood samples for hematology were collected
in tubes containing EDTA as an anticoagulant. Hematology parameters were determined using
an Advia 120 hematology analyzer (Bayer Diagnostics Division, Tarrytown, NY). Blood for
clinical chemistry determinations was collected in tubes without anticoagulant, allowed to clot,
and centrifuged and then the serum was harvested. Except as noted, clinical chemistry
parameters were determined using a Hitachi 911TM chemistry analyzer (Roche Diagnostics). For
osteocalcin and parathyroid hormone, serum was stored frozen at −20°C until analysis. Serum
osteocalcin was measured using a Rat-MIDTM Osteocalcin ELISA (Nordic Bioscience
Diagnostics, Herlev, Denmark). Serum parathyroid hormone was measured using an Intact PTH
Enzyme Immunoassay Kit (ALPCO Diagnostics, Salem, NH).
At study termination (week 26), right and left femurs were collected from the Group B rats for
determination of calcium, ash, and moisture. Covance Laboratories, Inc., determined bone
moisture by measuring weight loss upon drying, calcium by inductively coupled plasma
emission spectrometry, and ash gravimetrically.
At the end of the study (week 25), samples were collected for sperm motility and vaginal
cytology evaluations on Group A rats. The parameters evaluated are listed in Table 2. Due to
inconsistent sample collection and slide staining, an assessment of estrous cyclicity could not be
made. Male animals were evaluated for sperm count and motility. The left testis and left
epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then
removed from the epididymal body (corpus epididymis) and weighed. Test yolk was applied to
slides and a small incision was made at the distal border of the cauda epididymis. The sperm
effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile
and nonmotile spermatozoa were counted for five fields per slide by two observers. Following
completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline
solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then

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heat fixed at 65°C. Sperm density was then determined microscopically with the aid of a
hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was
determined by removing the tunica albuginea and homogenizing the left testis in phosphate-
buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei
were counted with a hemacytometer.
Necropsies were performed on all Group A animals at study termination (week 25). The heart,
right kidney, liver, lung, right ovary, parathyroid gland, right testis, thymus, thyroid gland and
parathyroid gland together, and uterus were weighed. Both tibias and both femurs were
collected; the lengths of both tibias and the left femur were measured. The right tibia and femur
were dehydrated in ethanol (70% to 100%) and infiltrated with glycol methacrylate. Tissues for
microscopic examination were fixed and preserved in 10% neutral buffered formalin (except
eyes were first fixed in Davidson’s solution), processed and trimmed, embedded in paraffin,
sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin. Complete
histopathologic examinations were performed by the study laboratory pathologist on 0% and 9%
rats. The kidney and liver of males and females and the parathyroid gland and prostate gland of
males were examined in all exposure groups. Table 2 lists the tissues and organs routinely
examined.
After a review of the laboratory reports and selected histopathology slides by a quality
assessment (QA) pathologist, the findings and reviewed slides were submitted to an NTP
Pathology Peer Review (PPR) coordinator for a second independent review. Any inconsistencies
in the diagnoses made by the study laboratory and QA pathologists were resolved by the NTP
pathology peer review process. Final diagnoses for reviewed lesions represent a consensus of the
PPR or a consensus between the study laboratory pathologist, NTP pathologist, QA
pathologist(s), and the PPR coordinator. Details of these review procedures have been described,
in part, by Maronpot and Boorman43 and Boorman et al.44.

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Table 2. Experimental Design and Materials and Methods in the Six-month Feed Study of Chitosan
Six-month Studies
Study Laboratory
Battelle Columbus Operations (Columbus, OH)
Strain and Species
Charles River Sprague Dawley [Crl:CD(SD)] rats
Animal Source
Charles River Laboratories (Portage, MI)
Time Held Before Study
Group A (core study): 14 (males) or 15 (females) days
Groups B and C (special studies): 12 (males) or 13 (females) days
Average Age When Study Began
7 to 8 weeks (Group A males and Groups B and C males and females)
8 to 9 weeks (Group A females)
Date of First Exposure
Group A: August 31 (males) or September 1 (females), 2006
Groups B and C: August 29 (males) or 30 (females), 2006
Duration of Exposure
Group A: 25 weeks
Groups B and C: 26 weeks
Date of Last Exposure
Group A: February 15 (males) or 16 (females), 2007
Groups B and C: February 20 (males) or 21 (females), 2007
Necropsy Dates
Group A: February 15 (males) or 16 (females), 2007
Groups B and C: February 20 (males) or 21 (females), 2007
Average Age at Necropsy
32 to 33 weeks (Group A females and Groups B and C males and females)
31 to 32 weeks (Group A males)
Size of Study Groups
10 males and 10 females
Method of Distribution
Animals were distributed randomly into groups of approximately equal initial mean body weights.
Animals per Cage
1
Method of Animal Identification
Tail tattoo
Diet
AIN-93M maintenance purified meal diet (Purina TestDiet, Richmond, IN), available ad libitum, changed twice
weekly
Water

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Six-month Studies
Tap water (Columbus, OH municipal supply) via automatic watering system (Edstrom Industries, Inc. Waterford,
WI), available ad libitum
Cages
Polycarbonate solid-bottom (Lab Products, Inc., Seaford, DE), changed weekly, rotated in rack every 2 weeks
Bedding
Irradiated hardwood bedding chips (P.J. Murphy Forest Products Corporation, Montville, NJ), changed weekly
Rack Filters
Spun-bonded polyester (Snow Filtration Company, Cincinnati, OH), changed every 2 weeks
Racks
Stainless steel (Lab Products, Inc), changed and rotated every 2 weeks
Animal Room Environment
Temperature: 72° ± 3°F
Relative humidity: 50% ± 15%
Room fluorescent light: 12 hours/day
Room air changes: 10/hour
Exposure Concentrations
0%, 1%, 3%, and 9% in feed, available ad libitum
Type and Frequency of Observation
Observed twice daily; Group A rats were weighed and clinical findings were recorded initially, on day 8, weekly
thereafter, and at the end of the study. Feed consumption was recorded weekly for Group A rats and during fecal
collection periods for Group C rats.
Method of Euthanasia
100% Carbon dioxide
Necropsy
Necropsies were performed on all Group A rats at the end of the study (week 25). Organs weighed were heart,
right kidney, liver, lung, right ovary, parathyroid gland, right testis, thymus, thyroid gland and parathyroid gland
together, and uterus. Lengths of both tibias and the left femur were measured.
Clinical Pathology
Blood was collected via the retroorbital plexus from all Group C rats on the first day of weeks 7, 13, 19, and 25 for
hematology (week 25 only) and clinical chemistry. Urine was collected from Group C rats for 24 hours beginning
the last day of weeks 6, 12, 18, and 24.
Hematology: hematocrit (auto and manual); hemoglobin concentration; erythrocyte, reticulocyte, and platelet
counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and
differentials
Clinical chemistry: urea nitrogen, creatinine, calcium, phosphorous, total protein, albumin, cholesterol,
triglycerides, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, bile acids,
total osteocalcin, and parathyroid hormone
Urinalysis: creatinine, volume, and deoxypyridinoline
Histopathology

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Six-month Studies
Histopathology was performed on 0% and 9% Group A rats. In addition to gross lesions and tissue masses, the
following tissues were examined: adrenal gland, bone (left femur and tibia) with marrow, brain, clitoral gland,
esophagus, eye, Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine
(duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and
mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate
gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis (with epididymis),
thymus, thyroid gland, trachea, urinary bladder, and uterus. The kidney and liver of males and females and the
parathyroid gland and prostate gland of males were also examined in the 1% and 3% groups.
Sperm Motility
At the end of the study, sperm samples were collected from male Group A rats for sperm count and motility
evaluations. The following parameters were evaluated: spermatid heads per gram testis and per testis, spermatid
heads per gram cauda and per cauda, and epididymal spermatozoal motility. The left cauda, left epididymis, and
left testis were weighed.
Digestion Studies
Feces were collected from Group C rats for 8 days beginning weeks 6, 12, 18, and 24 and analyzed for calcium,
fat, and moisture. Fecal calcium and fat content were compared to that in feed samples collected during the same
time period to produce values for fat digested and calcium absorbed.
Serum and Hepatic Vitamins
Blood was collected from the retroorbital plexus of Groups B and C rats on the first day of weeks 7, 13, 19, and 25
(Group C), and 26 (Group B). At study termination (week 26), liver samples were collected from Groups B and C
rats. Blood and liver samples were analyzed for vitamins A, E, 1,25 (OH)2 D, and/or K1.
Bone Analysis
At study termination (week 26), right and left femurs were collected from Group B rats, and calcium, ash, and
moisture levels were measured.

Statistical Methods
Calculation and Analysis of Lesion Incidences
The incidences of lesions are presented in Appendix A as the numbers of animals bearing such
lesions at a specific anatomic site and the numbers of animals with that site examined
microscopically. The Fisher exact test45, a procedure based on the overall proportion of affected
animals, was used to determine significance.

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between
dosed and control groups in the analysis of continuous variables. Organ and body weight data,
which historically have approximately normal distributions, were analyzed with the parametric
multiple comparison procedures of Dunnet46 and Williams47; 48. Hematology, clinical chemistry,
urinalysis, serum and liver vitamin concentrations, digestive and bone parameters, spermatid,
and epididymal spermatozoal data, which have typically skewed distributions, were analyzed
using the nonparametric multiple comparison methods of Shirley49 (as modified by Williams50)
and Dunn51. Jonckheere’s test52 was used to assess the significance of the dose-related trends and
to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for
pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s
or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon

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and Massey53 were examined by NTP personnel, and implausible values were eliminated from
the analysis.

Quality Assurance Methods

The 6-month study was conducted in compliance with Food and Drug Administration Good
Laboratory Practice Regulations54. In addition, as records from the 6-month study were
submitted to the NTP Archives, this study was audited retrospectively by an independent QA
contractor. Separate audits covered completeness and accuracy of the pathology data, pathology
specimens, final pathology tables, and a draft of this NTP Toxicity Study Report. Audit
procedures and findings are presented in the reports and are on file at NIEHS. The audit findings
were reviewed and assessed by NTP staff, and all comments were resolved or otherwise
addressed during the preparation of this Toxicity Study Report.

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Results
Six-month Study
All male and female Group A rats survived to the end of the study (Table 3); however, five rats
from Groups B and C died, often after seizures that occurred near the time of blood collection,
with the cause of death undetermined. There were no treatment-related clinical findings in
Group A animals, although 13 animals from Groups B and C (10 from the 9% group, one from
the 3% group, and two from the 1% group) were observed with seizures either during or after the
18-week blood collections. Seizures were not noted at any other time point. Body weights and
feed consumption were measured in Group A rats, and mean body weights of exposed males and
females were not significantly different from those of the control groups (Table 3 and Figure 2).
Feed consumption by 3% and 9% Group A males was greater than that by the controls, but the
increase may not be accurate due to observed food spillage possibly due to poor palatability
resulting in feed being wasted (Table G-1). Dietary concentrations of 1%, 3%, and 9% resulted
in average daily doses of approximately 450, 1,500, and 5,200 mg chitosan/kg body
weight per day to males and 650, 1,800, and 6,000 mg/kg per day to females, respectively.

Table 3. Survival, Body Weights, and Feed Consumption of Group A Rats in the Six-month Feed
Study of Chitosana
Change in
Initial Final Weight Feed Feed
b Final Body Body
Concentration Survival Body Relative to Consumption Consumption
Weight (g) Weight
Weight (g) Controls (%) Week 1 Week 25
(g)
Male
0% 10/10 238 ± 5 669 ± 20 432 ± 18 22.2 21.2
1% 10/10 243 ± 6 702 ± 21 459 ± 17 105 23.8 20.4
3% 10/10 242 ± 6 687 ± 23 445 ± 21 103 23.6 24.7
9% 10/10 243 ± 6 612 ± 17 369 ± 17 91 21.4 27.3
Female
0% 10/10 175 ± 3 338 ± 11 162 ± 12 17.7 16.4
1% 10/10 173 ± 2 335 ± 13 162 ± 12 99 22.3 20.3
3% 10/10 177 ± 4 328 ± 11 151 ± 9 97 17.3 17.1
9% 10/10 177 ± 2 301 ± 13 124 ± 12 89 16.9 18.8
aWeights and weight changes are given as mean ± standard error. Feed consumption is expressed as grams per animal per day.
Differences in weights and weight changes from the control group are not significant by Dunnett’s test.
bNumber of animals surviving at 25 weeks/number initially in group.

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Figure 2. Growth Curves for Group A Rats Exposed to Chitosan in Feed for Six Months

Hematology data for Group C rats are listed in Table B-1. Compared to the control group, mild
significant increases (4% to 6%) in automated hematocrit, hemoglobin concentration, mean cell
volume, and mean cell hemoglobin were observed in 9% males; manual hematocrit and
erythrocyte count were similar to those of the controls. These changes may be due to biological
variability and are likely not toxicologically relevant. All other differences from control values in
the male and female hematology data were mild or sporadic and not considered toxicologically
significant.

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Clinical chemistry data for Group C rats are listed in Table 4 and Table B-1. Both the 9% male
and female rats had significantly decreased cholesterol values (26% to 48%), compared to the
controls, at all time points. Triglycerides values were also significantly decreased in the 9% male
(47% to 57%) and female (30%) rats, but not at every time point. Phosphorus levels were
significantly decreased in the 9% male rats at weeks 13, 19, and 25 (12% to 18%); a decrease
also occurred in the 3% males at week 13 (14%). Similarly, phosphorus levels were significantly
decreased in the 3% and 9% females at weeks 13 (20% and 16%, respectively) and 25 (9% and
19%, respectively). A mild, but statistically significant, decrease (4%) in calcium concentration
was observed in 9% males at weeks 19 and 25. Alanine aminotransferase (ALT) activity, a
marker of hepatocellular injury, was mildly but significantly elevated at week 25 in the 9% male
rats (104%) and in the 3% and 9% female rats (28% and 88%, respectively). However, sorbitol
dehydrogenase (another marker of hepatocellular injury) was not significantly increased relative
to the controls, and hepatocellular changes associated with increases in ALT were not observed
microscopically. Thus, the toxicologic significance of the increases in ALT is uncertain. Urea
nitrogen was mildly increased in the 9% males (23%) and females (15%) at week 25. Minimal to
mild significant alterations were also observed in several other parameters. These alterations
were inconsistent or within the range of biological variability.
Total osteocalcin (a marker of bone turnover) and parathyroid hormone levels were analyzed in
Group C rats and were occasionally elevated throughout the study. Total osteocalcin was
significantly elevated in the 9% males (38%) at week 25, while parathyroid hormone levels were
significantly elevated in 9% males (96%) at Week 19 and in 9% females (56%) at week 25
(Table 4 and Table B-1).
Urine deoxypyridinoline/creatinine ratios were calculated at weeks 7, 13, 19, and 25 for both
males and females in Group C and were mostly unchanged (Table 4 and Table B-1). A
significant increase, compared to the control group, occurred at week 25 in the 9% males (28%).
In females, minimal increases and decreases occurred inconsistently across all time points with a
significant increase at week 7 in the 9% group (42%) and significant decreases at weeks 13
(26%) and 19 (20%) in the 1% group compared to controls.
To calculate the deoxypyridinoline/creatinine ratios, urine volume, urine creatinine
concentrations, and urine deoxypyridinoline concentrations were measured at weeks 7, 13, 19,
and 25. Urine volume was significantly decreased in various male and female exposure groups
throughout the study, but most consistently in the 9% chitosan group (approximately 40% to
60%). Increases in urine creatinine concentration tended to parallel the decreases in urine volume
indicating proper kidney function.

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Table 4. Selected Clinical Chemistry and Urinalysis Data for Group C Rats in the Six-month Feed
Study of Chitosana
0% 1% 3% 9%
Male
Clinical Chemistry
n 10 10 10 10
Calcium (mg/dL)
Week 13 12.6 ± 0.1 12.5 ± 0.1 12.3 ± 0.2 12.4 ± 0.2
Week 19 12.5 ± 0.1 12.3 ± 0.2 12.3 ± 0.1 12.0 ± 0.1*
Week 25 12.1 ± 0.1 12.1 ± 0.2 12.0 ± 0.1 11.6 ± 0.1*
Phosphorus (mg/dL)
Week 13 8.4 ± 0.3 8.1 ± 0.3 7.2 ± 0.3** 7.4 ± 0.4*
Week 19 8.2 ± 0.4 7.7 ± 0.2 7.4 ± 0.3 6.7 ± 0.2**
Week 25 6.9 ± 0.3 6.8 ± 0.2 6.7 ± 0.1 5.8 ± 0.3**
Cholesterol (mg/dL)
Week 7 82 ± 5 75 ± 8 80 ± 6 53 ± 3**
Week 13 95 ± 7 84 ± 8 90 ± 7 53 ± 2**
Week 19 101 ± 6 87 ± 10 94 ± 8 59 ± 4**
Week 25 95 ± 6 81 ± 8 90 ± 6 49 ± 4**
Triglycerides (mg/dL)
Week 7 202 ± 28 234 ± 43 226 ± 30 88 ± 15*
Week 13 198 ± 33 202 ± 38 195 ± 24 86 ± 8**
Week 19 180 ± 26 218 ± 43 210 ± 29 95 ± 13*
Week 25 173 ± 18 207 ± 30 218 ± 24 109 ± 13
Alanine aminotransferase (IU/L)
Week 25 28 ± 3 29 ± 2 29 ± 1 57 ± 2**
Sorbitol dehydrogenase (IU/L)
Week 25 17 ± 3 17 ± 2 15 ± 1 14 ± 1
Total osteocalcin (ng/mL)
Week 7 445.7 ± 17.2 439.8 ± 15.8 441.8 ± 18.2 520.4 ± 22.6
Week 13 306.2 ± 13.0 289.7 ± 28.6 245.4 ± 37.9 372.6 ± 23.4
Week 19 239.4 ± 12.4 225.7 ± 10.6 181.6 ± 26.8 269.2 ± 20.9
Week 25 158.3 ± 10.0 168.1 ± 11.6 145.9 ± 22.7 218.3 ± 14.6*
Parathyroid hormone (ng/mL)
Week 7 1.882 ± 0.137 1.643 ± 0.449 1.838 ± 0.348 1.521 ± 0.368
Week 13 2.343 ± 0.350 2.763 ± 0.479 3.215 ± 0.537 2.433 ± 0.222
Week 19 1.879 ± 0.186 3.101 ± 0.475 2.710 ± 0.365 3.679 ± 0.361**
Week 25 2.668 ± 0.475 2.924 ± 0.276 3.981 ± 0.349 2.848 ± 0.506

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 Chitosan, NTP TOX 93

0% 1% 3% 9%
Urinalysis
n
Week 7 10 9 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 25 10 10 10 10
Volume (mL)
Week 7 8.3 ± 0.8 7.5 ± 1.5 6.4 ± 0.8 5.0 ± 1.1*
Week 13 7.9 ± 1.0 4.6 ± 0.3** 5.1 ± 0.4* 4.5 ± 0.5**
Week 19 10.7 ± 1.6 4.0 ± 0.4** 5.3 ± 0.7* 5.6 ± 0.6
Week 25 8.6 ± 1.2 5.4 ± 0.6* 6.1 ± 0.8* 5.1 ± 0.6**
Deoxypyridinoline/creatinine (nmol/mg)
Week 7 1.810 ± 0.135 1.889 ± 0.148 1.810 ± 0.159 1.920 ± 0.160
Week 13 0.910 ± 0.035 0.890 ± 0.031 0.930 ± 0.040 0.960 ± 0.078
Week 19 0.530 ± 0.050 0.550 ± 0.034 0.570 ± 0.042 0.660 ± 0.048
Week 25 0.430 ± 0.030 0.470 ± 0.030 0.480 ± 0.020 0.550 ± 0.027**
Female
Clinical Chemistry
n
Week 7 10 10 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 25 10 9 10 10
Calcium (mg/dL)
Week 13 12.9 ± 0.2 13.1 ± 0.1 12.7 ± 0.2 12.5 ± 0.2
Week 19 12.9 ± 0.1 13.1 ± 0.2 12.8 ± 0.1 12.5 ± 0.1
Week 25 12.7 ± 0.3 12.8 ± 0.1 12.7 ± 0.2 12.3 ± 0.2
Phosphorus (mg/dL)
Week 13 8.1 ± 0.5 7.4 ± 0.4 6.5 ± 0.4** 6.8 ± 0.3*
Week 19 8.4 ± 0.4 8.2 ± 0.5 8.1 ± 0.3 7.4 ± 0.5
Week 25 6.8 ± 0.2 6.4 ± 0.2 6.2 ± 0.3* 5.5 ± 0.3**
Cholesterol (mg/dL)
Week 7 80 ± 6 81 ± 8 67 ± 4 59 ± 4**
Week 13 92 ± 8 86 ± 7 73 ± 5 58 ± 4**
Week 19 107 ± 7 105 ± 9 91 ± 8 67 ± 5**
Week 25 94 ± 7 108 ± 5 96 ± 8 63 ± 4**

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 Chitosan, NTP TOX 93

0% 1% 3% 9%
Triglycerides (mg/dL)
Week 7 88 ± 12 130 ± 48 81 ± 8 86 ± 14
Week 13 125 ± 10 163 ± 30 140 ± 23 88 ± 23*
Week 19 143 ± 15 181 ± 32 137 ± 18 90 ± 13
Week 25 188 ± 31 231 ± 44 245 ± 31 158 ± 35
Alanine aminotransferase (IU/L)
Week 25 25 ± 3 28 ± 3 32 ± 2** 47 ± 4**
Sorbitol dehydrogenase (IU/L)
Week 25 17 ± 3 17 ± 2 19 ± 2 16 ± 1
Total osteocalcin (ng/mL)
Week 7 293.6 ± 19.4 287.5 ± 21.2 282.1 ± 34.7 316.7 ± 23.5
Week 13 197.9 ± 22.6 202.3 ± 15.4 184.4 ± 19.4 234.2 ± 14.5
Week 19 158.1 ± 18.3 184.8 ± 13.2 166.7 ± 24.7 210.1 ± 16.0
Week 25 107.9 ± 18.6 97.1 ± 7.1 96.0 ± 16.2 148.8 ± 15.1
Parathyroid hormone (ng/mL)
Week 7 0.995 ± 0.150b 1.156 ± 0.176 1.092 ± 0.182 1.023 ± 0.146
Week 13 1.506 ± 0.203 1.734 ± 0.194 1.925 ± 0.306 1.767 ± 0.212
Week 19 1.406 ± 0.232 1.994 ± 0.353 1.845 ± 0.418 1.673 ± 0.223
b
Week 25 1.471 ± 0.189 1.628 ± 0.220 1.818 ± 0.224 2.301 ± 0.212*
Urinalysis
n
Week 7 10 10 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 25 10 9 10 9
Volume (mL)
Week 7 8.2 ± 1.2 8.5 ± 1.0 5.4 ± 0.8 3.4 ± 0.3**
Week 13 6.4 ± 0.7 6.5 ± 0.6 4.1 ± 0.8* 2.9 ± 0.5**
Week 19 7.7 ± 1.2 8.1 ± 1.4 5.0 ± 0.8 3.4 ± 0.5**
Week 25 8.2 ± 1.5 9.1 ± 1.5 5.8 ± 0.9 3.7 ± 0.5**
Deoxypyridinoline/creatinine (nmol/mg)
Week 7 1.620 ± 0.128 1.240 ± 0.129 1.940 ± 0.229 2.300 ± 0.182*
Week 13 0.580 ± 0.039 0.430 ± 0.037** 0.540 ± 0.034 0.570 ± 0.042
Week 19 0.450 ± 0.017 0.360 ± 0.016* 0.440 ± 0.016 0.520 ± 0.020
Week 25 0.340 ± 0.043 0.222 ± 0.022 0.340 ± 0.027 0.411 ± 0.026
*Significantly different (P ≤ 0.05) from the control group by Dunn’s or Shirley’s test.
**P ≤ 0.01.
aData are presented as mean ± standard error. Statistical tests were performed on unrounded data.
bn = 9.

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 Chitosan, NTP TOX 93

Serum and hepatic vitamin concentrations were measured in Group B rats (Table 5 and
Table C-1). Exposure concentration-dependent decreases were observed in serum vitamin A
concentrations starting at week 13 in the male rats. The decreases reached statistical significance
at weeks 13 (27%), 19 (26%), and 26 (29%) in 9% males and at weeks 13 (15%) and 26 (16%) in
3% males. Females were less affected with significant decreases observed in the 9% group at
weeks 19 (18%) and 26 (21%). Exposure concentration-dependent decreases were also observed
in serum vitamin E concentrations in male rats at all time points. The decreases were statistically
significant at all time points in 3% (33% to 42%) and 9% males (79% to 82%) and in 1% males
at week 13 (17%), with the 9% group measuring between 18% to 21% that of control values
throughout the study. Females were less affected with significant decreases in serum vitamin E
levels observed in the 9% group (approximately 60%) only (all time points). Hepatic vitamin E
concentrations were significantly decreased at week 26 in 3% and 9% males (48% and 87%,
respectively) and 9% females (80%). In the 9% group, levels of hepatic vitamin E measured only
13% and 20% of control values in the males and females, respectively. Serum concentrations of
1,25 (OH)2 vitamin D were significantly increased in 9% males (105% to 142%) and females
(100% to 180%) at weeks 7, 19, and 26 compared to the control groups. Results of plasma
hepatic vitamin K concentrations in Group C rats are not discussed or presented, as many
samples were below the level of quantification.

Table 5. Serum and Hepatic Vitamin Concentration Data for Group B Rats in the Six-month Feed
Study of Chitosana
0% 1% 3% 9%
Male
n
Week 7 9 10 10 10
Week 13 9 10 10 10
Week 19 9 10 10 10
Week 26 9 10 10 8
Serum vitamin A (µg/mL)
Week 7 0.532 ± 0.021 0.506 ± 0.033 0.513 ± 0.026 0.453 ± 0.018
Week 13 0.561 ± 0.024 0.499 ± 0.019 0.476 ± 0.022* 0.410 ± 0.009**
Week 19 0.533 ± 0.028 0.506 ± 0.031 0.475 ± 0.019 0.392 ± 0.014**
Week 26 0.476 ± 0.019 0.444 ± 0.024 0.398 ± 0.017** 0.336 ± 0.026**
Serum 1,25 (OH)2 vitamin D (pg/mL)
Week 7 124.4 ± 19.6 163.3 ± 21.7 183.2 ± 26.9 297.4 ± 41.0**
Week 13 70.1 ± 7.3 57.4 ± 5.3 77.3 ± 4.4 86.1 ± 8.5
Week 19 20.6 ± 2.8 21.7 ± 6.1 22.9 ± 2.2 42.3 ± 3.1**b
Week 26 27.7 ± 3.4c 28.0 ± 4.3 36.1 ± 4.6b 66.9 ± 11.9**
Serum vitamin E (µg/mL)
Week 7 19.33 ± 1.43 15.38 ± 1.29 12.92 ± 0.48** 4.14 ± 0.23**
Week 13 21.08 ± 1.61 17.45 ± 1.06* 12.27 ± 0.86** 4.33 ± 0.27**

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 Chitosan, NTP TOX 93

0% 1% 3% 9%
Week 19 20.59 ± 1.61 16.19 ± 0.96 12.86 ± 0.42** 4.07 ± 0.32**
Week 26 19.66 ± 1.66 17.35 ± 1.37 12.35 ± 0.61** 3.59 ± 0.65**
Liver vitamin E (µg/g)
Week 26 66.8 ± 16.2 55.0 ± 6.8 34.6 ± 2.2** 8.5 ± 0.8**
Female
n
Week 7 10 10 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 26 10 10 9 10
Serum vitamin A (µg/mL)
Week 7 0.272 ± 0.011 0.253 ± 0.007 0.260 ± 0.012 0.266 ± 0.012
Week 13 0.308 ± 0.020 0.295 ± 0.011 0.309 ± 0.019 0.281 ± 0.018
Week 19 0.283 ± 0.014 0.271 ± 0.015 0.291 ± 0.012 0.231 ± 0.010*
Week 26 0.316 ± 0.015 0.302 ± 0.014 0.294 ± 0.018 0.249 ± 0.010**
Serum 1,25 (OH)2 vitamin D (pg/mL)
Week 7 104.0 ± 15.1 96.7 ± 10.9 111.0 ± 8.7 208.1 ± 18.2**
Week 13 60.6 ± 7.5 60.7 ± 7.9 69.3 ± 11.0 110.1 ± 16.9
Week 19 11.6 ± 1.6 12.6 ± 1.7 15.8 ± 1.4 31.4 ± 3.2**
Week 26 19.2 ± 2.2 20.7 ± 4.2 28.6 ± 6.5 53.7 ± 5.8**
Serum vitamin E (µg/mL)
Week 7 18.65 ± 0.71 20.08 ± 0.87 18.38 ± 0.85 6.99 ± 0.58**
Week 13 19.81 ± 1.41 20.85 ± 1.06 20.19 ± 1.20 7.48 ± 0.38**
Week 19 21.02 ± 1.76 19.74 ± 1.75 19.86 ± 1.08 7.37 ± 0.57**
Week 26 20.94 ± 1.56 23.43 ± 1.66 22.23 ± 1.75 7.28 ± 0.64**
Liver vitamin E (µg/g)
Week 26 84.5 ± 8.9 97.1 ± 10.1 82.0 ± 11.8 17.2 ± 3.2**
*Significantly different (P ≤ 0.05) from the control group by Dunn’s or Shirley’s test.
**Significantly different (P ≤ 0.01) from the control group by Shirley’s test.
aData are presented as mean ± standard error. Statistical tests were performed on unrounded data.
b
n = 9.
cn = 7.

Digestive parameters were calculated for Group C rats and are listed in Table 6. Compared to the
control groups, percent fat digested was significantly decreased at week 6 in 9% males (28%)
and females (14%), during week 12 in 3% and 9% males (8% and 33%, respectively), during
week 18 in 9% males (20%) and females (10%), and during week 24 in all exposed groups of
males and females (up to 32%). Calcium absorption was significantly increased in 9% females
during weeks 12 (55%) and 24 (154%). Fecal weight was significantly increased in 3% and 9%
males (up to 170%) and females (up to 126%) during each collection period and in 1% females

22
 Chitosan, NTP TOX 93

during weeks 12, 18, and 24 (18% to 29%). Fecal moisture was significantly increased in 9%
males and females at all time points (10% to 15%), in 3% males (4%) at week 6, and in 3%
females (7%) at weeks 12 and 18.
Male rats did not display any changes in testis or epididymis weights or sperm parameters,
indicating that chitosan did not exhibit the potential to be a reproductive toxicant in male rats
(Table E-1).

Table 6. Digestive Data for Group C Rats in the Six-month Feed Study of Chitosana
0% 1% 3% 9%
Male
n
Weeks 6–7 10 10 10 10
Weeks 12–13 10 10 10 10
Weeks 18–19 10 10 10 9
Weeks 24–25 10 10 10 10
Fat digested (%)
Weeks 6–7 97.04 ± 0.40 97.55 ± 0.22 94.37 ± 0.84 69.55 ± 3.01**
Weeks 12–13 94.79 ± 0.46 93.36 ± 0.83 87.08 ± 0.68** 63.50 ± 2.40**
Weeks 18–19 97.56 ± 0.58 98.48 ± 0.19 95.87 ± 0.70 77.59 ± 1.83**
Weeks 24–25 97.01 ± 0.19 95.61 ± 0.32** 92.14 ± 0.87** 66.18 ± 3.24**
Calcium absorbed (%)
Weeks 6–7 31.69 ± 1.84 34.57 ± 4.05 27.54 ± 1.83 33.01 ± 1.59
Weeks 12–13 19.81 ± 3.36 14.73 ± 0.76 18.42 ± 3.25 28.01 ± 2.69
Weeks 18–19 13.33 ± 4.33 18.42 ± 5.43 3.64 ± 2.62 11.11 ± 1.35
Weeks 24–25 2.93 ± 1.54 5.14 ± 1.08 0.70 ± 1.57 9.46 ± 1.88
Fecal weight (g)
Weeks 6–7 21.42 ± 0.68 21.01 ± 1.93 31.33 ± 0.90** 52.39 ± 2.85**
Weeks 12–13 24.32 ± 1.68 27.70 ± 1.37 32.84 ± 1.73** 47.59 ± 4.30**
Weeks 18–19 23.11 ± 1.25 22.67 ± 1.85 33.30 ± 1.72** 62.38 ± 3.67**b
Weeks 24–25 26.43 ± 1.12 25.75 ± 0.73 37.17 ± 1.11** 56.35 ± 3.45**
Fecal moisture (%)
Weeks 6–7 45.0 ± 0.5 42.0 ± 1.6 46.8 ± 0.4* 51.0 ± 0.8**
Weeks 12–13 46.8 ± 2.0 49.0 ± 0.8 48.8 ± 0.6 53.6 ± 0.8**
Weeks 18–19 47.7 ± 1.1 45.3 ± 1.8 49.1 ± 0.7 54.8 ± 1.5**b
Weeks 24–25 47.2 ± 0.6 45.7 ± 0.5 49.3 ± 0.7 53.1 ± 0.8**

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 Chitosan, NTP TOX 93

0% 1% 3% 9%
Female
n
Weeks 6–7 10 10 10 9
Weeks 12–13 10 10 10 10
Weeks 18–19 10 10 10 10
Weeks 24–25 8 9 10 10
Fat digested (%)
Weeks 6–7 96.47 ± 0.49 95.53 ± 1.30 95.46 ± 0.66 83.23 ± 2.69**
Weeks 12–13 97.12 ± 1.58 98.54 ± 0.99 97.27 ± 1.16 91.95 ± 2.70
Weeks 18–19 99.17 ± 0.18 97.52 ± 0.50 97.15 ± 1.24 89.61 ± 2.53**
Weeks 24–25 98.66 ± 0.08 97.68 ± 0.39** 96.79 ± 0.49** 86.73 ± 1.55**
Calcium absorbed (%)
Weeks 6–7 31.44 ± 2.35 24.42 ± 2.54 24.36 ± 2.50 32.29 ± 1.69
Weeks 12–13 14.84 ± 1.76 17.03 ± 3.11 17.96 ± 1.22 23.02 ± 2.39*
Weeks 18–19 8.96 ± 3.00 9.78 ± 1.98 0.47 ± 3.37 13.07 ± 1.65
Weeks 24–25 5.65 ± 2.84 9.23 ± 2.74 8.25 ± 1.59 14.50 ± 1.40*
Fecal weight (g)
Weeks 6–7 14.37 ± 0.91 15.76 ± 0.60 19.85 ± 1.64** 32.61 ± 1.67**b
Weeks 12–13 15.37 ± 0.60 18.41 ± 1.28* 21.11 ± 1.07** 30.83 ± 2.78**
Weeks 18–19 16.30 ± 0.86 19.23 ± 0.97* 25.21 ± 1.42** 36.58 ± 2.41**
Weeks 24–25 16.01 ± 0.92b 20.66 ± 1.14** 24.85 ± 1.19** 35.78 ± 2.27**
Fecal moisture (%)
Weeks 6–7 45.3 ± 1.1 45.3 ± 0.4 47.3 ± 0.8 50.0 ± 0.9**b
Weeks 12–13 45.9 ± 0.7 47.5 ± 1.0 49.3 ± 0.5** 52.7 ± 1.0**
Weeks 18–19 46.1 ± 1.1 47.2 ± 0.4 49.5 ± 0.9** 53.0 ± 0.7**
b
Weeks 24–25 47.2 ± 0.6 48.4 ± 1.4 49.2 ± 0.6 52.6 ± 0.9**
*Significantly different (P ≤ 0.05) from the control group by Shirley’s test.
**P ≤ 0.01.
aData are presented as mean ± standard error. Statistical tests were performed on unrounded data.
bn = 10.

Bone parameters in Groups A and B rats were generally unaffected by chitosan exposure
(Table C-2). Bone moisture was significantly increased, relative to the control group, in 9%
females (7%).
The absolute and relative liver weights of Group A 9% males and females were significantly less
(22% and 21% lower, respectively) than those of the respective control groups (Table 7 and
Table D-1). The absolute and relative thymus weights of Group A 3% and 9% males and 9%
females were significantly less than those of the controls (Table D-1).

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 Chitosan, NTP TOX 93

There was a significant decrease in the incidence of periportal fatty change of the liver in
Group A female rats in the 9% group compared to the control group and decreases in 1% and 3%
females that resulted in a negative trend (Table 7 and Table A-2). In male rats, there were
decreases in the incidences of periportal fatty change in the 1% and 9% groups, and the severities
were decreased in the 3% and 9% groups (Table 7 and Table A-1). Fatty change was
characterized by hepatocytes with clear vacuoles (lipid), mostly located within the periportal
region of the liver (zone 1) (Figure 3).

Table 7. Liver Parameter Data for Group A Rats in the Six-month Feed Study of Chitosan
0% 1% 3% 9%
Male
na 10 10 10 10
Necropsy body wt 669 ± 20 702 ± 21 687 ± 23 612 ± 17
Liver weightb
Absolute 25.19 ± 0.87 24.87 ± 1.35 23.74 ± 1.51 19.53 ± 0.71##
Relative 37.662 ± 0.731 35.321 ± 1.179 34.345 ± 1.411# 31.933 ± 0.817##
Periportal, Fatty Changec 6 (1.7)d 3 (1.7) 6 (1.3) 3 (1.0)
Female
n 10 10 10 10
Necropsy body wt 338 ± 11 335 ± 13 328 ± 11 301 ± 13
Liver weight
Absolute 12.54 ± 0.82 12.47 ± 0.39 11.85 ± 0.29 9.85 ± 0.20##
Relative 36.900 ± 1.502 37.341 ± 0.444 36.346 ± 0.904 33.036 ± 0.910#
Periportal, Fatty Change 7 (1.1) 4 (1.0) 4 (1.0) 0**
#Significantly different (P ≤ 0.05) from the control group by Williams’ or Dunnett’s test.
##Significantly different (P ≤ 0.01) from the control group by Williams’ test.

**Significantly different (P ≤ 0.01) from the control group by the Fisher exact test.
aNumber of animals with liver weighed and with liver examined microscopically.
bLiver weights (absolute weights) and body weights are given in grams; Liver-weight-to-body-weight ratios (relative weights) are

given as mg liver weight/g body weight (mean ± standard error).

cNumber of animals with lesion.
dAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

Hepatocytes contained large, well-defined, single round vacuoles (macrovesicular) within each
cell that displaced the nuclei and cytoplasm to the cell periphery (Figure 4) and can be compared
with a liver lacking fatty change (Figure 5).

25
 Chitosan, NTP TOX 93

Figure 3. Section of the Liver from a Control Male Sprague Dawley Rat from the Six-month Feed
Study of Chitosan with a Moderate Degree of Fatty Change (H&E)

There is a predominant periportal distribution of affected hepatocytes.

Figure 4. Higher Magnification of Figure 3 (H&E)

The fatty change is characterized by round, discrete vacuoles within hepatocytes that displace the nuclei and cytoplasm to the
periphery.

26
 Chitosan, NTP TOX 93

Figure 5. Section of the Liver with a Lack of Fatty Change from a Male Sprague Dawley Rat
Exposed to 9% Chitosan in Feed for Six Months (H&E)

There is some minimal vacuolization within many hepatocytes. The vacuoles lack distinct round borders and the nuclei are
centrally located, consistent with glycogen accumulation.

27
 Chitosan, NTP TOX 93

Discussion
Human exposure to chitosan occurs primarily through consumption of dietary supplements, as
chitosan is marketed as a fiber-like supplement to increase satiation and promote weight loss
through inhibition of fat absorption16. The acute toxicity of chitosan has previously been
examined in human studies (12 days or up to 8 weeks) evaluating the effectiveness of chitosan as
a weight-loss supplement, and the results from these studies demonstrated no observable toxicity
following oral administration of chitosan32-34. However, there is indication of serum vitamin and
bone mineral depletion following consumption of chitosan in rats26. Therefore, NTP conducted
6-month feed studies to evaluate the effects of dietary chitosan on bone metabolism, fat-soluble
vitamin levels, and dietary fat and calcium absorption, as well as general toxicity in Charles
River Sprague Dawley rats.
Feed concentrations of 1%, 3%, and 9% chitosan, which resulted in average daily doses of
approximately 450, 1,500, and 5,200 mg chitosan/kg body weight per day to males and 650,
1,800, and 6,000 mg/kg per day to females, were selected based on existing data from animal
studies24; 26. The 9% concentration is higher than the typical 5% NTP concentration limit, but the
9% diet was considered to be nutritionally adequate. The AIN-93M feed was selected for this
study over the NTP-2000 feed based on the high levels of fat-soluble vitamins and higher total
fat content found in the NTP-2000 feed. The NTP-2000 feed contains almost double the amount
of required fat-soluble vitamins and has a higher fat content (7% to 8%) than the AIN-93M feed
(4%)41; 42. One of the primary rationales for this chitosan study was the potential for decreases in
fat-soluble vitamin concentrations, and therefore utilizing a diet with lower levels of preexisting
vitamins and a lower fat content was ideal to avoid confounding potential results.
The animals used in this study were split into three groups, the core group, Group A, and two
special study groups, Groups B and C. Different parameters were evaluated in each group,
which, while allowing for the collection of extensive endpoints, meant that only 10 animals were
examined per endpoint instead of 30, as there was no crossover of analyses between the groups.
Multiple endpoints were evaluated at multiple time points (6, 12, 18, and 24 weeks) in Group C
rats to determine effects on fat absorption. Treatment-related decreases in percentage fat
digestion of 20% to 33% in males and 5% to 14% in females relative to control, were
consistently observed in the 9% group with effects also noted in males in the 3% group
(decreases of 2% to 8%). Stronger responses were observed in males relative to females.
Additionally, fecal weight was significantly increased in 1% females at weeks 12, 18, and 24
(19%, 18%, and 29%, respectively), and in 3% (35% to 56%) and 9% (96% to 170%) males and
females relative to controls at all time points. These data suggest that consumption of chitosan
reduced the absorption of fat in the feed, resulting in increased fecal weight due to fat being
excreted. Similar results have been observed in other studies. Deuchi et al.55 reported that rats fed
deacetylated chitosan had decreased fat digestion; as the degree of deacetylation increased, fat
digestibility decreased. The chitosan used by Deuchi et al.55 was 70% to 90% deacetylated,
which is a level very similar to the chitosan (86.5% deacetylated) used in the current study.
Gallaher et al.12 demonstrated that male Wistar rats exposed to 10% chitosan in AIN-93 feed had
increased fecal fat excretion and dry fecal weight and decreased cholesterol absorption relative to
control rats, similar to what was observed in the current study.

28
 Chitosan, NTP TOX 93

Due to the high percentage of chitosan in the feed of the 9% group, it is possible that the
observed decreases in percentage fat digested were due to bulk chitosan in the feces confounding
the amount of fat actually being excreted. Misrepresented fecal weights would alter the
calculated amount of fat excreted in the feces, which would subsequently affect the calculation
of percentage fat digested. The observed increases in fecal weight could also be attributed to an
increase in the percentage fecal moisture, which was significantly increased in both males and
females in the 3% and 9% groups. In Group A, there were decreases, albeit not significant, in
mean body weights of 9% males and females (decreases of 9% and 11%, respectively), but
overall there were no significant changes in the body weights of rats exposed to chitosan; the
mean body weights of exposed animals were similar to those of control animals. Considering the
large decrease in percent fat digested, combined with the significant increase in fecal weight
observed in 9% males and females, it would be expected that mean body weights would
significantly decrease due to more fat being excreted than digested. The slight mean body weight
decrease observed in this study could be due in part to excretion of bulk chitosan, but regardless,
the magnitude of increase in fecal fat excretion as well as the decrease in hepatic periportal fatty
change still indicates a treatment-related response.
Consistent significant decreases in cholesterol levels were observed in 9% male and female rats;
triglycerides levels were also affected but not as consistently as cholesterol. Decreases in
cholesterol were consistent with many other studies and not an unexpected finding, as chitosan is
well known to have a cholesterol lowering effect in rats14; 56-59. The mechanism by which
chitosan lowers cholesterol is still controversial, but recent studies indicate that chitosan, acting
as a weak anion exchange resin, reduces cholesterol by causing a decrease in its absorption in the
small intestine and by inducing increases in bile acid excretion11-13. With bile acid excretion,
plasma or liver cholesterol is utilized to maintain the bile acid pool12. Alternatively, the
cholesterol lowering effects of chitosan may be related to an increase in viscosity of intestinal
contents, which entrap fat and prevent lipolysis, or this mechanism may be in addition to
chitosan’s ability to bind bile acids13-15.
Along with an inhibition in dietary fat absorption and decreases in serum lipids there were also
treatment-related decreases in the levels of fat-soluble vitamins A and E. Serum and liver
vitamin E levels were substantially affected, being 62% to 87% lower in the 9% males and
females. These findings are similar to those of Deuchi et al.26 where decreases in serum and liver
vitamin E levels were observed after 14 days of consuming a 5% chitosan feed. In this same
study, liver vitamin A levels were decreased, but vitamin A serum levels were unchanged. Bile
and lipids are needed for the absorption of dietary vitamins A and E, as both must be
incorporated into intestinal micelles for their absorption60. Thus, it is highly plausible that the
decrease in dietary fat absorption, including cholesterol, led to the decreases in serum and liver
concentrations of these vitamins. It is also possible that, by some unknown mechanism, chitosan
may enhance vitamin A or E requirements in the peripheral tissues.
There were no histologic changes associated with the observed decreases in vitamin levels;
however, the decreases were significant enough to suggest nutritional inadequacies. The long-
term effects of vitamin A and vitamin E deficiencies are well-known60-63, and it is unknown what
deficiency-related effects would have been observed had these decreased levels been maintained
for a longer period of time. When circulating levels of vitamin E, specifically α-tocopherol, are
depleted, tissue damage can occur. Vitamin E depletion in humans has subsequently been
correlated with anemia, disruption of normal growth, decreased responses to infection, and

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pregnancy concerns62. Vitamin A is essential in numerous biological processes and pathways,

including growth, vision development, immune function, and metabolism. Severe vitamin A
deficiency (VAD) results in disruption of normal tissue function and is associated with childhood
blindness, anemia, and depressed responses to infection; VAD during a severe infection may
result in death61-63. While the long-term effects of vitamin deficiency in rodents are not as well
understood, the available literature on human deficiencies suggests that the decreases in
vitamin A and E observed in this study may be detrimental over time.
In contrast to decreases in vitamins A and E, 1,25 (OH)2 vitamin D (bioactive vitamin D) levels
were significantly elevated in 9% male and female rats. Vitamin D’s main function is to help
maintain normal calcium and phosphorus levels by regulating the intestinal absorption of these
minerals from the diet. In addition to the increased 1,25 (OH)2 vitamin D levels, significant
decreases in serum phosphorus were also seen in male and female rats. Although intestinal
absorption of phosphorus was not measured in this study, chitosan has been observed by others
to cause a significant reduction in intestinal phosphorus absorption64. Low phosphorus
concentrations stimulate 1,25 (OH)2 vitamin D production by the kidney, therefore the increased
levels of 1,25 (OH)2 vitamin D observed in this study may be the result of the low phosphorus
levels. Increased levels of 1,25 (OH)2 vitamin D can cause an increase in intestinal absorption of
calcium regardless of serum calcium levels. Significant elevation in intestinal absorption of
calcium was observed sporadically in the female rats, but serum calcium levels were relatively
stable. This effect is most likely due to a loss of calcium through the urine, which has been
observed in other chitosan feed studies64; 65 and is known to occur in cases of hypophosphatemia-
induced elevations in 1,25 (OH)2 vitamin D due to Fanconi’s syndrome66. The reported urinary
calcium loss in chitosan feed studies may be compensatory or directly induced by the chitosan.
Significant decreases in urine volume were observed in various male and female groups, but
most consistently in the 9% group where decreases of 40% to 58% of the control group volume
were observed. As the urine volumes decreased, urine creatinine concentrations were seen to
increase significantly. This is consistent with proper renal function. The most likely cause of the
decrease in urine volume is decreased consumption of water, although water consumption was
not measured, so this cannot be certain. However, the mild increases in urea nitrogen in the
9% male and female rats at 25 weeks (the only time point measured) supports decreased water
consumption (i.e., mild dehydration). Water retention in the intestine may have contributed to the
decreases in urine volume, as fecal moisture was mildly increased in some of the treatment
groups, although it is highly unlikely this would be the primary cause and no diarrhea was
observed.
There was a significant decrease in the occurrence of periportal fatty change, or lipid
accumulation, in the livers of 9% females relative to the controls, and this negative trend was
maintained in both 1% and 3% females, although not significantly. In male rats, the incidences of
periportal fatty change were decreased in both 1% and 9% groups and the severities were
decreased in both the 3% and 9% groups. The decrease in lipid accumulation was inconsistent
between male and female rats in the 9% exposure groups, as a more severe decrease was
observed in the 9% female rats (100% lower) compared to the 9% male rats (50% lower) relative
to the respective controls. The morphologic features observed during this study (periportal
hepatocytes with large, single, well-defined intracytoplasmic vacuoles displacing the nucleus),
were consistent with the intracytoplasmic lipid accumulation that is associated with fatty
change67. During normal function, fatty acids circulate between the liver and adipose tissue,

30
 Chitosan, NTP TOX 93

which maintains a balance of triglycerides between the two locations. When this balance
becomes skewed, hepatic fatty acids can accumulate as small vacuoles in the hepatocytes and
progress over time into larger globules67; 68.
Lipid accumulation in the liver can occur via multiple mechanisms, including 1) increased
synthesis of fatty acids, 2) increased uptake of fatty acids from adipose tissue and/or the diet, 3)
improper removal of fatty acids from the liver, or 4) decreased oxidation of fatty acids69. Diet
and nutritional status can also influence lipid accumulation68; 70. Singh et al.71 demonstrated that
albino rats administered vitamin A orally for 2 days had increased hepatic lipid accumulation. In
the present study, there were treatment-related decreases in hepatic vitamin A and E in both male
and female rats, which could have contributed to the loss of periportal lipid accumulation
observed in the animals fed 9% chitosan. Lipid accumulation in the liver can also occur due to
imbalanced uptake of lipids from the blood and secretion of lipoproteins from the hepatocytes72.
In this chitosan study, the fatty change (lipid accumulation) observed was periportal, or in
Zone 1. Zone 1 is closest to the incoming vasculature and receives the majority of oxygenated
blood, and Zone 1 hepatocytes are generally resistant to the effects of nutritional deficiencies73.
Therefore, the decrease in fatty change observed in rats fed 9% chitosan could be an adaptive
response to the vitamin and mineral depletion noted in this study.
The incidences and severities of fatty change in both male and female control animals was
particularly high (6/10, males; 7/10, females; average severity 1.7 and 1.1, respectively),
suggesting that the Charles River Sprague Dawley rats used in this study may have a normally
high level of hepatic periportal lipid accumulation. Figure 3, Figure 4, and Figure 5, included in
this report, are well representative of the observations made in this study, as the increased
severity of periportal fatty change in control animals was a strong response.
Absolute and relative liver weights of male and female rats were significantly decreased in
animals fed 9% chitosan relative to control animals. As described above, there were decreases in
the incidence of periportal fatty change in all exposed animals, particularly in the female rats fed
9% chitosan. The decrease in liver weights observed in the 9% animals could be due to the loss
of fat accumulation in the livers, which would alter the weight of the organs.
The absolute and relative thymus weights of 3% and 9% males and 9% females were also
significantly decreased relative to those of control groups. The thymus is extremely sensitive to
toxic compounds and similar stressors, and alterations in thymus weight can be an indicator of
apoptosis and organ atrophy in response to a toxic insult. Nutritional status can cause a decrease
in thymus weight, in particular vitamin, mineral, and fatty acid deficiencies74. In the current
study, male and female rats fed 9% chitosan had depleted levels of serum vitamin A and E, liver
vitamin E, and serum cholesterol and triglycerides, indicating nutritional inadequacies. The
observations from this chitosan study, combined with what is known about the thymus, suggest
that exposure to chitosan may have induced reductions in thymus weight secondary to nutritional
deficiencies.
Results of this study did not support chitosan as a cause of bone resorption. Significant elevation
of parathyroid hormone levels occurred occasionally and inconsistently, while calcium levels
were relatively stable. Calcium was mildly, but significantly, decreased at only two time points
in male groups by no more than 4%. Additionally, serum total osteocalcin and urinary
deoxypyridinoline level, both biomarkers of bone turnover, while occasionally significantly

31
 Chitosan, NTP TOX 93

elevated, lacked any consistent increases over time or between sexes. In fact, deoxypyridinoline
was significantly decreased at some time points. Lastly, bone calcium, bone length, and the
histology findings of this study did not support calcium loss from the bone.
Although bone parameters were unaffected by chitosan exposure, a limitation of this study may
be that the time frame of the study was not extensive enough to adequately evaluate bone loss.
Rats are generally not considered skeletally mature until 10 months of age, and the long bones in
rats can continue to grow until 30 months of age, making it difficult to observe any loss of bone
before that point75. In a study of female Charles River Sprague Dawley rats, Wronski et al.76
observed closed growth plates in the tibias of 15-month-old animals. In a separate study, Fukuda
and Iida77 noted that natural decreases in bone mineral density did not begin until 15 months of
age in female Wistar rats. Also, standard osteoporosis studies using rat models commonly utilize
ovariectomized animals, which mimics the conditions of menopause and generally increases
rates of bone remodeling and bone loss. Ovariectomized SHRSP rats fed 10% chitosan alongside
a low calcium diet exhibited decreased bone mineral density and increased femur stiffness64.
Following ovariectomy, bone loss in the femurs, specifically the femoral neck, is still not
observed until a minimum of 30 days postprocedure75. Therefore, given the time frame of the
study there was reduced likelihood of observing any osteologic changes possibly induced by
chitosan exposure.
There were no treatment-related clinical findings in the core, Group A animals, but there were
instances of seizures in Groups B and C animals. Thirteen animals from Groups B and C (two
1%, one 3%, and ten 9%) were observed with seizures either during or after the 18-week blood
collection. Seizures were not noted at any other time point. Similarly, there was no treatment-
related mortality in the Group A animals, but five animals from Groups B and C died, often after
seizures, near the time of blood collection. Cause of death was undetermined for these animals.
While there was no clear connection between chitosan treatment and the incidence of seizures,
there was an exposure concentration-related increase in the occurrence of seizures. Therefore, it
is possible that chitosan exposure may have induced the increased rate of seizures observed in
this study.
Under the conditions of the 6-month feed study of chitosan, male and female rats fed 3% and 9%
chitosan in the diet had significantly decreased levels of serum vitamin A and serum and hepatic
vitamin E and increased levels of serum 1,25 (OH)2 vitamin D. Consumption of high levels of
chitosan decreased percentage fat digestion and increased fecal weight and moisture, as well as
reduced levels of phosphorous, cholesterol, and triglycerides. Female rats exposed to 9%
chitosan also had significant liver weight and histologic changes. Based on the above results, the
lowest-observed-effect level for chitosan exposure was 1% (approximately equivalent to
450 mg/kg) in male and 9% (approximately equivalent to 6,000 mg/kg) in female rats.

32
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References
1. Kean T, Thanou M. Biodegradation, biodistribution and toxicity of chitosan. Adv Drug Del
Rev. 2010; 62(1):3-11. http://dx.doi.org/10.1016/j.addr.2009.09.004
2. Rinaudo M. Chitin and chitosan: Properties and applications. Prog Polym Sci. 2006;
31(7):603-632. http://dx.doi.org/10.1016/j.progpolymsci.2006.06.001
3. Kubota N, Eguchi Y. Facile preparation of water-soluble N-acetylated chitosan and molecular
weight dependence of its water-solubility. Polym J. 1997; 29(2):123.
http://dx.doi.org/10.1295/polymj.29.123
4. Peniston Q, Johnson E, inventors. Process for the manufacture of chitosan. United States
patent 4,195,175; 1980.
5. Hirano S. Chitin biotechnology applications. Biotechnol Annu Rev. 1996; 2:237-258.
http://dx.doi.org/10.1016/S1387-2656(08)70012-7
6. Wedmore I, McManus JG, Pusateri AE, Holcomb JB. A special report on the chitosan-based
hemostatic dressing: Experience in current combat operations. J Trauma Acute Care Surg. 2006;
60(3):655-658. http://dx.doi.org/10.1097/01.ta.0000199392.91772.44
7. Songjiang Z, Lixiang W. Amyloid-beta associated with chitosan nano-carrier has favorable
immunogenicity and permeates the BBB. AAPS PharmSciTech. 2009; 10(3):900.
http://dx.doi.org/10.1208/s12249-009-9279-1
8. Felt O, Buri P, Gurny R. Chitosan: A unique polysaccharide for drug delivery. Drug Dev Ind
Pharm. 1998; 24(11):979-993. http://dx.doi.org/10.3109/03639049809089942
9. Lang G, Wendel H, Konrad E, inventors. Cosmetics composition based upon chitosan
derivatives, new chitosan derivatives as well as processes for production thereof. United States
patent 4,528,283; 1985.
10. Carvalho T, Lussi A. Combined effect of a fluoride-, stannous-and chitosan-containing
toothpaste and stannous-containing rinse on the prevention of initial enamel erosion–abrasion. J
Dent. 2014; 42(4):450-459. http://dx.doi.org/10.1016/j.jdent.2014.01.004
11. Ebihara K, Schneeman BO. Interaction of bile acids, phospholipids, cholesterol and
triglyceride with dietary fibers in the small intestine of rats. J Nutr. 1989; 119(8):1100-1106.
http://dx.doi.org/10.1093/jn/119.8.1100
12. Gallaher CM, Munion J, Hesslink Jr R, Wise J, Gallaher DD. Cholesterol reduction by
glucomannan and chitosan is mediated by changes in cholesterol absorption and bile acid and fat
excretion in rats. J Nutr. 2000; 130(11):2753-2759. http://dx.doi.org/10.1093/jn/130.11.2753
13. Liu J, Zhang J, Xia W. Hypocholesterolaemic effects of different chitosan samples in vitro
and in vivo. Food Chem. 2008; 107(1):419-425.
http://dx.doi.org/10.1016/j.foodchem.2007.08.044

33
 Chitosan, NTP TOX 93

14. Ikeda I, Sugano M, Yoshida K, Sasaki E, Iwamoto Y, Hatano K. Effects of chitosan

hydrolyzates on lipid absorption and on serum and liver lipid concentration in rats. J Agric Food
Chem. 1993; 41(3):431-435. http://dx.doi.org/10.1021/jf00027a016
15. Kanauchi O, Deuchi K, Imasato Y, Shizukuishi M, Kobayashi E. Mechanism for the
inhibition of fat digestion by chitosan and for the synergistic effect of ascorbate. Biosci
Biotechnol Biochem. 1995; 59(5):786-790. http://dx.doi.org/10.1271/bbb.59.786
16. General Nutrition Centers Inc. GNC Total LeanTM chitosan with glucomannan. 2015.
http://www.gnc.com/GNC-Total-Lean-Chitosan-with-
Glucomannan/product.jsp?productId=2459379 [Accessed: June, 2016]
17. Vitamin World Inc. Chitosan 500 mg. 2015. http://www.vitaminworld.com/fiber/chitosan-
500mg-0070004945.html [Accessed: June, 2016]
18. Chae SY, Jang M-K, Nah J-W. Influence of molecular weight on oral absorption of water
soluble chitosans. J Control Release. 2005; 102(2):383-394.
http://dx.doi.org/10.1016/j.jconrel.2004.10.012
19. Yang Y, Hu W, Wang X, Gu X. The controlling biodegradation of chitosan fibers by N-
acetylation in vitro and in vivo. J Mater Sci Mater Med. 2007; 18(11):2117-2121.
http://dx.doi.org/10.1007/s10856-007-3013-x
20. Funkhouser JD, Aronson NN. Chitinase family GH18: Evolutionary insights from the
genomic history of a diverse protein family. BMC Evol Biol. 2007; 7(1):96.
http://dx.doi.org/10.1186/1471-2148-7-96
21. Qin C, Gao J, Wang L, Zeng L, Liu Y. Safety evaluation of short-term exposure to
chitooligomers from enzymic preparation. Food Chem Toxicol. 2006; 44(6):855-861.
http://dx.doi.org/10.1016/j.fct.2005.11.009
22. Vahouny GV, Satchithanandam S, Cassidy MM, Lightfoot FB, Furda I. Comparative effects
of chitosan and cholestyramine on lymphatic absorption of lipids in the rat. Am J Clin Nutr.
1983; 38(2):278-284. http://dx.doi.org/10.1093/ajcn/38.2.278
23. Fukada Y, Kimura K, Ayaki Y. Effect of chitosan feeding on intestinal bile acid metabolism
in rats. Lipids. 1991; 26(5):395-399.
24. Landes D, Bough W. Effects of chitosan—a coagulating agent for food processing wastes—
in the diets of rats on growth and liver and blood composition. Bull Environ Contam Toxicol.
1976; 15(5):555-563. http://dx.doi.org/10.1007/BF01685704
25. Tanaka Y, Tanioka S-i, Tanaka M, Tanigawa T, Kitamura Y, Minami S, Okamoto Y,
Miyashita M, Nanno M. Effects of chitin and chitosan particles on BALB/c mice by oral and
parenteral administration. Biomaterials. 1997; 18(8):591-595. http://dx.doi.org/10.1016/S0142-
9612(96)00182-2
26. Deuchi K, Kanauchi O, Shizukuishi M, Kobayashi E. Continuous and massive intake of
chitosan affects mineral and fat-soluble vitamin status in rats fed on a high-fat diet. Biosci
Biotechnol Biochem. 1995; 59(7):1211-1216. http://dx.doi.org/10.1271/bbb.59.1211

34
 Chitosan, NTP TOX 93

27. Fukui K, Nakamura K, Shirai M, Hirano A, Takatsu H, Urano S. Long-term vitamin E-

deficient mice exhibit cognitive dysfunction via elevation of brain oxidation. J Nutr Sci
Vitaminol (Tokyo). 2015; 61(5):362-368. http://dx.doi.org/10.3177/jnsv.61.362
28. Oliveros LB, Domeniconi MA, Vega VA, Gatica LV, Brigada AM, Gimenez MS. Vitamin A
deficiency modifies lipid metabolism in rat liver. Br J Nutr. 2007; 97(2):263-272.
http://dx.doi.org/10.1017/S0007114507182659
29. Naito Y, Tago K, Nagata T, Furuya M, Seki T, Kato H, Morimura T, Ohara N. A 90-day ad
libitum administration toxicity study of oligoglucosamine in F344 rats. Food Chem Toxicol.
2007; 45(9):1575-1587. http://dx.doi.org/10.1016/j.fct.2007.02.018
30. Hirano S, Iwata M, Yamanaka K, Tanaka H, Toda T, Inui H. Enhancement of serum
lysozyme activity by injecting a mixture of chitosan oligosaccharides intravenously in rabbits.
Agric Biol Chem. 1991; 55(10):2623-2625. https://doi.org/10.1080/00021369.1991.10871007
31. Rao SB, Sharma CP. Use of chitosan as a biomaterial: Studies on its safety and hemostatic
potential. J Biomed Mater Res. 1997; 34(1):21-28. http://dx.doi.org/10.1002/(SICI)1097-
4636(199701)34:1<21::AID-JBM4>3.0.CO;2-P
32. Gades MD, Stern JS. Chitosan supplementation and fecal fat excretion in men. Obes Res.
2003; 11(5):683-688. http://dx.doi.org/10.1038/oby.2003.97
33. Gades MD, Stern JS. Chitosan supplementation and fat absorption in men and women. J Am
Diet Assoc. 2005; 105(1):72-77. http://dx.doi.org/10.1016/j.jada.2004.10.004
34. Tapola NS, Lyyra ML, Kolehmainen RM, Sarkkinen ES, Schauss AG. Safety aspects and
cholesterol-lowering efficacy of chitosan tablets. J Am Coll Nutr. 2008; 27(1):22-30.
http://dx.doi.org/10.1080/07315724.2008.10719671
35. Takahashi M, Inoue K, Yoshida M, Morikawa T, Shibutani M, Nishikawa A. Lack of chronic
toxicity or carcinogenicity of dietary N-acetylglucosamine in F344 rats. Food Chem Toxicol.
2009; 47(2):462-471. http://dx.doi.org/10.1016/j.fct.2008.12.002
36. Cheng Q, Zhang J, Xia W. Prenatal and developmental effect of high molecular weight
chitosan (HMWCS) to mice. Regul Toxicol Pharmacol. 2013; 65(3):294-303.
http://dx.doi.org/10.1016/j.yrtph.2013.01.003
37. Hu Y-L, Qi W, Han F, Shao J-Z, Gao J-Q. Toxicity evaluation of biodegradable chitosan
nanoparticles using a zebrafish embryo model. Int J Nanomed. 2011; 6:3351.
38. Domard A, Rinaudo M. Preparation and characterization of fully deacetylated chitosan. Int J
Biol Macromol. 1983; 5(1):49-52. http://dx.doi.org/10.1016/0141-8130(83)90078-8
39. Hirai A, Odani H, Nakajima A. Determination of degree of deacetylation of chitosan by 1 H
NMR spectroscopy. Polym Bull. 1991; 26(1):87-94. http://dx.doi.org/10.1007/BF00299352
40. Reeves PG, Nielsen FH, Fahey Jr GC. AIN-93 purified diets for laboratory rodents: Final
report of the American Institute of Nutrition ad hoc writing committee on the reformulation of
the AIN-76A rodent diet. J Nutr. 1993; 123:1939-1951. http://dx.doi.org/10.1093/jn/123.11.1939

35
 Chitosan, NTP TOX 93

41. Rao GN. New nonpurified diet (NTP-2000) for rodents in the National Toxicology Program's
toxicology and carcinogenesis studies. J Nutr. 1997; 127(5):842S-846S.
http://dx.doi.org/10.1093/jn/127.5.842S
42. Reeves PG. Components of the AIN-93 diets as improvements in the AIN-76A diet. J Nutr.
1997; 127(5):838S-841S. http://dx.doi.org/10.1093/jn/127.5.838S
43. Maronpot R, Boorman G. Interpretation of rodent hepatocellular proliferative alterations and
hepatocellular tumors in chemical safety assessment. Toxicol Pathol. 1982; 10(2):71-78.
http://dx.doi.org/10.1177/019262338201000210
44. Boorman GA, Montgomery CA, Jr., Eustis SL, Wolfe MJ, McConnell EE, Hardisty JF.
Quality assurance in pathology for rodent carcinogenicity studies. In: Milman HA, Weisburger
EK, editors. Handbook of Carcinogen Testing. Park Ridge, NJ: Noyes Publications; 1985. p.
345-357.
45. Gart JJ, Chu KC, Tarone RE. Statistical issues in interpretation of chronic bioassay tests for
carcinogenicity. J Natl Cancer Inst. 1979; 62(4):957-974.
46. Dunnett CW. A multiple comparison procedure for comparing several treatments with a
control. J American Stat Assoc. 1955; 50(272):1096-1121.
http://dx.doi.org/10.1080/01621459.1955.10501294
47. Williams D. A test for differences between treatment means when several dose levels are
compared with a zero dose control. Biometrics. 1971; 27(1):103-117.
http://dx.doi.org/10.2307/2528930
48. Williams D. The comparison of several dose levels with a zero dose control. Biometrics.
1972; 28(2):519-531. http://dx.doi.org/10.2307/2556164
49. Shirley E. A non-parametric equivalent of Williams' test for contrasting increasing dose
levels of a treatment. Biometrics. 1977; 33(2):386-389. http://dx.doi.org/10.2307/2529789
50. Williams D. A note on Shirley's nonparametric test for comparing several dose levels with a
zero-dose control. Biometrics. 1986; 42(1):183-186. http://dx.doi.org/10.2307/2531254
51. Dunn OJ. Multiple comparisons using rank sums. Technometrics. 1964; 6(3):241-252.
http://dx.doi.org/10.1080/00401706.1964.10490181
52. Jonckheere A. A distribution-free k-sample test against ordered alternatives. Biometrika.
1954; 41:133-145. http://dx.doi.org/10.1093/biomet/41.1-2.133
53. Dixon W, Massey F. Introduction to statistical analysis. New York, NY: McGraw Hill Book
Company Inc; 1957. http://dx.doi.org/10.2307/2332898
54. Code of Federal Regulations (CFR). 21:Part 58.
55. Deuchi K, Kanauchi O, Imasato Y, Kobayashi E. Effect of the viscosity or deacetylation
degree of chitosan on fecal fat excreted from rats fed on a high-fat diet. Biosci Biotechnol
Biochem. 1995; 59(5):781-785. http://dx.doi.org/10.1271/bbb.59.781

36
 Chitosan, NTP TOX 93

56. Sugano M, Fujikawa T, Hiratsuji Y, Hasegawa Y. Hypocholesterolemic effects of chitosan in

cholesterol-fed rats. Nutr Rep Int. 1978; 18:531-537.
57. Sugano M, Fujikawa T, Hiratsuji Y, Nakashima K, Fukuda N, Hasegawa Y. A novel use of
chitosan as a hypocholesterolemic agent in rats. Am J Clin Nutr. 1980; 33(4):787-793.
http://dx.doi.org/10.1093/ajcn/33.4.787
58. Chiang M-T, Yao H-T, Chen H-C. Effect of dietary chitosans with different viscosity on
plasma lipids and lipid peroxidation in rats fed on a diet enriched with cholesterol. Biosci
Biotechnol Biochem. 2000; 64(5):965-971. http://dx.doi.org/10.1271/bbb.64.965
59. Hossain S, Rahman A, Kabir Y, Shams A, Afros F, Hashimoto M. Effects of shrimp
(Macrobracium rosenbergii)-derived chitosan on plasma lipid profile and liver lipid peroxide
levels in normo- and hypercholesterolaemic rats. Clin Exp Pharmacol Physiol. 2007; 34:170-176.
http://dx.doi.org/10.1111/j.1440-1681.2007.04568.x
60. Rucker R, Morris J, Fascetti A. Vitamins In: Kaneko J, Harvey J, Bruss M, editors. Clinical
biochemistry of domestic animals. Burlington, MA: Academic Press; 2008. p. 695-730.
http://dx.doi.org/10.1016/B978-0-12-370491-7.00023-4
61. Sommer A. Vitamin A deficiency and clinical disease: An historical overview. J Nutr. 2008;
138(10):1835-1839. http://dx.doi.org/10.1093/jn/138.10.1835
62. Traber MG. Vitamin E inadequacy in humans: Causes and consequences. Adv Nutr. 2014;
5(5):503-514. http://dx.doi.org/10.3945/an.114.006254
63. Wiseman EM, Bar-El Dadon S, Reifen R. The vicious cycle of vitamin A deficiency: A
review. Crit Rev Food Sci Nutr. 2017; 57(17):3703-3714.
http://dx.doi.org/10.1080/10408398.2016.1160362
64. Yang C-Y, Oh T-W, Nakajima D, Maeda A, Naka T, Kim C-S, Igawa S, Ohta F. Effects of
habitual chitosan intake on bone mass, bone-related metabolic markers and duodenum CaBP
D9K mRNA in ovariectomized SHRSP rats. J Nutr Sci Vitaminol (Tokyo). 2002; 48(5):371-378.
http://dx.doi.org/10.3177/jnsv.48.371
65. Wada M, Nishimura Y, Watanabe Y, Takita T, Innami S. Accelerating effect of chitosan
intake on urinary calcium excretion by rats. Biosci Biotechnol Biochem. 1997; 61(7):1206-1208.
http://dx.doi.org/10.1271/bbb.61.1206
66. Tieder M, Arie R, Modai D, Samuel R, Weissgarten J, Liberman UA. Elevated serum 1, 25-
dihydroxyvitamin D concentrations in siblings with primary Fanconi's syndrome. N Engl J Med.
1988; 319(13):845-849. http://dx.doi.org/10.1056/NEJM198809293191307
67. Thoolen B, Maronpot RR, Harada T, Nyska A, Rousseaux C, Nolte T, Malarkey DE,
Kaufmann W, Küttler K, Deschl U et al. Proliferative and nonproliferative lesions of the rat and
mouse hepatobiliary system. Toxicol Pathol. 2010; 38(7_suppl):5S-81S.
http://dx.doi.org/10.1177/0192623310386499
68. Hassan K, Bhalla V, El Regal ME, A-Kader HH. Nonalcoholic fatty liver disease: A
comprehensive review of a growing epidemic. World J Gastroenterol. 2014; 20(34):12082.
http://dx.doi.org/10.3748/wjg.v20.i34.12082

37
 Chitosan, NTP TOX 93

69. Sozio MS, Liangpunsakul S, Crabb D. The role of lipid metabolism in the pathogenesis of
alcoholic and nonalcoholic hepatic steatosis. Semin Liver Dis. 2010; 30(04):378-390.
http://dx.doi.org/10.1055/s-0030-1267538
70. Greaves P. Liver and pancreas In: Greaves P, editor. Histopatology of Preclinical Toxicity
Studies, 3rd ed. Oxford, UK: Elsevier; 2007. p. 457-569. http://dx.doi.org/10.1016/B978-
044452771-4/50010-9
71. Singh VN, Singh M, Venkitasubramanian T. Early effects of feeding excess vitamin A:
Mechanism of fatty liver production in rats. J Lipid Res. 1969; 10(4):395-401.
72. Kucera O, Cervinkova Z. Experimental models of non-alcoholic fatty liver disease in rats.
World J Gastroenterol. 2014; 20(26):8364. http://dx.doi.org/10.3748/wjg.v20.i26.8364
73. Jungermann K, Katz N. Functional specialization of different hepatocyte populations.
Physiol Rev. 1989; 69(3):708-764. http://dx.doi.org/10.1152/physrev.1989.69.3.708
74. Pearse G. Histopathology of the thymus. Toxicol Pathol. 2006; 34(5):515-547.
http://dx.doi.org/10.1080/01926230600978458
75. Lelovas PP, Xanthos TT, Thoma SE, Lyritis GP, Dontas IA. The laboratory rat as an animal
model for osteoporosis research. Comp Med. 2008; 58(5):424-430.
76. Wronski T, Dann L, Scott K, Cintron M. Long-term effects of ovariectomy and aging on the
rat skeleton. Calcif Tissue Int. 1989; 45(6):360-366. http://dx.doi.org/10.1007/BF02556007
77. Fukuda S, Iida H. Age-related changes in bone mineral density, cross-sectional area and the
strength of long bones in the hind limbs and first lumbar vertebra in female Wistar rats. J Vet
Med Sci. 2004; 66:755-760.

38
 Chitosan, NTP TOX 93

Appendix A. Summary of Lesions in Rats in the Six-month

Feed Study of Chitosan

Tables
Table A-1. Summary of the Incidence of Nonneoplastic Lesions in Group A Male Rats in
the Six-month Feed Study of Chitosan .................................................................... A-2
Table A-2. Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in
Group A Female Rats in the Six-month Feed Study of Chitosan ............................ A-4

A-1
 Chitosan, NTP TOX 93

Table A-1. Summary of the Incidence of Nonneoplastic Lesions in Group A Male Rats in the Six-
month Feed Study of Chitosana
0% 1% 3% 9%
Disposition Summary
Animals initially in study 10 10 10 10
Survivors
Terminal euthanasia 10 10 10 10
Animals examined microscopically 10 10 10 10
Alimentary System
Liver (10) (10) (10) (10)
Degeneration, cystic 0 0 0 1 (10%)
Hematopoietic cell proliferation 2 (20%) 3 (30%) 3 (30%) 6 (60%)
Inflammation, chronic active 10 (100%) 10 (100%) 10 (100%) 9 (90%)
Periportal, fatty change 6 (60%) 3 (30%) 6 (60%) 3 (30%)
Pancreas (10) (0) (0) (10)
Basophilic focus 1 (10%) – – 0
Inflammation 2 (20%) – – 1 (10%)
Stomach, forestomach (10) (0) (0) (10)
Epithelium, hyperplasia 3 (30%) – – 1 (10%)
Cardiovascular System
Blood vessel (10) (0) (0) (10)
Inflammation 0 – – 1 (10%)
Heart (10) (0) (0) (10)
Cardiomyopathy 5 (50%) – – 3 (30%)
Mineralization 0 – – 1 (10%)
Endocrine System
Adrenal cortex (10) (1) (0) (10)
Vacuolization cytoplasmic 0 0 – 1 (10%)
Parathyroid gland (10) (10) (10) (10)
Hyperplasia 1 (10%) 0 0 0
Pituitary gland (10) (0) (0) (10)
Cyst 1 (10%) – – 0
Thyroid gland (10) (0) (0) (10)
C-cell, hyperplasia 0 – – 1 (10%)
General Body System
None – – – –

A-2
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Genital System
Preputial gland (10) (0) (0) (10)
Inflammation 0 – – 1 (10%)
Inflammation, chronic active 0 – – 2 (20%)
Prostate (10) (10) (10) (10)
Inflammation 8 (80%) 9 (90%) 10 (100%) 10 (100%)
Testes (10) (0) (0) (10)
Mineralization 0 – – 1 (10%)
Hematopoietic System
Lymph node, mandibular (10) (0) (0) (10)
Infiltration cellular, plasma cell 1 (10%) – – 2 (20%)
Spleen (10) (0) (0) (10)
Hematopoietic cell proliferation 5 (50%) – – 2 (20%)
Thymus (10) (0) (0) (10)
Atrophy 1 (10%) – – 0
Integumentary System
Skin (10) (0) (0) (10)
Hemorrhage 0 – – 1 (10%)
Mineralization 0 – – 1 (10%)
Ulcer 0 – – 1 (10%)
Musculoskeletal System
Skeletal muscle (0) (0) (0) (1)
Inflammation, granulomatous – – – 1 (100%)
Nervous System
None – – – –
Respiratory System
Lung (10) (0) (0) (10)
Hemorrhage 2 (20%) – – 0
Inflammation, chronic active 2 (20%) – – 4 (40%)
Nose (10) (0) (0) (10)
Inflammation 1 (10%) – – 0
Goblet cell, hyperplasia 0 – – 1 (10%)
Special Senses System – – –
Eye (10) (1) (0) (10)
Choroid, fibrosis 0 1 (100%) – 0
Lens, cataract 0 1 (100%) – 0

A-3
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Harderian gland (10) (0) (0) (10)
Hyperplasia 0 – – 1 (10%)
Infiltration cellular, lymphocyte 2 (20%) – – 1 (10%)
Urinary System
Kidney (10) (10) (10) (10)
Infarct 0 0 1 (10%) 0
Mineralization 2 (20%) 4 (40%) 3 (30%) 5 (50%)
Nephropathy 9 (90%) 9 (90%) 9 (90%) 9 (90%)
Cortex, cyst 1 (10%) 0 0 0
Pelvis, dilatation 2 (20%) 0 1 (10%) 0
Pelvis, inflammation 1 (10%) 0 0 0
Urinary bladder (10) (0) (0) (10)
Transitional epithelium, 0 – – 1 (10%)
hyperplasia
aNumber of animals examined microscopically at the site and the number of animals with lesion.

Table A-2. Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Group A Female
Rats in the Six-month Feed Study of Chitosana
0% 1% 3% 9%
Disposition Summary
Animals initially in study 10 10 10 10
Survivors
Terminal euthanasia 10 10 10 10
Animals examined microscopically 10 10 10 10
Alimentary System
Liver (10) (10) (10) (10)
Hematopoietic cell proliferation 1 (10%) 1 (10%) 2 (20%) 1 (10%)
Inflammation, chronic active 9 (90%) 9 (90%) 9 (90%) 10 (100%)
Periportal, fatty change 7 (70%) 4 (40%) 4 (40%) 0
Pancreas (10) (0) (0) (10)
Atrophy 0 – – 1 (10%)
Inflammation 1 (10%) – – 0
Inflammation, chronic active 0 – – 1 (10%)
Cardiovascular System
Heart (10) (0) (0) (10)
Cardiomyopathy 1 (10%) – – 0
Endocrine System
Pituitary gland (10) (0) (0) (10)

A-4
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Rathke’s cleft, hyperplasia 1 (10%) – – 0
General Body System
None – – – –
Genital System
Clitoral gland (10) (0) (0) (10)
Inflammation, chronic active 2 (20%) – – 0
Hematopoietic System
Spleen (10) (0) (0) (10)
Hematopoietic cell proliferation 1 (10%) – – 0
Thymus (10) (0) (0) (10)
Atrophy 1 (10%) – – 0
Integumentary System
Mammary gland (10) (0) (0) (10)
Adenoma 0 – – 1 (10%)
Musculoskeletal System
None – – – –
Nervous System
Brain (10) (0) (0) (10)
Developmental malformation 1 (10%) – – 0
Respiratory System
Lung (10) (0) (0) (10)
Mineralization 0 – – 1 (10%)
Alveolar epithelium, hyperplasia 0 – – 1 (10%)
Alveolus, infiltration cellular, 2 (20%) – – 0
histiocyte
Artery, mineralization 1 (10%) – – 1 (10%)
Nose (10) (0) (0) (10)
Goblet cell, hyperplasia 1 (10%) – – 0
Special Senses System
Harderian gland (10) (0) (0) (10)
Infiltration cellular, lymphocyte 1 (10%) – – 1 (10%)
Urinary System
Kidney (10) (10) (10) (10)
Mineralization 8 (80%) 8 (80%) 5 (50%) 6 (60%)
Nephropathy 5 (50%) 6 (60%) 5 (50%) 0
aNumber of animals examined microscopically at the site and the number of animals with lesion.

A-5
 Chitosan, NTP TOX 93

Appendix B. Clinical Pathology Results

Tables
Table B-1. Hematology, Clinical Chemistry, and Urinalysis Data for Group C Rats in the
Six-month Feed Study of Chitosan............................................................................B-2

B-1
 Chitosan, NTP TOX 93

Table B-1. Hematology, Clinical Chemistry, and Urinalysis Data for Group C Rats in the Six-
month Feed Study of Chitosana
0% 1% 3% 9%
Male
Hematology
n 10 10 10 10
Hematocrit (auto) (%)
Week 25 45.5 ± 0.4 47.1 ± 0.5 46.3 ± 0.4 47.4 ± 0.6*
Hematocrit (manual) (%)
Week 25 47.2 ± 0.5b 48.2 ± 0.5 47.6 ± 0.5 48.9 ± 0.6
Hemoglobin (g/dL)
Week 25 14.9 ± 0.2 15.4 ± 0.2 15.2 ± 0.1 15.7 ± 0.2**
Erythrocytes (10 /μL) 6

Week 25 8.44 ± 0.08 8.59 ± 0.12 8.43 ± 0.08 8.45 ± 0.12

Reticulocytes (103/μL)
Week 25 186.0 ± 14.3 138.4 ± 6.3** 157.7 ± 7.2 139.3 ± 9.1**
Mean cell volume (fL)
Week 25 53.9 ± 0.4 54.9 ± 0.6 54.9 ± 0.2 56.1 ± 0.6*
Mean cell hemoglobin (pg)
Week 25 17.6 ± 0.2 18.0 ± 0.2 18.0 ± 0.1 18.6 ± 0.2**
Mean cell hemoglobin concentration (g/dL)
Week 25 32.7 ± 0.2 32.7 ± 0.1 32.9 ± 0.2 33.2 ± 0.2
Platelets (10 /μL)
3

Week 25 916 ± 52 824 ± 26 921 ± 19 973 ± 36

Leukocytes (10 /μL)
3

Week 25 10.62 ± 0.98 9.39 ± 0.94 7.38 ± 0.69 9.54 ± 0.91

Segmented neutrophils (103/μL)
Week 25 2.04 ± 0.38 1.48 ± 0.24 1.06 ± 0.12 1.76 ± 0.39
Lymphocytes (10 /μL) 3

Week 25 8.02 ± 0.64 7.42 ± 0.74 6.01 ± 0.62 7.44 ± 0.65

Monocytes (10 /μL)3

Week 25 0.31 ± 0.05 0.32 ± 0.05 0.20 ± 0.02 0.19 ± 0.03

Basophils (103/μL)
Week 25 0.04 ± 0.01 0.04 ± 0.01 0.02 ± 0.00 0.03 ± 0.01
Eosinophils (10 /μL)
3

Week 25 0.21 ± 0.05 0.15 ± 0.03 0.09 ± 0.01* 0.11 ± 0.03

B-2
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Clinical Chemistry
n 10 10 10 10
Urea nitrogen (mg/dL)
Week 25 12.4 ± 0.6 12.1 ± 0.5 12.7 ± 0.5 15.3 ± 0.9**
Creatinine (mg/dL)
Week 25 0.62 ± 0.01 0.64 ± 0.02 0.62 ± 0.01 0.64 ± 0.02
Calcium (mg/dL)
Week 13 12.6 ± 0.1 12.5 ± 0.1 12.3 ± 0.2 12.4 ± 0.2
Week 19 12.5 ± 0.1 12.3 ± 0.2 12.3 ± 0.1 12.0 ± 0.1*
Week 25 12.1 ± 0.1 12.1 ± 0.2 12.0 ± 0.1 11.6 ± 0.1*
Phosphorus (mg/dL)
Week 13 8.4 ± 0.3 8.1 ± 0.3 7.2 ± 0.3** 7.4 ± 0.4*
Week 19 8.2 ± 0.4 7.7 ± 0.2 7.4 ± 0.3 6.7 ± 0.2**
Week 25 6.9 ± 0.3 6.8 ± 0.2 6.7 ± 0.1 5.8 ± 0.3**
Total protein (g/dL)
Week 25 7.4 ± 0.1 7.2 ± 0.1 7.3 ± 0.1 6.9 ± 0.1*
Albumin (g/dL)
Week 19 4.8 ± 0.1 4.6 ± 0.1 4.7 ± 0.1 4.5 ± 0.0*
Week 25 4.8 ± 0.1 4.7 ± 0.1 4.8 ± 0.1 4.6 ± 0.0
Cholesterol (mg/dL)
Week 7 82 ± 5 75 ± 8 80 ± 6 53 ± 3**
Week 13 95 ± 7 84 ± 8 90 ± 7 53 ± 2**
Week 19 101 ± 6 87 ± 10 94 ± 8 59 ± 4**
Week 25 95 ± 6 81 ± 8 90 ± 6 49 ± 4**
Triglycerides (mg/dL)
Week 7 202 ± 28 234 ± 43 226 ± 30 88 ± 15*
Week 13 198 ± 33 202 ± 38 195 ± 24 86 ± 8**
Week 19 180 ± 26 218 ± 43 210 ± 29 95 ± 13*
Week 25 173 ± 18 207 ± 30 218 ± 24 109 ± 13
Alanine aminotransferase (IU/L)
Week 25 28 ± 3 29 ± 2 29 ± 1 57 ± 2**
Alkaline phosphatase (IU/L)
Week 7 134 ± 7 134 ± 7 138 ± 8 137 ± 16
Week 13 100 ± 6 95 ± 6 102 ± 6 82 ± 5
Week 19 91 ± 11 87 ± 7 84 ± 4 72 ± 7
Week 25 85 ± 7 83 ± 7 82 ± 5 64 ± 5*

B-3
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0% 1% 3% 9%
Creatine kinase (IU/L)
Week 25 192 ± 29 205 ± 27 233 ± 23 245 ± 20
Sorbitol dehydrogenase (IU/L)
Week 25 17 ± 3 17 ± 2 15 ± 1 14 ± 1
Bile acids (µmol/L)
Week 25 9.6 ± 2.3 6.4 ± 2.7 2.4 ± 0.2** 4.3 ± 0.8
Total osteocalcin (ng/mL)
Week 7 445.7 ± 17.2 439.8 ± 15.8 441.8 ± 18.2 520.4 ± 22.6
Week 13 306.2 ± 13.0 289.7 ± 28.6 245.4 ± 37.9 372.6 ± 23.4
Week 19 239.4 ± 12.4 225.7 ± 10.6 181.6 ± 26.8 269.2 ± 20.9
Week 25 158.3 ± 10.0 168.1 ± 11.6 145.9 ± 22.7 218.3 ± 14.6*
Parathyroid hormone (ng/mL)
Week 7 1.882 ± 0.137 1.643 ± 0.449 1.838 ± 0.348 1.521 ± 0.368
Week 13 2.343 ± 0.350 2.763 ± 0.479 3.215 ± 0.537 2.433 ± 0.222
Week 19 1.879 ± 0.186 3.101 ± 0.475 2.710 ± 0.365 3.679 ± 0.361**
Week 25 2.668 ± 0.475 2.924 ± 0.276 3.981 ± 0.349 2.848 ± 0.506
Urinalysis
n
Week 7 10 9 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 25 10 10 10 10
Creatinine (mg/dL)
Week 7 192.5 ± 15.1 227.2 ± 30.7 269.4 ± 33.6 254.9 ± 37.2
Week 13 249.4 ± 25.1 360.7 ± 19.5* 350.3 ± 22.5* 334.0 ± 35.8
Week 19 204.3 ± 20.4 394.2 ± 32.5** 345.1 ± 26.0** 302.5 ± 26.6
Week 25 254.1 ± 27.4 374.8 ± 25.6* 345.9 ± 27.1 325.4 ± 36.0
Volume (mL)
Week 7 8.3 ± 0.8 7.5 ± 1.5 6.4 ± 0.8 5.0 ± 1.1*
Week 13 7.9 ± 1.0 4.6 ± 0.3** 5.1 ± 0.4* 4.5 ± 0.5**
Week 19 10.7 ± 1.6 4.0 ± 0.4** 5.3 ± 0.7* 5.6 ± 0.6
Week 25 8.6 ± 1.2 5.4 ± 0.6* 6.1 ± 0.8* 5.1 ± 0.6**
Deoxypyridinoline (nmol/L)
Week 7 3,396.0 ± 268.0 4,210.0 ± 643.0 4,917.0 ± 826.0 4,754.0 ± 761.0
Week 13 2,185.1 ± 188.9 3,197.3 ± 148.3* 3,233.8 ± 218.0* 3,129.1 ± 296.5*
Week 19 1,084.9 ± 158.9 2,209.6 ± 246.3** 1,963.0 ± 200.5* 1,994.9 ± 214.3*

B-4
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Week 25 1,083.5 ± 145.9 1,699.3 ± 139.6* 1,658.3 ± 136.7* 1,750.8 ± 167.6*
Deoxypyridinoline/creatinine (nmol/mg)
Week 7 1.810 ± 0.135 1.889 ± 0.148 1.810 ± 0.159 1.920 ± 0.160
Week 13 0.910 ± 0.035 0.890 ± 0.031 0.930 ± 0.040 0.960 ± 0.078
Week 19 0.530 ± 0.050 0.550 ± 0.034 0.570 ± 0.042 0.660 ± 0.048
Week 25 0.430 ± 0.030 0.470 ± 0.030 0.480 ± 0.020 0.550 ± 0.027**
Female
Hematology
n 10 9 10 10
Hematocrit (auto) (%)
Week 25 45.5 ± 1.0 44.9 ± 0.9 44.5 ± 0.8 45.2 ± 0.9
Hematocrit (manual) (%)
Week 25 47.4 ± 1.1 46.9 ± 1.0 46.5 ± 0.8 46.6 ± 0.9
Hemoglobin (g/dL)
Week 25 15.2 ± 0.4 15.0 ± 0.3 15.0 ± 0.3 15.1 ± 0.3
Erythrocytes (10 /μL) 6

Week 25 8.16 ± 0.19 8.17 ± 0.18 8.01 ± 0.11 8.10 ± 0.15

Reticulocytes (103/μL)
Week 25 135.2 ± 14.6 109.2 ± 6.1 109.6 ± 7.7 129.5 ± 14.6
Mean cell volume (fL)
Week 25 55.8 ± 0.6 55.0 ± 0.3 55.6 ± 0.3 55.8 ± 0.8
Mean cell hemoglobin (pg)
Week 25 18.7 ± 0.2 18.4 ± 0.1 18.7 ± 0.1 18.7 ± 0.3
Mean cell hemoglobin concentration (g/dL)
Week 25 33.5 ± 0.1 33.4 ± 0.2 33.6 ± 0.2 33.4 ± 0.2
Platelets (10 /μL)
3

Week 25 791 ± 43 798 ± 40 848 ± 38 1,024 ± 51**

Leukocytes (10 /μL)
3

Week 25 6.62 ± 0.92 3.66 ± 0.49* 5.72 ± 0.87 4.92 ± 0.58

Segmented neutrophils (103/μL)
Week 25 1.15 ± 0.24 0.53 ± 0.10* 0.67 ± 0.11 0.67 ± 0.11
Lymphocytes (10 /μL) 3

Week 25 5.09 ± 0.79 2.93 ± 0.41 4.78 ± 0.77 4.06 ± 0.49

Monocytes (10 /μL)3

Week 25 0.24 ± 0.04 0.13 ± 0.02 0.18 ± 0.03 0.11 ± 0.02*

B-5
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Basophils (103/μL)
Week 25 0.02 ± 0.00 0.01 ± 0.00* 0.02 ± 0.01 0.01 ± 0.00
Eosinophils (103/μL)
Week 25 0.12 ± 0.02 0.06 ± 0.01 0.08 ± 0.01 0.07 ± 0.02
Clinical Chemistry
n
Week 7 10 10 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 25 10 9 10 10
Urea nitrogen (mg/dL)
Week 25 14.2 ± 1.4 14.1 ± 0.7 15.2 ± 0.7 16.3 ± 0.7*
Creatinine (mg/dL)
Week 25 0.65 ± 0.02 0.68 ± 0.01 0.70 ± 0.01 0.69 ± 0.02
Calcium (mg/dL)
Week 13 12.9 ± 0.2 13.1 ± 0.1 12.7 ± 0.2 12.5 ± 0.2
Week 19 12.9 ± 0.1 13.1 ± 0.2 12.8 ± 0.1 12.5 ± 0.1
Week 25 12.7 ± 0.3 12.8 ± 0.1 12.7 ± 0.2 12.3 ± 0.2
Phosphorus (mg/dL)
Week 13 8.1 ± 0.5 7.4 ± 0.4 6.5 ± 0.4** 6.8 ± 0.3*
Week 19 8.4 ± 0.4 8.2 ± 0.5 8.1 ± 0.3 7.4 ± 0.5
Week 25 6.8 ± 0.2 6.4 ± 0.2 6.2 ± 0.3* 5.5 ± 0.3**
Total protein (g/dL)
Week 25 8.2 ± 0.2 9.0 ± 0.1** 8.6 ± 0.2 8.4 ± 0.2
Albumin (g/dL)
Week 19 5.9 ± 0.2 6.2 ± 0.1 5.9 ± 0.2 5.7 ± 0.1
Week 25 5.8 ± 0.2 6.5 ± 0.2* 6.2 ± 0.1 6.2 ± 0.1
Cholesterol (mg/dL)
Week 7 80 ± 6 81 ± 8 67 ± 4 59 ± 4**
Week 13 92 ± 8 86 ± 7 73 ± 5 58 ± 4**
Week 19 107 ± 7 105 ± 9 91 ± 8 67 ± 5**
Week 25 94 ± 7 108 ± 5 96 ± 8 63 ± 4**
Triglycerides (mg/dL)
Week 7 88 ± 12 130 ± 48 81 ± 8 86 ± 14
Week 13 125 ± 10 163 ± 30 140 ± 23 88 ± 23*
Week 19 143 ± 15 181 ± 32 137 ± 18 90 ± 13

B-6
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Week 25 188 ± 31 231 ± 44 245 ± 31 158 ± 35
Alanine aminotransferase (IU/L)
Week 25 25 ± 3 28 ± 3 32 ± 2** 47 ± 4**
Alkaline phosphatase (IU/L)
Week 7 102 ± 7 99 ± 5 99 ± 7 95 ± 10
Week 13 57 ± 4 63 ± 4 71 ± 7 59 ± 5
Week 19 49 ± 5 53 ± 3 55 ± 6 46 ± 6
Week 25 46 ± 4 44 ± 2 51 ± 6 44 ± 7
Creatine kinase (IU/L)
Week 25 258 ± 44 193 ± 46 210 ± 50 225 ± 26
Sorbitol dehydrogenase (IU/L)
Week 25 17 ± 3 17 ± 2 19 ± 2 16 ± 1
Bile acids (µmol/L)
Week 25 10.7 ± 1.8 8.2 ± 1.1 32.0 ± 14.1 10.8 ± 1.1
Total osteocalcin (ng/mL)
Week 7 293.6 ± 19.4 287.5 ± 21.2 282.1 ± 34.7 316.7 ± 23.5
Week 13 197.9 ± 22.6 202.3 ± 15.4 184.4 ± 19.4 234.2 ± 14.5
Week 19 158.1 ± 18.3 184.8 ± 13.2 166.7 ± 24.7 210.1 ± 16.0
Week 25 107.9 ± 18.6 97.1 ± 7.1 96.0 ± 16.2 148.8 ± 15.1
Parathyroid hormone (ng/mL)
Week 7 0.995 ± 0.150b 1.156 ± 0.176 1.092 ± 0.182 1.023 ± 0.146
Week 13 1.506 ± 0.203 1.734 ± 0.194 1.925 ± 0.306 1.767 ± 0.212
Week 19 1.406 ± 0.232 1.994 ± 0.353 1.845 ± 0.418 1.673 ± 0.223
b
Week 25 1.471 ± 0.189 1.628 ± 0.220 1.818 ± 0.224 2.301 ± 0.212*
Urinalysis
n
Week 7 10 10 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 25 10 9 10 9
Creatinine (mg/dL)
Week 7 98.2 ± 14.7 107.1 ± 12.3 206.3 ± 55.2* 192.0 ± 8.3**
Week 13 144.7 ± 14.8 139.5 ± 15.9 247.5 ± 30.9* 241.6 ± 29.0**
Week 19 142.0 ± 19.7 137.7 ± 19.4 196.3 ± 23.6 230.3 ± 19.2**
Week 25 179.8 ± 59.7 120.3 ± 24.1 184.5 ± 20.5 217.9 ± 23.4*

B-7
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Volume (mL)
Week 7 8.2 ± 1.2 8.5 ± 1.0 5.4 ± 0.8 3.4 ± 0.3**
Week 13 6.4 ± 0.7 6.5 ± 0.6 4.1 ± 0.8* 2.9 ± 0.5**
Week 19 7.7 ± 1.2 8.1 ± 1.4 5.0 ± 0.8 3.4 ± 0.5**
Week 25 8.2 ± 1.5 9.1 ± 1.5 5.8 ± 0.9 3.7 ± 0.5**
Deoxypyridinoline (nmol/L)
Week 7 1,622.0 ± 328.0 1,378.0 ± 295.0 4,130.0 ± 1,109.0* 4,423.0 ± 355.0**
Week 13 875.5 ± 129.6 587.0 ± 68.1 1,364.0 ± 215.9 1,421.6 ± 267.0
Week 19 666.3 ± 106.9 487.7 ± 68.5 894.9 ± 122.1 1,212.3 ± 107.4**
Week 25 565.7 ± 178.2 250.4 ± 47.1 625.1 ± 83.7 891.5 ± 114.1*
Deoxypyridinoline/creatinine (nmol/mg)
Week 7 1.620 ± 0.128 1.240 ± 0.129 1.940 ± 0.229 2.300 ± 0.182*
Week 13 0.580 ± 0.039 0.430 ± 0.037** 0.540 ± 0.034 0.570 ± 0.042
Week 19 0.450 ± 0.017 0.360 ± 0.016* 0.440 ± 0.016 0.520 ± 0.020
Week 25 0.340 ± 0.043 0.222 ± 0.022 0.340 ± 0.027 0.411 ± 0.026
*Significantly different (P ≤ 0.05) from the control group by Dunn’s or Shirley’s test.
**P ≤ 0.01.
aData are presented as mean ± standard error. Statistical tests were performed on unrounded data.
bn = 9.

B-8
 Chitosan, NTP TOX 93

Appendix C. Vitamin Concentration and Bone Parameter

Results

Tables
Table C-1. Serum and Hepatic Vitamin Concentration Data for Group B Rats in the Six-
month Feed Study of Chitosan ..................................................................................C-2
Table C-2. Bone Data for Groups A and B Rats in the Six-month Feed Study of Chitosan .......C-3

C-1
 Chitosan, NTP TOX 93

Table C-1. Serum and Hepatic Vitamin Concentration Data for Group B Rats in the Six-month
Feed Study of Chitosana
0% 1% 3% 9%
Male
n
Week 7 9 10 10 10
Week 13 9 10 10 10
Week 19 9 10 10 10
Week 26 9 10 10 8
Serum vitamin A (µg/mL)
Week 7 0.532 ± 0.021 0.506 ± 0.033 0.513 ± 0.026 0.453 ± 0.018
Week 13 0.561 ± 0.024 0.499 ± 0.019 0.476 ± 0.022* 0.410 ± 0.009**
Week 19 0.533 ± 0.028 0.506 ± 0.031 0.475 ± 0.019 0.392 ± 0.014**
Week 26 0.476 ± 0.019 0.444 ± 0.024 0.398 ± 0.017** 0.336 ± 0.026**
Serum 1,25 (OH)2 vitamin D (pg/mL)
Week 7 124.4 ± 19.6 163.3 ± 21.7 183.2 ± 26.9 297.4 ± 41.0**
Week 13 70.1 ± 7.3 57.4 ± 5.3 77.3 ± 4.4 86.1 ± 8.5
Week 19 20.6 ± 2.8 21.7 ± 6.1 22.9 ± 2.2 42.3 ± 3.1**b
Week 26 27.7 ± 3.4c 28.0 ± 4.3 36.1 ± 4.6b 66.9 ± 11.9**
Serum vitamin E (µg/mL)
Week 7 19.33 ± 1.43 15.38 ± 1.29 12.92 ± 0.48** 4.14 ± 0.23**
Week 13 21.08 ± 1.61 17.45 ± 1.06* 12.27 ± 0.86** 4.33 ± 0.27**
Week 19 20.59 ± 1.61 16.19 ± 0.96 12.86 ± 0.42** 4.07 ± 0.32**
Week 26 19.66 ± 1.66 17.35 ± 1.37 12.35 ± 0.61** 3.59 ± 0.65**
Liver vitamin A (µg/g)
Week 26 57.4 ± 17.6 29.9 ± 2.5 39.6 ± 3.1 31.4 ± 3.7
Liver vitamin E (µg/g)
Week 26 66.8 ± 16.2 55.0 ± 6.8 34.6 ± 2.2** 8.5 ± 0.8**
Female
n
Week 7 10 10 10 10
Week 13 10 10 10 10
Week 19 10 10 10 10
Week 26 10 10 9 10
Serum vitamin A (µg/mL)
Week 7 0.272 ± 0.011 0.253 ± 0.007 0.260 ± 0.012 0.266 ± 0.012
Week 13 0.308 ± 0.020 0.295 ± 0.011 0.309 ± 0.019 0.281 ± 0.018
Week 19 0.283 ± 0.014 0.271 ± 0.015 0.291 ± 0.012 0.231 ± 0.010*
Week 26 0.316 ± 0.015 0.302 ± 0.014 0.294 ± 0.018 0.249 ± 0.010**

C-2
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Serum 1,25 (OH)2 vitamin D (pg/mL)
Week 7 104.0 ± 15.1 96.7 ± 10.9 111.0 ± 8.7 208.1 ± 18.2**
Week 13 60.6 ± 7.5 60.7 ± 7.9 69.3 ± 11.0 110.1 ± 16.9
Week 19 11.6 ± 1.6 12.6 ± 1.7 15.8 ± 1.4 31.4 ± 3.2**
Week 26 19.2 ± 2.2 20.7 ± 4.2 28.6 ± 6.5 53.7 ± 5.8**
Serum vitamin E (µg/mL)
Week 7 18.65 ± 0.71 20.08 ± 0.87 18.38 ± 0.85 6.99 ± 0.58**
Week 13 19.81 ± 1.41 20.85 ± 1.06 20.19 ± 1.20 7.48 ± 0.38**
Week 19 21.02 ± 1.76 19.74 ± 1.75 19.86 ± 1.08 7.37 ± 0.57**
Week 26 20.94 ± 1.56 23.43 ± 1.66 22.23 ± 1.75 7.28 ± 0.64**
Liver vitamin A (µg/g)
Week 26 65.2 ± 5.4 58.9 ± 5.0 62.3 ± 6.3 60.3 ± 4.8
Liver vitamin E (µg/g)
Week 26 84.5 ± 8.9 97.1 ± 10.1 82.0 ± 11.8 17.2 ± 3.2**
*Significantly different (P ≤ 0.05) from the control group by Dunn’s or Shirley’s test.
**Significantly different (P ≤ 0.01) from the control group by Shirley’s test.
aData are presented as mean ± standard error. Statistical tests were performed on unrounded data.
bn = 9.
cn = 7.

Table C-2. Bone Data for Groups A and B Rats in the Six-month Feed Study of Chitosana
0% 1% 3% 9%
n 10 10 10 10
Male
Bone calcium (%) 23.79 ± 0.21b 23.95 ± 0.22 23.92 ± 0.30 23.74 ± 0.11c
Bone ash (%) 45.33 ± 0.79b 45.24 ± 0.67 45.83 ± 0.52 43.46 ± 0.62c
Bone moisture (%) 29.90 ± 0.49b 30.30 ± 0.44 29.72 ± 0.36 31.79 ± 0.62c
Left femur length (mm) 43.96 ± 0.34 44.33 ± 0.30 44.10 ± 0.30 43.42 ± 0.37
Left tibia length (mm) 48.00 ± 0.37 48.27 ± 0.36 47.95 ± 0.37 47.57 ± 0.41
Right tibia length (mm) 48.06 ± 0.32 48.41 ± 0.41 47.95 ± 0.33 47.57 ± 0.43
Female
Bone calcium (%) 24.65 ± 0.17 24.96 ± 0.20 24.77 ± 0.23b 24.84 ± 0.12
Bone ash (%) 47.07 ± 0.58 47.14 ± 0.57 47.44 ± 0.46b 45.87 ± 0.44
Bone moisture (%) 28.40 ± 0.54 28.45 ± 0.45 28.53 ± 0.49b 30.37 ± 0.37**
Left femur length (mm) 36.65 ± 0.21 36.75 ± 0.17 36.73 ± 0.28 36.37 ± 0.26
Left tibia length (mm) 40.56 ± 0.28 40.25 ± 0.23 40.62 ± 0.40 40.10 ± 0.24
Right tibia length (mm) 40.53 ± 0.30 40.42 ± 0.24 40.74 ± 0.42 40.12 ± 0.21
**Significantly different (P ≤ 0.01) from the control group by Shirley’s test.
aData are presented as mean ± standard error. Statistical tests were performed on unrounded data. Bone content data are from

Group B rats at week 26 and bone lengths are from Group A rats at week 25.
bn = 9.
cn = 8.

C-3
 Chitosan, NTP TOX 93

Appendix D. Organ Weights and Organ-Weight-to-Body-

Weight Ratios

Tables
Table D-1. Organ Weights and Organ-Weight-to-Body-Weight Ratios for Group A Rats
in the Six-month Feed Study of Chitosan ................................................................ D-2

D-1
 Chitosan, NTP TOX 93

Table D-1. Organ Weights and Organ-Weight-to-Body-Weight Ratios for Group A Rats in the Six-
month Feed Study of Chitosana
0% 1% 3% 9%
n 10 10 10 10
Male
Necropsy body wt 669 ± 20 702 ± 21 687 ± 23 612 ± 17
Heart
Absolute 1.82 ± 0.07 1.81 ± 0.06 1.86 ± 0.08 1.77 ± 0.06
Relative 2.723 ± 0.089 2.589 ± 0.070 2.710 ± 0.091 2.904 ± 0.085
R. Kidney
Absolute 2.04 ± 0.04 2.04 ± 0.04 2.11 ± 0.06 1.88 ± 0.04*
Relative 3.068 ± 0.088 2.920 ± 0.047 3.093 ± 0.088 3.093 ± 0.094
Liver
Absolute 25.19 ± 0.87 24.87 ± 1.35 23.74 ± 1.51 19.53 ± 0.71**
Relative 37.662 ± 0.731 35.321 ± 1.179 34.345 ± 1.411* 31.933 ± 0.817**
Lung
Absolute 2.49 ± 0.11 2.77 ± 0.09 2.62 ± 0.08 2.53 ± 0.14
Relative 3.738 ± 0.163 3.949 ± 0.095 3.841 ± 0.138 4.120 ± 0.160
R. Testis
Absolute 1.696 ± 0.054 1.778 ± 0.046 1.726 ± 0.062 1.750 ± 0.028
Relative 2.555 ± 0.108 2.546 ± 0.078 2.534 ± 0.107 2.883 ± 0.104
Thymus
Absolute 0.763 ± 0.045 0.727 ± 0.065 0.606 ± 0.063* 0.489 ± 0.032**
Relative 1.147 ± 0.071 1.030 ± 0.077 0.888 ± 0.091* 0.797 ± 0.045**
Thyroid gland and parathyroid gland
Absolute 0.033 ± 0.003 0.034 ± 0.002 0.034 ± 0.002 0.031 ± 0.002
Relative 0.049 ± 0.004 0.048 ± 0.003 0.050 ± 0.003 0.051 ± 0.003
Parathyroid gland
Absolute 0.0012 ± 0.0001 0.0010 ± 0.0001 0.0011 ± 0.0001 0.0011 ± 0.0001
Relative 0.002 ± 0.000 0.001 ± 0.000 0.002 ± 0.000 0.002 ± 0.000
Female
Necropsy body wt 338 ± 11 335 ± 13 328 ± 11 301 ± 13
Heart
Absolute 1.14 ± 0.03 1.09 ± 0.02 1.15 ± 0.03 1.03 ± 0.02**
Relative 3.393 ± 0.121 3.295 ± 0.094 3.515 ± 0.100 3.473 ± 0.134
R. Kidney
Absolute 1.12 ± 0.04 1.10 ± 0.02 1.13 ± 0.03 1.01 ± 0.03

D-2
 Chitosan, NTP TOX 93

0% 1% 3% 9%
Relative 3.311 ± 0.085 3.311 ± 0.095 3.465 ± 0.108 3.399 ± 0.104
Liver
Absolute 12.54 ± 0.82 12.47 ± 0.39 11.85 ± 0.29 9.85 ± 0.20**
Relative 36.900 ± 1.502 37.341 ± 0.444 36.346 ± 0.904 33.036 ± 0.910*
Lung
Absolute 1.83 ± 0.06 1.80 ± 0.08 1.81 ± 0.05 1.65 ± 0.05
Relative 5.463 ± 0.181 5.396 ± 0.170 5.552 ± 0.202 5.557 ± 0.281
R. Ovary
Absolute 0.054 ± 0.005 0.049 ± 0.005 0.057 ± 0.005 0.056 ± 0.007
Relative 0.161 ± 0.015 0.147 ± 0.015 0.179 ± 0.021 0.190 ± 0.026
Thymus
Absolute 0.436 ± 0.033 0.400 ± 0.036 0.383 ± 0.023 0.302 ± 0.021**
Relative 1.284 ± 0.081 1.188 ± 0.083 1.169 ± 0.062 1.000 ± 0.047**
Thyroid gland and parathyroid gland
Absolute 0.028 ± 0.002 0.027 ± 0.002 0.035 ± 0.002 0.031 ± 0.002
Relative 0.084 ± 0.005 0.082 ± 0.007 0.106 ± 0.007 0.104 ± 0.008
Parathyroid gland
Absolute 0.0007 ± 0.0001 0.0009 ± 0.0001 0.0008 ± 0.0001 0.0008 ± 0.0001
Relative 0.002 ± 0.000 0.003 ± 0.000* 0.002 ± 0.000 0.003 ± 0.000*
Uterus
Absolute 0.657 ± 0.052 0.744 ± 0.060 0.714 ± 0.038 0.789 ± 0.096
Relative 1.980 ± 0.186 2.252 ± 0.191 2.184 ± 0.104 2.650 ± 0.329
*Significantly different (P ≤ 0.05) from the control group by Williams’ or Dunnett’s test.
**P ≤ 0.01.
aOrgan weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights)

are given as mg organ weight/g body weight (mean ± standard error).

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 Chitosan, NTP TOX 93

Appendix E. Reproductive Tissue Evaluations

Tables
Table E-1. Summary of Reproductive Tissue Evaluations for Group A Male Rats in the
Six-month Feed Study of Chitosan ............................................................................ E-2

E-1
 Chitosan, NTP TOX 93

Table E-1. Summary of Reproductive Tissue Evaluations for Group A Male Rats in the Six-month
Feed Study of Chitosana
0% 1% 3% 9%
n 10 10 10 10
Weights (g)
Necropsy body wt 669 ± 20 702 ± 21 687 ± 23 612 ± 17
L. Cauda epididymis 0.2013 ± 0.0073 0.2134 ± 0.0079 0.2281 ± 0.0167 0.2072 ± 0.0103
L. Epididymis 0.6874 ± 0.0184 0.7047 ± 0.0274 0.7398 ± 0.0175 0.6402 ± 0.0165
L. Testis 1.7349 ± 0.0423 1.8209 ± 0.0478 1.7922 ± 0.0619 1.7900 ± 0.0333
Spermatid measurements
Spermatid heads (106/testis) 207.79 ± 18.44 183.39 ± 9.19 238.70 ± 20.45 175.57 ± 8.43
Spermatid heads (106/g testis) 120.38 ± 11.23 101.50 ± 5.84 135.54 ± 14.11 98.05 ± 4.29
Epididymal spermatozoal measurements
Sperm motility (%) 86.0 ± 0.37 86.1 ± 0.46 85.9 ± 0.46 85.8 ± 0.47
Sperm (106/cauda epididymis) 169.25 ± 14.82 182.38 ± 8.81 160.75 ± 12.63 157.63 ± 12.41
6
Sperm (10 /g cauda epididymis) 833 ± 52 856 ± 33 711 ± 33 760 ± 46
aData are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (body and
tissue weights) or Dunn’s test (spermatid and epididymal spermatozoal measurements).

E-2
 Chitosan, NTP TOX 93

Appendix F. Chemical Characterization and Dose

Formulation Studies

Table of Contents
F.1. Procurement and Characterization of Chitosan .................................................................... F-2
F.2. Preparation and Analysis of Dose Formulations .................................................................. F-3

Tables
Table F-1. Gel Permeation Chromatography Systems Used in the Six-month Feed Study
of Chitosan ................................................................................................................. F-3
Table F-2. Preparation and Storage of Dose Formulations in the Six-month Feed Study of
Chitosan ..................................................................................................................... F-4
Table F-3. Results of Analyses of Dose Formulations Administered to Rats in the
Six-month Feed Study of Chitosan ............................................................................ F-4

Figures
Figure F-1. Infrared Absorption Spectrum of Chitosan ............................................................... F-5
Figure F-2. Proton Nuclear Magnetic Resonance Spectrum of Chitosan .................................... F-5

F-1
 Chitosan, NTP TOX 93

F.1. Procurement and Characterization of Chitosan

Chitosan was obtained from Vanson HaloSource, Inc. (Redmond, WA), in one lot (02-ASSF-
0715), which was used in the 6-month study. Identity, purity, and stability analyses were
conducted by the analytical chemistry laboratory at Midwest Research Institute (MRI) (Kansas
City, MO) and by the study laboratory at Battelle Columbus Operations (Columbus, OH).
Reports on analyses performed in support of the chitosan studies are on file at the National
Institute of Environmental Health Sciences.
The test article, an off-white powder, was identified as chitosan by the analytical chemistry
laboratory using infrared (IR) and proton nuclear magnetic resonance (NMR) spectroscopy and
by the study laboratory using IR spectroscopy. The percentage of deacetylation of the test article,
determined by proton NMR, ranged from 85.97% to 87.17%, with an average of 86.5%. All
spectra were consistent with the literature spectra38; 39, and with the Sadtler spectral database.
Representative IR and NMR spectra are presented in Figure F-1 and Figure F-2, respectively.
The moisture content for lot 02-ASSF-0715 was determined by the analytical chemistry
laboratory using weight loss on drying in a 110°C oven for 24 hours; the inorganic content was
determined on the dried test article by ashing at 500°C for 4 hours. Viscosity was determined at
approximately 22.5°C using a Brookfield viscometer fitted with an SC4-18/R13 spindle at a
speed of 30 rpm. Lot 02-ASSF-0715 was characterized by the analytical chemistry laboratory
using gel permeation chromatography (GPC) with refractive index (RI) detection using system A
(Table F-1) to find the most abundant molecular weight. Samples were prepared by transferring
approximately 75 mg of the test article into a vial, and adding a 25 mL aliquot of diluent; vials
®
were sealed with Teflon -lined septa and crimp caps, allowed to stand for 2 hours at ambient
temperature, swirled by hand, and placed on a rotary shaker for at least 1 hour. Standards
containing a total of six molecular weight dextran markers with known peak molecular
weights (Mp) (4,400, 21,400, 43,500, 196,000, 277,000, and 3,900,000 Mp) were prepared;
approximately 10 mg of each marker (3 mg of 3,900,000 Mp marker) and 10 mL of diluent were
®
pipetted into vials, sealed with Teflon -lined septa and crimp caps, allowed to stand for a least
2 hours (the 3,900,000 marker was allowed to stand overnight) at ambient temperature to
dissolve the standards, then swirled to mix prior to analysis.
For lot 02-ASSF-0715, weight loss on drying indicated 4.50% water, the average inorganic
content by ashing was determined to be 2.13%, and viscosity was 81.3 centipoise. GPC/RI
indicated one major peak and the determined molecular weight of the bulk chemical ranged from
62,755 to 87,343 daltons (Da). This resulted in an average molecular weight of 81,644 g/mol, or
approximately 82 kDa, classifying the test article as a low molecular weight chitosan (LMWCS).
A sample of chitosan was submitted to Covance Laboratories, Inc. (Madison, WI), for nutritional
and contaminant testing using standard methods. For lot 02-ASSF-0715, levels of organochlorine
and organophosphorous pesticides, nitrosamines, and aflatoxins were below the detection limits
of the analytical methods. The purity of lot 02-ASSF-0715 was estimated to be approximately
94% based on the analysis of moisture and inorganic content. Taken together, these data
indicated that the test article was chitosan.

F-2
 Chitosan, NTP TOX 93

To ensure stability, the test article was stored in sealed amber glass vials at room temperature.
Reanalysis of the test article was performed during the study by the study laboratory using
GPC/RI by system B, and no degradation of the test article was detected.

F.2. Preparation and Analysis of Dose Formulations

The dose formulations were prepared approximately monthly by mixing chitosan with feed
(Table F-2). Dose formulations were stored in lined plastic buckets sealed with lids and stored at
−30°C to −15°C for up to 42 days.
Homogeneity studies of approximately 0.5% and 9% formulations (5,046 and 90,049 µg/g,
respectively) and stability studies of an approximately 0.5% (5,046 µg/g) formulation were
performed by the analytical chemistry laboratory using GPC/RI by system C (Table F-1). Two
peaks were attributed to chitosan with retention times of approximately 6.9 minutes and
12.1 minutes, respectively. Chitosan quantitation was based on the larger polymeric components
of the first peak only because vehicle components co-eluted with the later oligomeric peak.
Homogeneity studies of 1% and 9% dose formulations (10 mg/g and 90 mg/g in feed,
respectively) were performed by the study laboratory using GPC/RI by system B. Homogeneity
was confirmed, and stability was confirmed for at least 42 days for dose formulations stored in
lined plastic buckets sealed with lids at temperatures up to room temperature and for at least
7 days under simulated animal room conditions.
Periodic analyses of the dose formulations of chitosan were performed by the study laboratory
using GPC/RI by system B. Of the dose formulations analyzed, all nine were within 10% of the
target concentrations (Table F-3). Animal room samples of dose formulations were also
analyzed; all three were within 10% of the target concentrations.

Table F-1. Gel Permeation Chromatography Systems Used in the Six-month Feed Study of
Chitosana
Detection System Column Solvent System
System A
Refractive index In series: NOVEMA 10,000 Å, 0.25% Trifluoroacetic acid, isocratic,
300 mm × 8 mm, 10 µm and NOVEMA flow rate 1.0 mL/minute
3,000 Å, 50 mm × 8 mm (guard) and
NOVEMA 3,000 Å, 300 mm × 8 mm,
10 µm (Polymer Standards Service
GmbH, Mainz, Germany)
System B
Refractive index In series: BioSep-SEC-S2000 145 Å, 1% Trifluoroacetic acid, isocratic, flow
300 mm × 4.6 mm, 5 µm and BioSep- rate 0.35 mL/minute
SEC-S3000 290 Å, 300 mm × 4.6 mm,
5 µm (Phenomenex, Torrance, CA)
System C
Refractive index In series: Alltech® Macrosphere 100 Å, 1% Trifluoroacetic acid, isocratic, flow
250 mm × 4.6 mm, 7 µm and Alltech® rate 0.5 mL/minute
Macrosphere 300 Å, 250 mm × 4.6 mm,
7 µm (Grace, Columbia, MD)

F-3
 Chitosan, NTP TOX 93

aThe liquid chromatographs were manufactured by Waters Corporation (Milford, MA) (System A), Agilent (Palo Alto, CA)
(System B), or Perkin Elmer (Boston, MA) (System C).

Table F-2. Preparation and Storage of Dose Formulations in the Six-month Feed Study of Chitosan
Six-month Feed Study
Preparation
The appropriate amounts of chitosan and AIN-93M feed (87 kg for 1% and 3% formulations and 79 kg for the
9% formulation) were weighed in tared stainless steel buckets and layered into a Patterson-Kelly twin-shell
blender. The chitosan beaker was rinsed twice with portions of the blank feed, added to the blender, and the
formulation was mixed for 15 minutes. The dose formulations were prepared approximately monthly.
Chemical Lot Number
02-ASSF-0715
Maximum Storage Time
42 days
Storage Conditions
Stored in plastic-lined 5 gallon plastic buckets sealed with lids at −30° to −15°C
Study Laboratory
Battelle Columbus Operations (Columbus, OH)

Table F-3. Results of Analyses of Dose Formulations Administered to Rats in the Six-month Feed
Study of Chitosan
Determined Difference from
Target Concentrationa
Date Prepared Date Analyzed Concentrationb Target
(mg/g)
(mg/g) (%)
August 15, 2006 August 17–18, 2006 10 9.1 −10
30 27.3 −9
90 83.5 −7
c
October 2–3, 2006 10 9.99 0
30 30.3 +1
90 92.4 +3
October 10, 2006 October 11–12, 2006 10 9.5 −5
30 27.0 −10
90 94.2 +5
January 2, 2007 January 2–3, 2007 10 10.6 +6
30 29.5 −2
90 94.3 +5
a10,30, and 90 mg/g are equivalent to 1%, 3%, and 9% chitosan concentrations, respectively.
bResults of duplicate analyses.
cAnimal room samples.

F-4
 Chitosan, NTP TOX 93

Figure F-1. Infrared Absorption Spectrum of Chitosan

Figure F-2. Proton Nuclear Magnetic Resonance Spectrum of Chitosan

F-5
 Chitosan, NTP TOX 93

Appendix G. Feed and Compound Consumption in the Six-

month Feed Study of Chitosan

Tables
Table G-1. Feed and Compound Consumption by Group A Male Rats in the Six-month
Feed Study of Chitosan ............................................................................................ G-2
Table G-2. Feed and Compound Consumption by Group A Female Rats in the Six-month
Feed Study of Chitosan ............................................................................................ G-3

G-1
 Chitosan, NTP TOX 93

Table G-1. Feed and Compound Consumption by Group A Male Rats in the Six-month Feed Study
of Chitosan
0% 1% 3% 9%

Week Body Body Body Body

Feeda Feed Doseb Feed Dose Feed Dose
Weight Weight Weight Weight
(g/day) (g/day) (mg/kg) (g/day) (mg/kg) (g/day) (mg/kg)
(g) (g) (g) (g)
1 22.2 238 23.8 243 980 23.6 242 2,929 21.4 243 7,931
2 21.6 297 23.1 308 750 23.0 303 2,278 26.9 265 9,137
3 22.8 346 24.0 359 668 25.4 354 2,156 27.3 307 8,002
4 22.6 388 23.5 404 582 24.9 398 1,877 26.7 350 6,872
5 22.3 421 23.5 438 537 25.8 436 1,774 27.4 388 6,355
6 21.5 446 21.7 465 467 24.6 464 1,591 26.4 413 5,759
7 23.1 475 23.5 493 477 25.7 491 1,570 27.8 442 5,662
8 22.3 496 24.1 513 470 25.2 514 1,471 27.1 464 5,259
9 22.3 514 24.1 535 450 25.1 534 1,411 26.7 483 4,980
10 21.8 529 23.2 554 419 25.1 548 1,373 26.2 498 4,735
11 21.8 543 23.3 570 409 25.5 566 1,353 25.5 511 4,493
12 21.6 554 23.1 585 395 24.5 579 1,270 26.4 521 4,557
13 22.3 563 22.6 598 378 24.8 584 1,274 26.9 527 4,595
14 21.5 578 22.9 612 374 25.7 602 1,280 26.4 544 4,371
15 21.7 587 22.7 622 365 25.5 613 1,249 26.7 557 4,315
16 22.2 597 22.7 631 360 27.0 620 1,306 28.5 565 4,542
17 23.4 607 23.7 645 367 28.0 634 1,325 29.2 575 4,569
18 23.6 614 24.6 657 375 28.4 646 1,320 29.0 584 4,468
19 22.3 624 23.0 667 345 26.6 657 1,214 27.8 595 4,202
20 23.6 633 23.2 677 343 25.4 664 1,148 27.4 600 4,112
21 23.8 643 23.4 689 340 24.8 670 1,110 28.5 606 4,230
22 24.1 653 23.1 700 330 25.7 677 1,139 26.9 612 3,959
23 22.6 665 21.3 707 301 25.7 686 1,125 25.4 615 3,715
24 21.5 666 19.6 704 278 24.9 689 1,084 25.4 612 3,738
25 21.2 20.4 24.7 27.3
Mean for Weeks
1–13 22.2 447 23.3 466 537 24.9 462 1,717 26.4 416 6,026
14–24 22.8 624 22.7 665 343 26.2 651 1,209 27.4 588 4,202
aGrams of feed consumed per animal per day.
bMilligrams of chitosan consumed per kilogram body weight per day.

G-2
 Chitosan, NTP TOX 93

Table G-2. Feed and Compound Consumption by Group A Female Rats in the Six-month Feed
Study of Chitosan
0% 1% 3% 9%

Week Body Body Body Body Dose

Feeda Feed Doseb Feed Dose Feed
Weight Weight Weight Weight (mg/kg)
(g/day) (g/day) (mg/kg) (g/day) (mg/kg) (g/day)
(g) (g) (g) (g)
1 17.7 175 22.3 173 1,286 17.3 177 2,940 16.9 177 8,606
2 15.1 199 15.4 198 779 15.6 197 2,381 17.6 191 8,298
3 15.8 220 16.2 217 747 15.0 214 2,102 16.9 206 7,371
4 16.2 233 16.7 229 728 15.6 231 2,023 17.3 221 7,044
5 16.0 248 17.4 241 722 15.9 242 1,974 17.3 234 6,649
6 14.7 258 17.5 252 694 14.7 251 1,759 16.4 243 6,067
7 15.9 266 17.9 262 683 15.2 259 1,759 16.7 248 6,069
8 15.9 274 17.3 268 645 15.5 267 1,739 16.7 259 5,810
9 15.9 281 17.7 276 641 15.9 274 1,740 16.7 266 5,656
10 15.9 287 18.2 284 641 15.7 281 1,678 16.6 268 5,577
11 15.2 294 17.4 289 601 15.5 286 1,624 16.2 274 5,329
12 15.8 300 17.5 295 594 15.9 292 1,632 15.7 279 5,071
13 15.3 305 16.1 300 537 16.9 298 1,699 16.2 281 5,192
14 15.3 312 16.8 303 555 16.3 304 1,609 16.5 285 5,218
15 14.7 316 16.2 307 528 16.7 309 1,623 16.8 288 5,258
16 16.2 320 19.1 311 615 17.7 314 1,694 19.2 291 5,940
17 15.9 325 18.2 314 581 16.9 315 1,611 18.0 293 5,537
18 16.4 327 19.5 317 615 17.8 318 1,679 19.1 296 5,803
19 17.8 330 19.6 321 610 18.9 321 1,768 18.6 299 5,607
20 17.3 328 21.7 324 670 18.8 321 1,757 19.2 297 5,819
21 18.2 335 21.1 332 636 19.0 330 1,725 19.1 302 5,688
22 18.5 339 19.7 337 584 19.2 336 1,712 18.9 306 5,554
23 17.4 343 17.5 340 515 17.3 339 1,532 17.7 306 5,201
24 16.1 345 17.2 340 506 15.6 339 1,381 16.7 309 4,863
25 16.4 20.3 17.1 18.8
Mean for Weeks
1–13 15.8 257 17.5 253 715 15.7 251 1,927 16.7 242 6,364
14–24 16.7 329 18.8 322 583 17.7 322 1,645 18.2 297 5,499
aGrams of feed consumed per animal per day.
bMilligrams of chitosan consumed per kilogram body weight per day.

G-3
 Chitosan, NTP TOX 93

Appendix H. Ingredients and Nutrient Composition in AIN-

93M Maintenance Purified Diet

Tables
Table H-1. Ingredients of AIN-93M Maintenance Purified Rodent Diet ................................... H-2
Table H-2. Vitamins, Minerals, and Nutrient Composition of AIN-93M Maintenance
Purified Rodent Diet ................................................................................................ H-2

H-1
 Chitosan, NTP TOX 93

Table H-1. Ingredients of AIN-93M Maintenance Purified Rodent Diet

Ingredients Percent by Weight
Corn starch 46.5692
Dextrin 15.5000
Casein (vitamin free) 14.0000
Sucrose 10.0000
Powdered cellulose 5.0000
Soybean oil 4.0000
AIN-93M mineral mix 3.5000
AIN-93M vitamin mix 1.0000
Choline bitartrate 0.2500
L-Cystine 0.1800
t-Butylhydroquinone 0.0008

Table H-2. Vitamins, Minerals, and Nutrient Composition of AIN-93M Maintenance Purified
Rodent Diet
Amount
Vitamins
A 4.00 IU/g
D3 (added) 1.00 IU/g
E 78.80 IU/g
K (as menadione) 0.75 ppm
Thiamine hydrochloride 6.00 ppm
Riboflavin 6.50 ppm
Niacin 30.00 ppm
Pantothenic acid 16.00 ppm
Folic acid 2.10 ppm
Pyridoxine 5.80 ppm
Biotin 0.20 ppm
B12 28.00 mcg/kg
Choline chloride 1,250.00 ppm
Ascorbic acid 0.00 ppm
Minerals
Calcium 0.50%
Phosphorus 0.31%
Potassium 0.36%
Magnesium 0.05%

H-2
 Chitosan, NTP TOX 93

Amount
Sodium 0.13%
Chlorine 0.20%
Fluorine 1.00 ppm
Iron 39.00 ppm
Zinc 35.00 ppm
Manganese 11.00 ppm
Copper 6.00 ppm
Cobalt 0.00 ppm
Iodine 0.21 ppm
Chromium 1.00 ppm
Molybdenum 0.14 ppm
Selenium 0.22 ppm
Typical Analysis
Protein 13.06%
Fat 4.00%
Fiber 5.00%
Carbohydrate 73.80%
Metabolizable energy 3.83%

H-3
 Chitosan, NTP TOX 93

Appendix I. Sentinel Animal Program

Table of Contents
I.1. Methods .................................................................................................................................. I-2
I.2. Results .................................................................................................................................... I-3

Tables
Table I-1. Laboratory Methods and Agents Tested for in the Sentinel Animal Program ............. I-2

I-1
 Chitosan, NTP TOX 93

I.1. Methods

Rodents used in the National Toxicology Program are produced in optimally clean facilities to
eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part
of the periodic monitoring of animal health that occurs during the toxicological evaluation of test
compounds. Under this program, the disease state of the rodents is monitored via sera from extra
(sentinel) animals in the study rooms. The sentinel animals and the study animals are subject to
identical environmental conditions. Furthermore, the sentinel animals come from the same
production source and weanling groups as the animals used for the studies of test compounds.
Blood samples were collected from each rat and allowed to clot and the serum was separated. All
samples were processed appropriately and tested for the presence of pathogens at BioReliance
Corporation (Rockville, MD) or the Research Animal Diagnostic Laboratory (RADIL),
University of Missouri, Columbia, MO. The laboratory methods and agents for which testing
was performed are tabulated below; the times at which samples were collected during the studies
are also listed.
Blood was collected from five rats per sex per time point, except at study termination when
blood was collected from four males and five females.

Table I-1. Laboratory Methods and Agents Tested for in the Sentinel Animal Program
Method and Test Time of Collection
ELISA
Kilham rat virus (KRV) 4 weeks
Pneumonia virus of mice (PVM) End of quarantine, 4 weeks, study termination
Rat coronavirus/sialodacryoadenitis virus (RCV/SDA) End of quarantine, 4 weeks, study termination
Rat parvovirus (RPV) 4 weeks
Sendai End of quarantine, 4 weeks, study termination
Toolan’s H-1 virus (H-1) 4 weeks
Immunofluorescence Assay
H-1 4 weeks
KRV 4 weeks
Parvovirus End of quarantine, 4 weeks, 6 weeks, study termination
RCV/SDA End of quarantine
RPV 4 weeks
Multiplex Fluorescent Immunoassay
H-1 6 weeks
KRV 6 weeks
Parvo NS-1 6 weeks
Rat minute virus 6 weeks
RPV 6 weeks

I-2
 Chitosan, NTP TOX 93

I.2. Results

A positive test result for parvovirus occurred in one animal at the 4-week timepoint; additional
testing of serum from this animal and other sentinel animals via other testing methodologies
deemed the original positive result to be a false positive. All other test results were negative for
rodent pathogens.

I-3
National Toxicology Program
NTP Central Data Management, MD EC-03
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park, NC 27709

http://ntp.niehs.nih.gov

ISSN 2378-8992