INTERNATIONAL COUNCIL FOR HARMONISATION OF TECHNICAL

REQUIREMENTS FOR PHARMACEUTICALS FOR HUMAN USE

ICH HARMONISED GUIDELINE

TESTING FOR CARCINOGENICITY OF

PHARMACEUTICALS
S1B(R1)

Final version

Adopted on 4 August 2022

This Guideline has been developed by the appropriate ICH Expert Working Group and has been subject
to consultation by the regulatory parties, in accordance with the ICH Process. At Step 4 of the Process
the final draft is recommended for adoption to the regulatory bodies of ICH regions.
 ICH S1B(R1)
Document History

New
First Codification
History Date
Codification November
2005
S1B Approval by the Steering Committee under 1 S1B
Step 2 and release for public consultation. May
1996

S1B Approval by the Steering Committee under 16 S1B

Step 4 and recommendation for adoption to July
the three ICH regulatory bodies. 1997

Current Step 4 version of the S1B(R1)

Code History Date

S1B(R1)* Endorsement by the Members of the ICH Assembly 10 May 2021
under Step 2 and release for public consultation.

S1B(R1)* Endorsement by the Regulatory Members of the ICH 4 August 2022

Assembly under Step 4 and integration with the S1B
Guideline.

*This addendum is complementary to the S1 Guidelines (S1A, S1B and S1C(R2)) and is not
intended to replace the existing S1B Guideline.

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 TESTING FOR CARCINOGENICITY OF PHARMACEUTICALS

TABLE OF CONTENTS

Part I:
TESTING FOR CARCINOGENICITY OF PHARMACEUTICALS

1. OBJECTIVE ................................................................................................................ 1
2. BACKGROUND........................................................................................................... 1
3. SCOPE OF THE GUIDELINE ................................................................................... 2
4. THE GUIDELINE........................................................................................................ 2
4.1 Preamble ......................................................................................................................... 2
4.2 Experimental approaches to testing for carcinogenic potential .....................................2
4.2.1 Choice of species for a long-term carcinogenicity study .....................................2
4.2.2 Additional in vivo tests for carcinogenicity ......................................................... 3
4.2.3 Considerations in the choice of short or medium term tests
for carcinogenicity. ............................................................................................. 3
5. MECHANISTIC STUDIES ........................................................................................ 3
5.1 Cellular changes ............................................................................................................ 3
5.2 Biochemical measurements ........................................................................................... 3
5.3 Considerations for additional genotoxicity testing........................................................ 4
5.4 Modified protocols ........................................................................................................ 4
6. GENERAL CONSIDERATIONS IN THE CHOICE OF AN APPROPRIATE
SPECIES FOR LONG TERM CARCINOGENICITY TESTING ....................... 4
6.1 Information from surveys on pharmaceuticals .............................................................. 4
6.2 Potential to study mechanisms. ..................................................................................... 4
6.3 Metabolic disposition ....................................................................................................5
6.4 Practicality ..................................................................................................................... 5
6.5 Testing in more than one species. ................................................................................. 5
6.6 Exceptions. .................................................................................................................... 5
7. EVALUATION OF CARCINOGENIC POTENTIAL ............................................ 5
NOTES ..................................................................................................................................6
ANNEX: OTHER ICH GUIDELINES CITED.................................................................7

i
Part II:
ADDENDUM TO TESTING FOR CARCINOGENICITY FOR
PHARMACEUTICALS

PREAMBLE ......................................................................................................................... 8
1. INTRODUCTION ...................................................................................................... 8
1.1 Scope of the Addendum ............................................................................................... 8
1.2 Purpose of the Addendum ............................................................................................ 8
1.3 Background .................................................................................................................. 8
2. A WEIGHT OF EVIDENCE APPROACH TO ASSESS THE HUMAN
CARCINOGENIC POTENTIAL OF PHARMACEUTICALS .............................. 9
2.1 Factors to Consider for a WoE Assessment................................................................ 10
2.2 Integration of WoE Factors for Assessing Human Carcinogenic Risk ...................... 11
2.3 Mouse Carcinogenicity Studies.................................................................................. 13
3. CLARIFICATION ON CRITERIA FOR HIGH DOSE SELECTION BASED ON
EXPOSURE FOR RASH2-TG MOUSE CARCINOGENICITY STUDIES ....... 13
REFERENCES ................................................................................................................... 14
APPENDIX: CASE STUDIES APPLYING THE WEIGHT OF EVIDENCE
APPROACH ....................................................................................................................... 16
 Part I:

TESTING FOR CARCINOGENICITY OF PHARMACEUTICALS

ICH Harmonised Tripartite Guideline

Having reached Step 4 of the ICH Process at the ICH Steering Committee meeting on
16 July 1997, this guideline is recommended for adoption
to the three regulatory parties to ICH

1. OBJECTIVE
This document provides guidance on approaches for evaluating the carcinogenic potential of
pharmaceuticals.

2. BACKGROUND
Historically, the regulatory requirements for the assessment of the carcinogenic potential of
pharmaceuticals in the three regions (E.U., Japan, U.S.) provided for the conduct of long-term
carcinogenicity studies in two rodent species, usually the rat and the mouse. Given the cost of
these studies and their extensive use of animals, it is in keeping with the mission of ICH to
examine whether this practice requiring long term carcinogenicity studies in two species could
be reduced without compromising human safety.
This guideline should be read in conjunction with other guidelines (see Annex), especially:
S1.A: Guideline on the Need for Carcinogenicity Studies of Pharmaceuticals.
S1.C: Dose Selection for Carcinogenicity Studies of Pharmaceuticals.
Long-term rodent carcinogenicity studies for assessing the carcinogenic potential of chemicals
(including pharmaceuticals) to humans are currently receiving critical examination. Since the
early 1970's, many investigations have shown that it is possible to provoke a carcinogenic
response in rodents by a diversity of experimental procedures, some of which are now
considered to have little or no relevance for human risk assessment. This guideline outlines
experimental approaches to the evaluation of carcinogenic potential that may obviate the
necessity for the routine conduct of two long-term rodent carcinogenicity studies for those
pharmaceuticals that need such evaluation. The relative individual contribution of rat and
mouse carcinogenicity studies and whether the use of rats or mice alone would result in a
significant loss of information on carcinogenicity relevant to human risk assessment has been
addressed by six surveys of the data for human pharmaceuticals. The surveys were those of the
International Agency for Research on Cancer (IARC), the U.S. Food and Drug Administration
(FDA), the U.S. Physicians’ Desk Reference (PDR), the Japanese Pharmaceutical
Manufacturers’ Association (JPMA), the EU Committee for Proprietary Medicinal Products
(CPMP), and the UK Centre for Medicines Research (CMR). The dimensions of these surveys
and the principal conclusions of the analyses can be found in the Proceedings of the Third
International Conference (1995) on Harmonization.
Positive results in long-term carcinogenicity studies that are not relevant to the therapeutic use
of a pharmaceutical present a dilemma to all parties: regulatory reviewers, companies

1
developing drugs and the public at large. The conduct of one long-term carcinogenicity study
(rather than two long term studies) would, in part, allow resources to be diverted to other
approaches to uncover potential carcinogenicity relevant to humans. A “weight of evidence”
approach, that is use of scientific judgment in evaluation of the totality of the data derived from
one long-term carcinogenicity study along with other appropriate experimental investigations,
enhances the assessment of carcinogenic risk to humans.

3. SCOPE OF THE GUIDELINE

The guideline embraces all pharmaceutical agents that need carcinogenicity testing as indicated
in Guideline S1A. For biotechnology-derived pharmaceuticals refer to Guideline S6.

4. THE GUIDELINE

4.1 Preamble
The strategy for testing the carcinogenic potential of a pharmaceutical is developed only after
the acquisition of certain key units of information, including the results of genetic toxicology
(Guidelines S2A and S2B), intended patient population, clinical dosage regimen (Guideline
S1A), pharmacodynamics in animals and in humans (selectivity, dose-response) (Guideline
S1C), and repeated-dose toxicology studies. Repeated-dose toxicology studies in any species
(including nonrodents) may indicate that the test compound possesses immunosuppressant
properties, hormonal activity, or other activity considered to be a risk factor for humans, and
this information should be considered in the design of any further studies for the assessment of
carcinogenic potential (see also Note 1).

4.2 Experimental approaches to testing for carcinogenic potential

Flexibility and judgment should be exercised in the choice of an approach which should be
influenced by the information cited in the above preamble. Given the complexity of the process
of carcinogenesis, no single experimental approach can be expected to predict the carcinogenic
potential of all pharmaceuticals for humans.

The basic principle:

The basic scheme comprises one long-term rodent carcinogenicity study, plus one other study
of the type mentioned in §4.2.2 that supplements the long term carcinogenicity study and
provides additional information that is not readily available from the long term assay.

4.2.1 Choice of species for a long-term carcinogenicity study

The species selected should be appropriate, based on considerations that include the
following:
(a) Pharmacology.
(b) Repeated-dose toxicology.
(c) Metabolism (see also Guidelines S1C and S3A).
(d) Toxicokinetics (see also Guidelines S1C, S3A, and S3B).
(e) Route of administration (e.g., less common routes such as dermal and inhalation).
In the absence of clear evidence favoring one species, it is recommended that the rat be
selected. This view is based on the factors discussed in §6.

2
4.2.2 Additional in vivo tests for carcinogenicity
Additional tests may be either (a) or (b) (see Note 2).
(a) Short or medium-term in vivo rodent test systems.
Possibilities should focus on the use of in vivo models providing insight into
carcinogenic endpoints. These may include models of initiation-promotion in
rodents, or models of carcinogenesis using transgenic or neonatal rodents (Note 3).
(b) A long-term carcinogenicity study in a second rodent species is still considered
acceptable (see § 4.2.1 for considerations).

4.2.3 Considerations in the choice of short or medium term tests for carcinogenicity.
Emphasis should be placed on selection of a test method that can contribute information
valuable to the overall “weight of evidence” for the assessment of carcinogenic potential.
The rationale for this choice should be documented and based on information available
at the time of method selection about the pharmaceutical such as pharmacodynamics and
exposure compared to human or any other information that may be relevant. This
rationale should include a scientific discussion of the strengths and weaknesses of the
method selected for the pharmaceutical(see Note 4).

5. MECHANISTIC STUDIES
Mechanistic studies are often useful for the interpretation of tumor findings in a carcinogenicity
study and can provide a perspective on their relevance to human risk assessment. The need for
or the design of an investigative study will be dictated by the particular properties of the drug
and/or the specific results from the carcinogenicity testing. Dose dependency and the
relationship to carcinogenicity study conditions should be evaluated in these investigational
studies. Suggestions include:

5.1 Cellular changes

Relevant tissues may be examined for changes at the cellular level using morphological,
histochemical, or functional criteria. As appropriate, attention may be directed to such changes
as the dose-relationships for apoptosis, cell proliferation, liver foci of cellular alteration, or
changes in intercellular communication.

5.2 Biochemical measurements

Depending on the putative mode of tumorigenic action, investigations could involve
measurements of:
• plasma hormone levels, e.g. T3/T4, TSH, prolactin
• growth factors
• binding to proteins such as 2-globulin
• tissue enzyme activity, etc.
In some situations, it may be possible to test a hypothesis of, for example, a hormone imbalance
with another study in which the imbalance has been, at least in part, compensated.

3
5.3 Considerations for additional genotoxicity testing
(see Guidelines S2A and S2B)
Additional genotoxicity testing in appropriate models may be invoked for compounds that were
negative in the standard test battery but which have shown effects in a carcinogenicity test with
no clear evidence for an epigenetic mechanism. Additional testing can include modified
conditions for metabolic activation in in vitro tests or can include in vivo tests measuring
genotoxic damage in target organs of tumor induction (e.g., DNA damage and repair tests, 32P-
postlabeling, mutation induction in transgenes).

5.4 Modified protocols

Modified protocols may be helpful to clarify the mode of tumorigenic action of the test
substance. Such protocols might include groups of animals to explore, for example, the
consequence of interrupted dosage regimens, or the reversibility of cellular changes after
cessation of dosing.

6. GENERAL CONSIDERATIONS IN THE CHOICE OF AN APPROPRIATE

SPECIES FOR LONG TERM CARCINOGENICITY TESTING
There are several general considerations which, in the absence of other clear indications,
suggest that the rat will normally be the species of choice for a long term carcinogenicity study.

6.1 Information from surveys on pharmaceuticals

In the six analyses, attention was given to data on genetic toxicology, tumor incidence, strain
of animal, route and dosage regimen, pharmacological or therapeutic activity, development
and/or regulatory status, and, if relevant, reason for termination of development. Inevitably,
there was considerable overlap of the data, but that is not necessarily an impediment to drawing
valid conclusions.
The main overall conclusions from the analysis were:
a. Although very few instances have been identified of mouse tumors being the sole reason
for regulatory action concerning a pharmaceutical, data from this species may have
contributed to a “weight of evidence” decision and in identifying agents that caused
tumors in two rodent species.
b. Of the compounds displaying carcinogenic activity in only one species, the number of
"rat-only" compounds was about double the number of "mouse-only" compounds,
implying in a simplistic sense that the rat is more "sensitive" than the mouse.
c. As with other surveys accessible in the literature, the data for pharmaceuticals were
dominated by the high incidence of rodent liver tumors. The high susceptibility of mouse
liver to nongenotoxic chemicals has been the subject of many symposia and workshops.
These have concluded that these tumors may not always have relevance to carcinogenic
risk in humans and can potentially be misleading.

6.2 Potential to study mechanisms

The carcinogenic activity of nongenotoxic chemicals in rodents is characterized by a high
degree of species, strain, and target organ specificity and by the existence of thresholds in the
dose-response relationship. Mechanistic studies in recent years have permitted the distinction
between effects that are specific to the rodent model and those that are likely to have relevance
for humans. Progress has often been associated with increased understanding of species and

4
tissue specificity. For example, receptor-mediated carcinogenesis is being recognized as of
growing importance. Most of these advances are being made in the rat, and only rarely in the
mouse.

6.3 Metabolic disposition

Neither rats nor mice would seem, on metabolic grounds, to be a priori generally more suitable
for the conduct of long term carcinogenicity studies. However, much attention is now being
given to pharmacokinetic-pharmacodynamic relationships and rapid progress is occurring in
knowledge of the P-450 isozymes that mediate the biotransformation of drugs. Most of this
research activity is confined to rats and humans. Therefore, in the near future at least, where
specific information on the P-450 isozymes involved in biotransformation is critical for the
evaluation it appears that mice would be less likely to provide this mechanistic information.

6.4 Practicality
Pertinent to the above two topics is the question of feasibility of investigative studies. Size
considerations alone put the mouse at a severe disadvantage when it comes to the taking of
serial blood samples, microsurgery/catheterization, and the weighing of organs. Blood
sampling often requires the sacrifice of the animals, with the result that many extra animals
may be needed when mice are subject to such investigations.

6.5 Testing in more than one species

Most of the currently available short and medium term in vivo models for carcinogenicity
testing involve the use of mice. In order to allow testing in more than one species for
carcinogenic potential, when this is considered important and appropriate, the rat will often be
used in the long term carcinogenicity study.

6.6 Exceptions
Despite the above considerations, there may be circumstances under which the mouse or
another rodent species could be justified on mechanistic, metabolic, or other grounds as being
a more appropriate species for the long term carcinogenicity study for human risk assessment
(c.f. §4.2.1). Under such circumstances it may still be acceptable to use the mouse as the short
term or medium term model.

7. EVALUATION OF CARCINOGENIC POTENTIAL

Evidence of tumorigenic effects of the drug in rodent models should be evaluated in light of the
tumor incidence and latency, the pharmacokinetics of the drug in the rodent models as
compared to humans, and data from any ancillary or mechanistic studies that are informative
with respect to the relevance of the observed effects to humans.
The results from any tests cited above should be considered as part of the overall “weight of
evidence” taking into account the scientific status of the test systems.

5
Notes
Note 1. Data from in vitro assays, such as a cell transformation assay, can be useful at the
compound selection stage.
Note 2. If the findings of a short or long-term carcinogenicity study and of genotoxicity tests
and other data indicate that a pharmaceutical clearly poses a carcinogenic hazard to
humans, a second carcinogenicity study would not usually be useful.
Note 3. Several experimental methods are under investigation to assess their utility in
carcinogenicity assessment. Generally, the methods should be based on mechanisms
of carcinogenisis that are believed relevant to humans and applicable to human risk
assessment. Such studies should supplement the long term carcinogenicity study and
provide additional information that is not readily available from the long term assay.
There should also be consideration given animal numbers, welfare and the overall
economy of the carcinogenic evaluation process. The following is a representative list
of some approaches that may meet these criteria and is likely to be revised in the light
of further information.
(a) The initiation-promotion model in rodent. One initiation-promotion model for the
detection of hepatocarcinogens (and modifiers of hepatocarcinogenicity) employs
an initiator, followed by several weeks of exposure to the test substance. Another
multi-organ carcinogenesis model employs up to five initiators followed by
several months of exposure to the test substance.
(b) Several transgenic mouse assays including the p53+/- deficient model, the Tg.AC
model, the TgHras2 model, the XPA deficient model, etc.
(c) The neonatal rodent tumorigenicity model.
Note 4. While there may be a number of approaches that will in general meet the criteria
described in Note 3 for use as the additional in vivo study, not all may be equally
suitable for a particular pharmaceutical. The following are examples of factors that
should be considered and addressed in the rationale:
1. Can results from the model provide new information not expected to be available
from the long-term study that is informative with respect to hazard identification
and/or risk assessment?
2. Can results from the model address concerns related to the carcinogenic process
arising from prior knowledge of the pharmaceutical or compounds with similar
structures and/or mechanisms of action? These concerns may include genotoxic,
mitogenic, promotional, or receptor-mediated effects, etc.
3. Does the metabolism of the pharmaceutical shown in the animal model affect the
evaluation of carcinogenic risk for humans?
4. Is adequate systemic or local exposure attained in relation to human exposure?
5. How extensively has the model been evaluated for its intended use? Prior to using
any new in vivo methods in testing the carcinogenic potential of pharmaceuticals
for humans, it is critical that the method be evaluated for its ability to contribute to
the weight of evidence assessment. Many experimental studies are in progress
(1997) to evaluate the new short or medium tests for carcinogenic potential. These
include selected pharmaceuticals with known potencies and known mechanism of
carcinogenic activity in rodents, and also putative human non-carcinogens. When
the results of these studies become available, it may be possible to offer clearer

6
 guidance on which of these tests have the most relevance for cancer assessment in
humans.

ANNEX: Other ICH Guidelines Cited

Guideline S2A: Notes for Guidance on Specific Aspects of Regulatory Genotoxicity Tests.
Guideline S2B: A Standard Battery of Genotoxicity Testing of Pharmaceuticals.
Guideline S3A: Notes for Guidance on Toxicokinetics. The Assessment of Systemic
Exposure in Toxicity Studies.
Guideline S3B: Guidance on Repeat-Dose Tissue Distribution Studies.
Guideline S6: Preclinical Testing of Biotechnology-derived Pharmaceuticals.

7
 Part II:
ADDENDUM TO TESTING FOR CARCINOGENICITY FOR PHARMACEUTICALS

PREAMBLE
This Addendum is to be used in close conjunction with ICH S1A Guideline on the Need for
Carcinogenicity Studies of Pharmaceuticals, S1B Testing for Carcinogenicity of
Pharmaceuticals, and S1C(R2) Dose Selection for Carcinogenicity Studies. The Addendum is
complementary to the S1 Guidelines.

1. INTRODUCTION

1.1 Scope of the Addendum

This Addendum applies to all pharmaceuticals that need carcinogenicity testing as described in
Guideline S1A. For biotechnology-derived pharmaceuticals, refer to Guideline S6(R1)
Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals.

1.2 Purpose of the Addendum

This Addendum expands the evaluation process for assessing human carcinogenic risk of
pharmaceuticals by introducing an additional approach that is not described in the original S1B
Guideline. This is an integrative approach that provides specific weight of evidence (WoE)
criteria that inform whether or not a 2-year rat study is likely to add value to a human
carcinogenicity risk assessment. The Addendum also adds a plasma exposure ratio-based
approach for setting the high dose in the rasH2-Tg mouse model,1 while all other aspects of the
recommendations for high dose selection in S1C(R2) Guideline still apply.
Application of this integrative approach reduces the use of animals in accordance with the 3R
(reduce/refine/replace) principles and shifts resources to focus on generating more scientific
mechanism-based carcinogenicity assessments, while continuing to promote safe and ethical
development of new pharmaceuticals.

1.3 Background
While the S1B Guideline calls for flexibility in considering approaches to address
pharmaceutical carcinogenicity testing, the basic paradigm generally recommends a long-term
rodent study which, in practice, is usually a 2-year study in rats, along with a second rodent
carcinogenicity study in mice (2-year or short-term study). Since publication of the ICH S1B

1
The rasH2-Tg mouse was developed in the laboratory of Tatsuji Nomura of the Central Institute for Experimental
Animals (1). The model is referred to in the S1B Guideline as the TgHras2 transgenic mouse. The official
nomenclature for the model is CByB6F1-Tg(HRAS)2Jic which is maintained by intercrossing C57BL/6JJic-
Tg(HRAS)2Jic hemizygous male mice with BALB/cByJJic female mice. The littermates derived from these
intercrosses are the transgenic rasH2-Tg mice with the tg/wt genotype, and the wild type rasH2-Wt mice with a
wt/wt genotype.
Since other short-term models mentioned in S1B have not gained significant use compared to rasH2-Tg mouse
over the past 20 years, pharmaceutical development experience with these models is far more limited. Therefore,
other short-term carcinogenicity models referred to in S1B would not qualify for a plasma exposure ratio-based
high dose selection.
It is appropriate to use wild-type rasH2-Wt littermates of rasH2-Tg mice for dose range-finding studies and for
generating exposure data.

8
Guideline, scientific advances toward elucidation of mechanisms of carcinogenicity, greater
understanding of the limitations of rodent models, and several retrospective analyses of
pharmaceutical datasets indicate that 2-year rat carcinogenicity studies might not add value to
human carcinogenicity risk assessment in some cases and the carcinogenic potential could have
been assessed adequately based on a comprehensive assessment of all available
pharmacological, biological, and toxicological data (2-9).
To determine whether the conclusions from these retrospective analyses could be confirmed in
a real-world setting (i.e., prior to knowledge of the 2-year rat carcinogenicity study outcomes),
a subsequent international prospective study was conducted under ICH S1(R1) Proposed
Change to Rodent Carcinogenicity Testing of Pharmaceuticals – Regulatory Notice Document.
The process and several status updates reporting results are posted and available at the ICH
website (10-14). Carcinogenicity assessment documents (CADs) and associated data from 2-
year rat carcinogenicity studies for 45 compounds were received and evaluated by regulatory
members of the ICH EWG. The conclusion from this prospective evaluation confirmed that an
integrated WoE approach could be used to adequately assess the human carcinogenic risk for
certain pharmaceuticals in lieu of conducting a 2-year rat study.2
In addition, an exposure ratio endpoint based on animal to human plasma Area Under the Curve
(AUC) for high dose selection in 2-year rodent studies as per ICH S1C(R2) has not been
globally accepted for use in the rasH2-Tg mouse study. Therefore, a comprehensive analysis
was conducted to assess exposures and outcomes in rasH2-Tg mouse studies from available
information (15). As described in Section 3, the results of this analysis indicate that a 50-fold
plasma AUC exposure ratio (rodent:human) is an adequate criterion for high dose selection.

2. A WEIGHT OF EVIDENCE APPROACH TO ASSESS THE HUMAN

CARCINOGENIC POTENTIAL OF PHARMACEUTICALS
Over the course of drug development, it is important for sponsors to develop a scientifically
robust strategy for carcinogenicity assessment that considers key biologic, pharmacologic, and
toxicologic information.
The integrative WoE assessment approach described in Sections 2.1 and 2.2 may support a
conclusion that the carcinogenic potential of the pharmaceutical in humans is:
• likely, such that a 2-year rat carcinogenicity study would not add value; or
• unlikely, such that a 2-year rat carcinogenicity study would not add value3; or
• uncertain, such that a 2-year rat carcinogenicity study would add value to human risk
assessment.

In cases where the WoE assessment leads to a conclusion of uncertainty regarding human
carcinogenicity potential, the approach described in S1B of conducting a long-term
carcinogenicity study together with an additional in vivo carcinogenicity study remains the most
appropriate strategy (Figure 1).

2
Methods and results of the ICH S1 prospective evaluation study will be summarized in a future publication.
3
A WoE assessment may indicate that a compound is likely to be carcinogenic in rats. The compound may not be
considered carcinogenic in humans if there is sufficient evidence that the mechanism of carcinogenicity is
irrelevant to humans.

9
Figure 1: Flow scheme outlining key steps and options in developing a carcinogenicity
assessment strategy and determining the added value of a 2-year rat study. Note that key
biologic, pharmacologic, and toxicologic information should be assessed even when taking the
ICH S1B approach that utilizes a 2-year rat study. When a sponsor decides to conduct a 2-year
rat study in accordance with ICH S1B, there is no obligation to seek concurrence with the Drug
Regulatory Agency (DRA). Refer to Sections 2.1 and 2.2 for additional detail.

2.1 Factors to Consider for a WoE Assessment

A WoE approach is based on a comprehensive assessment of the totality of data relevant to
carcinogenic potential available from public sources and from relevant drug development
studies. These factors include, but are not limited to:
1) data that inform carcinogenic potential based on drug target biology and the primary
pharmacologic mechanism of the parent compound and major human metabolites; this
includes drug target distribution in rats and humans along with the pharmacologic
activity and potency of the parent compound and major metabolites in these species;
available information from genetically engineered models; human genetic association
studies; cancer gene databases; and carcinogenicity information on class effects, if
available,
2) results from secondary pharmacology screens for the parent compound and major
metabolites that inform selectivity and off-target potential, especially those that inform
carcinogenic risk (e.g., binding to nuclear receptors),

10
 3) histopathology data4 from repeated-dose toxicity studies completed with the compound,
with particular emphasis on the 6-month rat study, including plasma exposure margin
assessments of parent drug and major metabolites,
4) evidence for hormonal perturbation5, including knowledge of drug target and
compensatory endocrine response mechanisms; weight, gross and microscopic changes
in endocrine and reproductive organs from repeated-dose toxicity studies; and relevant
results from reproductive toxicology studies, if available,
5) genetic toxicology study data using criteria from ICH S2(R1) Genotoxicity Testing and
Data Interpretation for Pharmaceuticals Intended for Human Use; equivocal genotoxicity
data that cannot be resolved in accordance with ICH S2(R1) recommendations increases
uncertainty with respect to the carcinogenic potential,
6) evidence of immune modulation in accordance with ICH S8 Immunotoxicity Studies for
Human Pharmaceuticals. Evidence of broad immunosuppression may provide sufficient
concern for human risk that would not be further informed by standard rat and mouse
carcinogenicity studies (16,17).

The above WoE factors may be sufficient to conclude whether or not a 2-year rat study would
add value to the assessment of human carcinogenic risk. However, where one or more WoE
factors may be inconclusive or indicate a concern for carcinogenicity, the sponsor can apply
investigative approaches that could address the uncertainty or inform human relevance of the
identified risk. Possible approaches may include, but are not limited to:
1) additional investigative studies or analyses of specimens collected from prior studies (e.g.,
special histochemical stains, molecular biomarkers, serum hormone levels, immune cell
function, in vitro or in vivo test systems, data from emerging technologies), and
2) clinical data generated to inform human mechanistic relevance at therapeutic doses and
exposures (e.g., urine drug concentrations and evidence of crystal formation, targeted
measurements of clinical plasma hormonal alterations, human imaging data).

A rasH2-Tg mouse study is not expected to be completed to support a WoE assessment.

However, if rasH2-Tg mouse study results are available, they should be included in the WoE
document.

2.2 Integration of WoE Factors for Assessing Human Carcinogenic Risk

An integrated analysis of the WoE factors described above should be used to determine whether
or not a 2-year rat study would contribute to the human carcinogenic risk assessment. While all

4
Histopathology findings from 6-month rat toxicity studies of particular interest for identifying carcinogenic
potential in a 2-year rat study include cellular hypertrophy, cellular hyperplasia, persistent tissue injury and/or
chronic inflammation, foci of cellular alteration, preneoplastic changes, and tumors. It is important to provide an
understanding of the likely pathogenesis, and/or address the human relevance of such findings. While the 6-month
rat toxicity study is the primary study to be used for assessing the likely outcome and value of conducting a 2-year
rat study, shorter-term rat studies can sometimes also provide histopathologic conclusions of value. Data from
long-term toxicity studies in non-rodents and mice may also be useful for providing additional context on the
human relevance of rat study findings (e.g., species-specific mechanistic differences) and whether there is value
in conducting a 2-year rat study.
5
Findings from rat toxicity studies suggesting hormonal perturbation may include microscopic changes in
endocrine or reproductive tissues of atrophy, hypertrophy, and hyperplasia and/or biologically significant
endocrine and reproductive organ weight changes which are not explained as findings secondary to processes such
as stress or altered body weight. Changes of this nature may be considered evidence of functional hormonal
perturbation even when changes in hormone levels are not documented. Such findings may be suggestive of
potential carcinogenic risk unless investigated for human relevance and demonstrated otherwise.

11
factors will contribute to the integrated analysis, the relative importance of each factor will vary
depending on the compound being considered (Figure 2).

Figure 2: Integration of key WoE factors and potential investigative approaches to further
inform on the value of conducting a 2-year rat study for assessment of human carcinogenic risk.
When all WoE attributes align towards the right side of the figure, a conclusion that a 2-year
rat study would not add value is more likely. Note that for the genotoxicity WoE factor a 2-year
rat study is less likely to be of value either in cases where there is no genotoxicity risk or in
cases with unequivocal genotoxicity risk. Similarly, for the immune modulation WoE factor, a
2-year rat study is less likely to be of value in cases where there are either no effects on the
immune system or in cases where there is broad immunosuppression.

A summary of key outcomes and examples based on the experience accrued during the ICH S1
study (S1(R1) Proposed Change to Rodent Carcinogenicity Testing of Pharmaceuticals –
Regulatory Notice Document) are provided in the Appendix, demonstrating how the WoE
factors could be integrated to determine the value of conducting a 2-year rat study for
assessment of human carcinogenic risk.
Experience from the ICH S1 study indicates that an established profile of other compound(s)
in a drug class contributes substantially to assessing human carcinogenic risk associated with
modulation of the pharmacologic target. Compounds with novel drug targets (i.e., first-in-class)
are, nevertheless, considered eligible for an integrative WoE assessment. For such compounds,
further evidence that there is no cause-for-concern in regard to target biology is needed to
compensate for the lack of precedent. Case 4 in the Appendix describes an example for a novel
target where a 2-year rat study was not considered to add value given sufficient evidence to
compensate for the lack of precedent. In this example, a cause-for-carcinogenic-concern was

12
not identified regarding drug target biology or compound selectivity, and no proliferative
changes in any organs or tissues were observed at a high multiple of exposure in the 6-month
study in rats (a pharmacologically relevant species).
When the WoE assessment supports a conclusion that conduct of a 2-year rat study does not
add value to the assessment of human carcinogenic risk, the sponsor should seek consultation
with the applicable DRA in accordance with the established regulatory consultation procedure
for that region. When a sponsor decides to conduct a 2-year rat study in accordance with ICH
S1B, there is no obligation to seek consultation with the DRA.

2.3 Mouse Carcinogenicity Studies

A carcinogenicity study in mice, either a 2-year study in a standard strain of mice or a short-
term study in a transgenic model as in ICH S1B, remains a recommended component of a
carcinogenicity assessment plan, even for those compounds for which the WoE assessment
indicates a 2-year rat study would not contribute significant value. Use of a transgenic model is
consistent with the 3R (reduce/refine/replace) principles and this model should be prioritized
unless there is a scientific rationale for conducting a 2-year study in mice.
There are cases where it may not be appropriate to conduct a mouse carcinogenicity study. As
one example, a mouse study may not be appropriate when the WoE evaluation strongly
indicates no carcinogenic risk to humans and the data indicate that only subtherapeutic and
pharmacologically inactive drug levels relative to human exposure can be achieved in the
mouse. As an additional example, when the WoE assessment indicates that a compound is likely
to be carcinogenic in humans, the conduct of a mouse study may not be appropriate.

3. CLARIFICATION ON CRITERIA FOR HIGH DOSE SELECTION BASED ON

EXPOSURE FOR RASH2-TG MOUSE CARCINOGENICITY STUDIES
A plasma exposure (AUC) ratio for high dose selection in the absence of dose limiting toxicity
or other criteria as outlined in ICH S1C(R2) has not been globally accepted as a dose-setting
criterion in the rasH2-Tg mouse model. A retrospective evaluation of available data from 53
compounds tested in this model determined that detection of compound-related tumors emerged
in all cases within a systemic rodent-to-human exposure ratio up to 50-fold (15). Based on this
analysis, it was concluded that a 50-fold plasma exposure ratio (rodent:human) is an adequate
criterion for high dose selection. Therefore, all criteria for selection of the high dose as specified
in S1C(R2) for 2-year rodent carcinogenicity studies are applicable to rasH2-Tg mice, including
a plasma exposure ratio, except that the plasma exposure ratio will be 50-fold in rasH2-Tg mice
rather than 25-fold as for 2-year studies conducted in standard strains of rodents.

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REFERENCES
(1) Saitoh A, Kimura M, Takahashi R, Yokoyama M, Nomura T, Izawa M et al. Most
tumors in transgenic mice with human c-Ha-ras gene contained somatically activated
transgenes. Oncogene 1990;5(8):1195-200.
(2) Van Oosterhout JPJ, Van der Laan JW, De Waal EJ, Olejniczak K, Hilgenfeld M,
Schmidt V et al. The utility of two rodent species in carcinogenic risk assessment of
pharmaceuticals in Europe. Reg Toxicol Pharmacol 1997;25:6-17.
(3) Contrera JF, Jacobs AC, DeGeorge JJ. Carcinogenicity testing and the evaluation of
regulatory requirements for pharmaceuticals. Reg Toxicol Pharmacol 1997;25:130-45.
(4) Reddy MV, Sistare FD, Christensen JS, DeLuca JG, Wollenberg GK, DeGeorge JJ. An
evaluation of chronic 6- and 12-month rat toxicology studies as predictors of 2-year
tumor outcome. Vet Pathol 2010;47:614–29.
(5) Sistare FD, Morton D, Alden C, Christensen J, Keller D, De Jonghe S et al. An analysis
of pharmaceutical experience with decades of rat carcinogenicity testing: support for a
proposal to modify current regulatory guidelines. Toxicol Pathol 2011;39:716-44.
(6) Alden CL, Lynn A, Bourdeau A, Morton D, Sistare FD, Kadambi VJ et al. A critical
review of the effectiveness of rodent pharmaceutical carcinogenesis testing in predicting
for human risk. Vet Pathol 2011;48:772-84.
(7) Friedrich A, Olejniczak K. Evaluation of carcinogenicity studies of medicinal products
for human use authorised via the European centralised procedure (1995-2009). Reg
Toxicol Pharmacol 2011;60:225-48.
(8) Van der Laan JW, Kasper P, Lima BS, Jones DR, Pasanen M. Critical analysis of
carcinogenicity study outcomes. Relationship with pharmacological properties. Crit
Rev Toxicol 2016;46:587-614.
(9) Van der Laan JW, Buitenhuis WHW, Wagenaar L, Soffers AEMF, Van Someren EP,
Krul CAM et al. Prediction of the carcinogenic potential of human pharmaceuticals
using repeated dose toxicity data and their pharmacological properties. Frontiers in
Medicine 2016;3:45. doi: 10.3389/fmed2016.00045
(10) Proposed Change to Rodent Carcinogenicity Testing of Pharmaceuticals –Regulatory
Notice Document. ICH, 2016. URL:
https://database.ich.org/sites/default/files/S1%28R1%29_EWG_RND.pdf (last
accessed 31 May 2022)
(11) The ICHS1 Regulatory Testing Paradigm of Carcinogenicity in Rats - Status Report
Introduction Background: The RND Hypothesis and the Prospective Evaluation Study.
ICH, 2016. URL:
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2016.pdf. (last accessed 31 May 2022)
(12) The ICHS1 Regulatory Testing Paradigm of Carcinogenicity in Rats: Status Report
December 2017. ICH, 2017. URL:
https://database.ich.org/sites/default/files/S1%28R1%29%20EWG_StatusReport_Dec
2017.pdf. (last accessed 31 May 2022)
(13) The ICHS1 Regulatory Testing Paradigm of Carcinogenicity in Rats: Status Report
2019. ICH, 2019. URL:

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 https://database.ich.org/sites/default/files/S1_StatusReport_2019_0802.pdf. (last
accessed 31 May 2022)
(14) The ICHS1 Regulatory Testing Paradigm of Carcinogenicity in Rats: Status Report
2021. ICH, 2021. URL:
https://database.ich.org/sites/default/files/S1_StatusReport_2021_0823.pdf. (last
accessed 31 May 2022)
(15) Hisada S, Tsubota K, Inoue K, Yamada H, Ikeda T, Sistare FD. Survey of tumorigenic
sensitivity in 6-month rasH2-Tg mice studies compared with 2-year rodent assays. J
Toxicol Pathol 2022;35:53–73.
(16) Bugelski PJ, Volk A, Walker MA, Krayer JH, Martin P, Descotes J. Critical review of
preclinical approaches to evaluate the potential of immunosuppressive drugs to
influence human neoplasia. Int J Toxicol 2010;29:435-66.
(17) Lebrec H, Brennan FR, Haggerty H, Herzyk D, Kamperschroer C, Maier CC et al.
HESI/FDA workshop on immunomodulators and cancer risk assessment: Building
blocks for a weight-of-evidence approach. Reg Toxicol Pharmacol 2016;75: 72-80.

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APPENDIX: CASE STUDIES APPLYING THE WEIGHT OF EVIDENCE
APPROACH
Preamble
One outcome of the ICH S1 study was the recognition that programs with the following WoE
attributes are more likely to support a conclusion that the results of a 2-year rat study would not
contribute value to human carcinogenicity risk assessment.
• Target biology is well-characterized and not associated with cellular pathways known
to be involved with human cancer development. Often, the pharmaceutical target was
non-mammalian (e.g., viral, microbial) and carcinogenicity data were available with
the pharmacologic drug class.
• No identified concerns from secondary pharmacology intended to inform off-target
potential for the pharmaceutical.
• Results from chronic toxicity studies indicate no hyperplastic, hypertrophic, atypical
cellular alterations, or degenerative/regenerative changes without adequate
explanation of pathogenesis or human relevance, indicative of no on- or off-target
potential of carcinogenic concern.
• No perturbation of endocrine and reproductive organs observed, or endocrine findings
adequately explained with respect to potential human relevance.
• The overall assessment of genotoxic potential is concluded to be negative based on
criteria from ICH S2(R1) Guidance.
• No evidence of immune modulation or immunotoxicity based on target biology and
repeat-dose toxicology studies.

Case studies are provided to illustrate the application of the WoE approach. These cases are
provided for illustrative purposes only and are not intended to be prescriptive nor to indicate
the sufficiency of data to support a WoE assessment. Cases 1 and 2 are examples of
pharmaceuticals for which the key WoE factors were integrated to conclude that a 2-year rat
study would not add value to the assessment of human carcinogenic risk. Case 3 describes how
data from the WoE factors were integrated to conclude that the carcinogenic potential for
humans was uncertain, and a 2-year rat carcinogenicity study would add value to the assessment
of human carcinogenic risk. Case 4 describes a pharmaceutical for which a 2-year rat
carcinogenicity study was concluded to not contribute value to the assessment of human
carcinogenic risk despite there being no data available for other compounds within the
pharmacologic class.

Case 1: An inhibitor of viral replication

Summary
Prospective WoE Assessment
• The carcinogenic potential in both rats and humans is unlikely such that a 2-year rat
study would not add value to the assessment of human carcinogenicity risk.
• The compound was sufficiently studied at high exposure margins and cause-for-
concern was not identified for any of the WoE factors.

2-year Rat Study Results

• No compound-related carcinogenicity findings.

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Supportive WoE Factors
Target Biology
• Non-mammalian (viral) target excludes intentional alteration of potential mammalian
carcinogenic pathways.
• No compound-related carcinogenicity findings in 2-year rat studies conducted with
other compounds with the same viral replication target.

Secondary Pharmacology
• No evidence of off-target interactions at drug concentrations up to 10 µM, including no
interaction with estrogen, androgen, glucocorticoid receptors.

Histopathology Data from Chronic Studies

Rat Study
• Chronic (6-month) toxicology study in Wistar rats dosed to saturation of absorption,
achieving up to a 31-fold margin to human exposure.
• No compound-related histopathologic findings observed in standard battery of tissues.

Non-rodent Study
• Chronic administration (9-month) to non-human primates identified bile duct
hyperplasia and hepatocellular hypertrophy, with reactive neutrophils and regenerative
hyperplasia. A No-Observed-Adverse-Effect-Level for these effects was identified
which provided a 5-fold margin to human exposure.
• Further evaluation in rats would not provide useful information, as similar findings were
not observed in the chronic rat study.

Hormonal Effects
• No compound-related findings on endocrine and reproductive organ weights or
histopathology.

Genotoxicity
• No evidence of genotoxic potential based on criteria from ICH S2(R1) Guidance.

Immune Modulation
• No compound-related changes in clinical pathology or histopathology of immune
tissues (e.g., lymph nodes, spleen, thymus, bone marrow).

Additional Investigations
• No data available

Case 2: An antagonist of a neuronal G-protein coupled receptor

Summary
Prospective WoE Assessment
• The carcinogenic potential is unlikely in humans but likely in rats through well-
recognized mechanisms shown to be human irrelevant, such that a 2-year rat study
would not add value to the assessment of human carcinogenic risk.

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 • The potential for rodent-specific liver and thyroid tumors was based on the
toxicology observed in the chronic rat study and on tumor outcome with the
pharmacological class. Hormonal effects due to target pharmacology occurred at
high multiples of human exposure and were not considered a human carcinogenic
risk. Fluorosis, a potential carcinogenic risk, was observed in rats due to release of
fluoride from the compound; however, release of fluoride from the compound was
not observed in humans.

2-year Rat Study Results

• The 2-year rat study demonstrated hepatocellular hypertrophy but no compound-
related carcinogenicity findings.

Supportive WoE Factors

Target Biology
• Predominate receptor expression in brain with lower expression in some peripheral
tissues, similar across species.
• Receptor activation increases adrenocorticotropic hormone (ACTH) release from
pituitary secondary to hypothalamic production of adrenocorticotropin-releasing
hormone.
• Target knock-out mice showed no findings related to carcinogenicity.
• A 2-year rat study with a comparable compound did not identify a carcinogenic effect
that could be ascribed to the intended pharmacological target (see secondary
pharmacology section for off-target effects).

Secondary Pharmacology
• Antagonist binding interaction identified for one off-target receptor with Ki 8-fold
higher than Cmax at maximum clinical dose. Known pharmacology of off-target
receptor not associated with tumorigenesis.
• Thyroid follicular cell adenoma/carcinoma was observed in a 2-year rat study with a
comparable compound which was associated with increased thyroid stimulating
hormone and ascribed to an off-target pathway related to drug metabolism.

Histopathology Data from Chronic Studies

Rat Study
• Increased liver hypertrophy and organ weight at 50-fold to 74-fold human exposure.
• Increased thyroid follicular hypertrophy at 170-fold to 670-fold human exposure.

Non-rodent Study
• Increased liver hypertrophy and organ weight at ~ 230-fold human exposure.

Hormonal Effects
• Reduced adrenal weight without histopathological correlates and reduced ACTH level
at > 74-fold human exposure in the 6-month rat study, consistent with inhibition of drug
target.
• Irregular estrous cycles and decreased pregnancy rate were observed at 60-fold human
exposure, and decreased numbers of corpora lutea, implantations, and live embryos
were observed at > 500-fold human exposure in a fertility study in rats. Considered

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 consistent with suppression of luteinizing hormone and gonadotropin release associated
with inhibition of the drug target.
• No treatment-related changes observed in reproductive organ weight or histopathology
in 6-month rat study.

Genotoxicity
• No evidence of genotoxic potential of parent or major human metabolite based on
criteria from ICH S2(R1) Guidance.

Immune Modulation
• No treatment-related changes in clinical pathology, lymphocyte subsets, or
histopathology of immune tissues (e.g., lymph nodes, spleen, thymus, bone marrow).

Additional Investigations
• Induction of CYP1A2 and CYP3A1 demonstrated.
• Bone and teeth fluorosis related to release of fluoride from the compound in rats and
demonstrated not to occur in humans.

Case 3: An inhibitor of a ubiquitously expressed serine/threonine kinase (novel target)

Summary
Prospective WoE Assessment
• The carcinogenic potential in humans is uncertain and a 2-year rat carcinogenicity
study would add value to the assessment of human carcinogenic risk.
• Carcinogenic uncertainty is related to the complex target pharmacology (e.g.,
inhibition of cellular apoptosis), the lack of precedent with the drug target, and
histopathological changes of concern with inadequate mechanistic explanation from
the 6-month rat study which are supported by similar findings in cynomolgus
monkeys. While the immune toxicology findings in monkeys (i.e., suppression of T
cell-dependent antigen response) contributed to the assessment of human
carcinogenicity risk, this finding was not expected to be further informed by a rat
carcinogenicity study.

2-year Rat Study Results

• Increased incidence, lethality, and reduced latency of pituitary tumors was observed
in both sexes and may be attributed to target pharmacology. The outcome of the 2-
year rat study contributed to the overall assessment of human carcinogenic risk.

Supportive WoE Factors

Target Biology
• Target activation by inflammation-related oxidative stress promotes cellular apoptosis
and is linked to control of cell proliferation; target inhibition suppresses apoptotic
signaling and impacts cell proliferation, theoretically promoting cancer growth.
• Drug target displays tissue-dependent roles in cancer development, both promotion and
suppression in animal models.
• No data available on tumor outcome from target inhibition in 2-year rodent or 6-month
transgenic mouse studies.

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Histopathology Data from Chronic Studies
Rat Study
• Increased incidence and severity of renal basophilic tubules, eosinophilic droplets, and
brown pigment in renal cortex starting at 14-fold human exposure. Human relevance of
lesions was not addressed.
• Chronic irritation of limiting ridge in non-glandular stomach at 39-fold human
exposure. Human relevance of lesions was not addressed.
• Increased liver weight without microscopic correlates.
Non-rodent Study
• In monkeys, gastrointestinal epithelial degeneration, necrosis, reactive hyperplasia,
ectasia, inflammation, and ulceration were observed at doses 12-fold human exposure.
• Increased incidence of renal tubule degeneration /regeneration, necrosis, dilation, and
vacuolation observed at 12-fold human exposure.

Hormonal Effects
• Increased adrenal weight and cortical hypertrophy in rats at 17-fold human exposure.
Human relevance of lesions was not addressed.

Genotoxicity
• No evidence of genotoxic potential of parent or major human metabolite based on
criteria from ICH S2(R1) Guidance.

Immune Modulation
• In monkeys, suppression of T cell-dependent antigen response occurred with no effect
on natural killer cell cytotoxicity or granulocyte function.
• Decreased lymphoid cellularity observed in spleen, thymus, lymph nodes at 12-fold
human exposure.

Additional investigations
• Increases in hepatic enzymes CYPs 1A, 3A, and 2B demonstrated.

Case 4: An inhibitor of a prostaglandin receptor (novel target)

Summary
Prospective WoE Assessment
• The carcinogenic potential in both rats and humans is unlikely such that a 2-year rat
study would not add value to the assessment of human carcinogenic risk.
• The drug target is not associated with a role in cancer development, histopathological
findings were not observed in the 6-month rat study at a > 50-fold margin of human
exposure. Secondary pharmacology also indicated high target selectivity for the
compound.

2-year Rat Study Results

• No compound-related carcinogenicity findings.

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Supportive WoE Factors
Target Biology
• Receptor activation on innate immune cells is associated with allergic inflammatory
responses and available data do not suggest a role in carcinogenesis.
• Knock-out mice lacking the drug target showed no histological abnormalities or effects
on immune function during one year of observation.

Secondary pharmacology
• Compound was at least 300-fold more selective for drug target when compared with
other receptors in the same class as well as for a sub-set of other receptors involved in
the inflammatory response.
• Compound was at least 2000-fold more selective for the drug target in a screen of
various receptors, ion channels, transporters, and enzymes.

Histopathology Data from Chronic Studies

Rat Study
• No proliferative changes observed in any organ or tissue at the highest dose tested
(~ 54-fold human exposure).

Non-rodent Study
• No proliferative changes in any organ or tissue at the highest dose tested (~ 45-fold
human exposure) in repeated-dose toxicity studies of up to 39 weeks.

Hormonal Effects
• No compound-related findings on endocrine and reproductive organ weights or
histopathology.

Genotoxicity
• No evidence of genotoxic potential based on criteria from ICH S2(R1) Guidance.

Immune Modulation
• In the 6-month rat toxicity study, there were no effects on immune function (including
in a T cell-dependent antibody response assay) or adverse effects on lymphocyte subsets
at the highest dose tested (~ 54-fold human exposure).

Additional Investigations
• No data available.

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