S1B(R1) Addendum  to S1B Testing for 
Carcinogenicity of Pharmaceuticals  
 
Guidance for Industry  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services  
Food and Drug Administration  
Center for  Drug Evaluation and  Research (CDER)  
Center for Biologics Evaluation and Research (CBER)  
 
November 2022  
ICH  
 
    
 
S1B(R1) Addendum  to S1B Testing  for 
Carcinogenicity of Pharmaceuticals  
 
Guidance for Industry  
 
 
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U.S. Department of Health and Human Services  
Food and Drug Administration  
Center for Drug Evaluation and Research (CDER)  
Center for Biologics Evaluation and Research (CBER)  
 
November 2022  
ICH 
  Contains Nonbinding Recommendations  
 
FOREWORD  
 
The  International Council for Harmonisation of Technical Requirements  for Pharmaceuticals for 
Human Use (ICH) has the mission of achieving greater regulatory harmonization worldwide to 
ensure that safe, effective, and high-quality medicines are developed, registered, and maintained 
in the most resource-efficient manner.   By harmonizing the  regulatory expectations in regions  
around the  world, ICH guidelines have substantially reduced duplicative clinical studies, 
prevented unnecessary animal studies, standardized safety reporting and marketing application 
submissions, and contributed to many other  improvements in the quality of global drug 
development and manufacturing and the products available  to patients.  
 
ICH is a consensus-driven process  that involves technical experts  from regulatory authorities and  
industry parties in detailed technical and science-based harmonization work that  results in the 
development of ICH guidelines.  The commitment to consistent adoption of these  consensus- 
based guidelines by regulators around the globe  is critical to  realizing the  benefits of safe,  
effective, and high-quality medicines for patients  as well as  for industry.  As a Founding 
Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a major  role  in the  
development of each of the ICH guidelines, which FDA then adopts and issues as guidance to 
industry.  
 
 
   
  Contains Nonbinding Recommendations  
 
TABLE OF CONTENTS  
 
 
PREAMBLE ..................................................................................................................................  1  
I.   INTRODUCTION (1) .......................................................................................................  1  
A.   Scope of the Addendum (1.1)  ........................................................................................................  1  
B.   Purpose of the Addendum (1.2)  ....................................................................................................  2  
C.   Background (1.3) ............................................................................................................................  2  
II.   A WEIGHT OF  EVIDENCE APPROACH TO ASSESS  THE HUMAN 
CARCINOGENIC POTENTIAL  OF PHARMACEUTICALS (2) ..........................................  3  
A.   Factors to Consider for a WoE Assessment (2.1)    ......................................................................  4  
B.   Integration of WoE Factors for Assessing Human Carcinogenic Risk (2.2)  ............................  6  
C.   Mouse Carcinogenicity Studies (2.3)  ............................................................................................  7  
III.   CLARIFICATION ON CRITERIA FOR HIGH DOSE SELECTION BASED  ON 
EXPOSURE FOR RASH2-TG MOUSE CARCINOGENICITY STUDIES (3)  .....................  7  
REFERENCES ..............................................................................................................................  9  
APPENDIX:  CASE STUDIES APPLYING  THE WEIGHT OF EVIDENCE APPROACH . 
  ............................................................................................................................................11  
  Contains Nonbinding Recommendations  
 
S1B(R1) Addendum  to S1B  Testing  for  Carcinogenicity of 
Pharmaceuticals  
Guidance for Industry1  
 
 
This guidance represents the current thinking  of the  Food and Drug Administration (FDA or  Agency) on 
this topic.  It  does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.   
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
PREAMBLE  
 
This Addendum is to be used in close conjunction with the  ICH guidances  for industry  S1A  The  
Need for Long-term Rodent  Carcinogenicity Studies of Pharmaceuticals2  (March  1996), S1B  
Testing for  Carcinogenicity of Pharmaceuticals  (July  1997), and S1C(R2) Dose Selection for  
Carcinogenicity Studies  (September 2008).   The Addendum is complementary to the  ICH  S1  
guidances for  industry.   
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current  thinking on a topic  and should be viewed only 
as recommendations, unless specific  regulatory or statutory requirements are cited.  The use of  
the word should  in Agency guidances means  that something is suggested or recommended, but  
not required.   
 
 
I.   INTRODUCTION  (1)3  
A.   Scope of the Addendum  (1.1)  
 
This Addendum applies to all pharmaceuticals that need carcinogenicity  testing as described in   
ICH S1A.  For biotechnology-derived pharmaceuticals, refer  to  the  ICH guidance  for industry  
S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals  (May 2012)).  
 
1  This  guidance  was  developed  within  the  Expert  Working Group (Safety)  of  the  International  Council  for  
Harmonisation  of  Technical  Requirements  for  Registration  of  Pharmaceuticals  for  Human Use (ICH) and  has  been  
subject  to consultation by  the  regulatory parties,  in accordance  with the  ICH  process.   This  document  has  been 
endorsed by  the  ICH Assembly  at Step 4  of  the  ICH  process,  August  2022.   At  Step 4  of  the  process,  the  final  draft  is  
recommended for  adoption to  the  regulatory  bodies  of  the  ICH  regions.  
2  We  update  guidances  periodically.   For  the  most  recent  version of  a  guidance,  check the  FDA guidance  web page  
at  https://www.fda.gov/regulatory-information/search-fda-guidance-documents.    
3  The  numbers  in parentheses  reflect  the  organizational  breakdown  of  the  document  endorsed by the ICH  Steering 
Committee  at  Step 4  of  the  ICH  process, August 2022.  
  1   Contains Nonbinding Recommendations  
 
B.   Purpose of the Addendum  (1.2)      
 
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  ICH 
S1B.  This is an integrative approach that provides specific weight of evidence (WoE)  criteria 
that inform whether  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,4  while all other aspects of the recommendations for 
high-dose selection in  ICH S1C(R2) 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.  
C.   Background (1.3)     
 
While ICH S1B 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 ICH  S1B, 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  
(Refs.  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 Document5. The process 
and several status updates reporting  results are posted and available at the ICH  website (Refs.  10-
14).  Carcinogenicity assessment documents and associated data from 2-year rat carcinogenicity  
studies for 45 compounds were received and evaluated by regulatory members of the ICH  Expert  
 
4  The rasH2-Tg mouse  was  developed in  the  laboratory  of  Tatsuji  Nomura  of  the  Central  Institute  for  Experimental  
Animals  (Ref.  1).   The  model  is  referred  to  in  ICH  S1B  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 ICH  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 ICH  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.  
5  Available  at  https://database.ich.org/sites/default/files/S1%28R1%29_EWG_RND.pdf  
  2   Contains Nonbinding Recommendations  
 
Working Group.  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.6  
 
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  (Ref.  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.  
II.   A WEIGHT OF  EVIDENCE APPROACH TO ASSESS  THE HUMAN 
CARCINOGENIC POTENTIAL  OF PHARMACEUTICALS  (2)  
 
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  
   Unlikely,  such that  a 2-year rat carcinogenicity study would not add value7  
   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).   
 
 
6  Methods  and results  of  the  ICH  S1 prospective  evaluation study will  be  summarized in  a  future  publication.  
7  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.  
  3   Contains Nonbinding Recommendations  
 
   
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.  
A.   Factors to  Consider for a WoE Assessment  (2.1)    
 
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)  histopathology data8  from repeated-dose toxicity 
 
8  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  
  4   Contains Nonbinding Recommendations  
 
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.  
(3)   Evidence for hormonal  perturbation,9  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.  
(4)   Genetic toxicology study data using criteria from  ICH guidance for  
industry S2(R1)  Genotoxicity Testing and Data Interpretation for  
Pharmaceuticals Intended for Human Use  (June 2012); equivocal  
genotoxicity data that cannot be resolved in accordance with ICH  S2(R1)  
recommendations increases uncertainty with respect to the carcinogenic 
potential  
(5)   Evidence of  immune modulation in accordance with ICH S8.  
Evidence of  broad immunosuppression may provide sufficient concern for  
human risk that would not be further informed by  standard rat and mouse  
carcinogenicity studies  (Refs.  16  and  17).  
 
The above WoE factors may be sufficient to conclude whether  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)  
(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.  
 
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.  
9  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.  
  5   Contains Nonbinding Recommendations  
 
However,  if rasH2-Tg mouse study results are available, they should be included  in the  WoE  
document.  
B.   Integration of WoE Factors for Assessing Human Carcinogenic Risk  (2.2)  
 
An  integrated analysis of the WoE factors described above  should be used to determine whether 
a 2-year rat  study would contribute  to the human carcinogenic risk assessment.  While  all 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 see S1(R1)  Proposed Change to Rodent  Carcinogenicity Testing of Pharmaceuticals    
Regulatory Notice Document)  are provided in the  Appendix,  demonstrating how the WoE factors  
  6   Contains Nonbinding Recommendations  
 
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 regarding  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 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.  
C.   Mouse Carcinogenicity Studies  (2.3)  
 
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.   
III.   CLARIFICATION ON CRITERIA FOR HIGH-DOSE SELECTION BASED  
ON EXPOSURE FOR RASH2-TG MOUSE CARCINOGENICITY STUDIES  
(3)  
 
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  
  7   Contains Nonbinding Recommendations  
 
criterion  for high-dose selection.  Therefore, all criteria  for selection of the high dose as specified  
in ICH 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.  
 
   
  8   Contains Nonbinding Recommendations  
 
REFERENCES  
 
(1)  Saitoh A, Kimura M, Takahashi R, Yokoyama M, Nomura  T, Izawa M, Sekiya T, 
Nishimura S, Katsuki M.  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, Bass R.  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;47:61429.  
(5)  Sistare FD, Morton D, Alden C, Christensen J,  Keller D, De Jonghe S, Storer R, Reddy 
M, Kraynak A, Trela B, Bienvenu J, Bjurstrom S, Bosmans V, Brewster D, Colman K, 
Dominick M, Evans J, Hailey J, Kinter L, Liu M, Mahrt C, Marien D, Myer J, Perry R, 
Potenta D, Roth A, Sherratt P, Singer T, Slim R, Soper K, Fransson-Steen R, Stoltz  J,  
Turner O, Turnquist S, van Heerden M, Woicke  J, DeGeorge J.  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, Silverman L.  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, Woutersen R.  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:  
  9   Contains Nonbinding Recommendations  
 
https://database.ich.org/sites/default/files/S1%28R1%29%20EWG_StatusReport_Mar201 
6.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_Dec201 
7.pdf. (last accessed 31  May 2022)  
(13)  The ICHS1 Regulatory Testing Paradigm of Carcinogenicity  in Rats: Status Report 
2019.  ICH, 2019. URL:  
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)  
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(17)  Lebrec H, Brennan FR, Haggerty H, Herzyk D, Kamperschroer C, Maier CC et al.  
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for a weight-of-evidence approach. Reg Toxicol Pharmacol  2016;75: 72-80.  
   
  10   Contains Nonbinding Recommendations  
 
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 guidance  for industry S2(R1)  Genotoxicity Testing and Data 
Interpretation for Pharmaceuticals Intended  for Human Use  (June 2012).  
   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.  
 
  11   Contains Nonbinding Recommendations  
 
2-year Rat Study Results   
   No compound-related  carcinogenicity findings.  
 
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 M, 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 guidance for  industry 
S2(R1)  Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for  
Human Use  (June 2012).  
 
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  
  12   Contains Nonbinding Recommendations  
 
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.  
   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   
  13   Contains Nonbinding Recommendations  
 
   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 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 guidance for  industry S2(R1)  Genotoxicity Testing and Data Interpretation for  
Pharmaceuticals Intended for Human Use  (June 2012)) .  
 
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  Cytochrome P450 (CYP)1A2 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  
  14   Contains Nonbinding Recommendations  
 
   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.  
 
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  guidance for  industry S2(R1)  Genotoxicity Testing and Data Interpretation for  
Pharmaceuticals Intended for Human Use  (June 2012).  
 
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.  
  15   Contains Nonbinding Recommendations  
 
 
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.  
 
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.  
  16   Contains Nonbinding Recommendations  
 
 
Genotoxicity  
   No evidence of genotoxic potential based on criteria from  ICH S2(R1) .  
 
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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