Guidance for Industry 
and Review Staff 
Recommended Approaches to 
Integration of Genetic 
Toxicology Study Results 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
January 2006 
Pharmacology and Toxicology 
 
 
   
Guidance for Industry 
and Review Staff 
Recommended Approaches to 
Integration of Genetic 
Toxicology Study Results 
 
 
 
Additional copies are available from: 
 
Office of Training and Communications 
Division of Drug Information, HFD-240 
Center for Drug Evaluation and Research 
Food and Drug Administration 
5600 Fishers Lane 
Rockville, MD  20857 
(Tel) 301-827-4573 
http://www.fda.gov/cder/guidance/index.htm 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
January 2006 
Pharmacology and Toxicology 
 
   
TABLE OF CONTENTS 
 
I.  INTRODUCTION.............................................................................................................1 
II.  BACKGROUND...............................................................................................................1 
III.  INTEGRATION OF GENETIC TOXICOLOGY STUDY RESULTS.......................2 
A.  Weight-of-Evidence Approach.....................................................................................................3 
B.  Mechanism of Action.....................................................................................................................3 
C.  Additional Supportive Studies......................................................................................................3 
REFERENCES..............................................................................................................................5 
  Contains Nonbinding Recommendations 
 
1
Guidance for Industry and Review Staff  
Recommended Approaches to Integration of  
Genetic Toxicology Study Results 
 
 
 
 
This guidance represents the Food and Drug Administrations (FDAs) current thinking on this topic.  It 
does not create or confer any rights for or on any person and does not operate to bind FDA or the public.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance.  
 
 
 
 
I.  INTRODUCTION  
 
The purpose of this guidance is to inform industry and the review staff in the Center for Drug 
Evaluation and Research (CDER) on how CDER views positive findings in genetic toxicology 
assays during drug development.  The guidance provides recommendations on how to proceed 
with clinical studies while ensuring the safety of study participants when results in genotoxicity 
studies suggest a potential cancer or genetic hazard.  This guidance pertains to pharmaceuticals 
administered through oral, intravenous, topical, and other routes, as appropriate.  
 
FDAs guidance documents, including this guidance, 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.  
 
 
II.  BACKGROUND 
 
The timing and conduct of genetic toxicology studies have been described in the ICH guidelines 
M3, S2A, and S2B.2  We recommend that these guidances be consulted and that this document 
be considered an adjunct guidance.   
                                                 
1 This guidance has been prepared by the Pharmacology Toxicology Coordinating Committee (PTCC) in the Office 
of New Drugs (OND) in the Center for Drug Evaluation and Research (CDER) of the Food and Drug 
Administration.  
 
2 ICH guidance for industry M3 Nonclinical Safety Studies for the Conduct of Human Clinical Trials for 
Pharmaceuticals, ICH guidance for industry S2A Specific Aspects of Regulatory Genotoxicity Tests for 
Pharmaceuticals, and ICH guidance for industry S2B Genotoxicity:  A Standard Battery for Genotoxicity Testing of 
Pharmaceuticals.  (http://www.fda.gov/cder/guidance/index.htm) 
  1 Contains Nonbinding Recommendations 
 
 
Risk for carcinogenesis is usually determined in rodent assays, either 2-year studies or shorter-
term studies using alternative models.3  A core battery of genetic toxicology studies has been 
accepted by industry and regulators through the International Conference on Harmonisation 
(ICH) consultative process.  These studies, which are designed to identify genotoxic hazard, 
include: 
  A test for gene mutation in bacteria;  
  An in vitro assessment of chromosomal damage using mammalian cells or an in vitro 
mouse lymphoma tk+/- assay; and 
  An in vivo test for chromosomal damage using rodent hematopoietic cells.  
 
The following discussion is based on current guidance documents.4  We recommend that results 
from in vitro genetic toxicology studies be available before the initiation of phase 1 trials.  
 
 
III.  INTEGRATION OF GENETIC TOXICOLOGY STUDY RESULTS  
 
The Agency takes into account the totality of safety data when considering whether it is safe to 
proceed with a clinical trial when there are positive genetic toxicology study results.  This 
consideration includes a thorough evaluation of all the genetic toxicology data and the nature of 
the proposed trial.  If the results of the genetic toxicology tests indicate a lack of genotoxic 
potential, then clinical trials can generally be undertaken in healthy subjects or patient 
populations with the proposed medical indication.   
 
Pharmaceuticals that give positive results in genetic toxicology assays but do not directly interact 
with DNA do not always present a significant in vivo risk.  In such cases, we recommend 
providing evidence of the mechanism of genotoxicity and relevance of the mechanism to 
anticipated in vivo exposure.  Alternatively, it is also appropriate to rule out mechanisms 
involving direct interaction with DNA (e.g., demonstration that a drug does not cause DNA 
alkylation or DNA strand breakage).  
 
Drugs known to directly damage DNA may be permitted to be used in patients with debilitating 
or life-threatening diseases, such as cancer, but should not be administered to healthy subjects.5 
 
If any of the three assays in the ICH genotoxicity standard battery are positive, then we 
recommend completing the fourth test in the ICH battery.  Equivocal studies should be repeated 
to determine the reproducibility of the results.  If a positive response is seen in one or more 
assays, sponsors should consider choosing from one or more of the following options. 
 
                                                 
3 ICH guidance for industry S1B Testing for Carcinogenicity of Pharmaceuticals.  
(http://www.fda.gov/cder/guidance/index.htm) 
 
4 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the CDER 
guidance Web page at http://www.fda.gov/cder/guidance/index.htm. 
 
5 ICH guidance for industry S2A Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.  
(http://www.fda.gov/cder/guidance/index.htm) 
  2 Contains Nonbinding Recommendations 
 
A.  Weight-of-Evidence Approach 
 
In some instances, after evaluation of all available data, the weight of evidence (WOE) suggests 
a lack of genotoxic hazard.  For example, a positive response is observed in one exposure 
regimen of an in vitro cytogenetics assay.  The positive result is seen only at the high dose, and 
the increase is within or just outside the range for historical control values for the solvent and 
cell line employed.  The WOE approach could indicate that although a small increase in the 
frequency of chromosomal aberrations is statistically significant, it lacks biological relevance.  
Contributing considerations could include (1) the level of cytotoxicity at which the response was 
seen, and (2) corroborating data from the same or complementary assays.  For example, a 
positive response seen with a short-term exposure without metabolic activation but not 
corroborated with the longer exposure at comparable levels of cytotoxicities would argue against 
the biological significance of the positive result.  Similarly, such a positive finding in an in vitro 
chromosomal aberration assay that is not corroborated by the matching exposure regimen of the 
mouse lymphoma assay could also call into question the significance of the positive finding.  If 
the WOE approach indicates a lack of genotoxic hazard, clinical studies could proceed provided 
the positive response is described in the investigators brochure and the informed consent form. 
 
B.  Mechanism of Action 
 
Positive results are sometimes satisfactorily explained by knowledge of the mechanism of action.  
For example, it has been demonstrated that in vitro clastogenic effects can result from 
excessively high osmolarity or low pH.  Positive responses elicited under such nonphysiologic 
exposure conditions are not relevant to human risk.  In addition, certain genotoxic responses are 
thought to have thresholds below which a hazard does not exist.  Agents that induce effects by 
indirect mechanisms (e.g., interference with metabolism of nucleotides and their precursors, 
damage to spindle proteins, inhibition of DNA synthesis, or inhibition of topoisomerase) can 
have thresholds for genotoxic effects.  In such cases, we recommend presenting evidence of the 
existence of a threshold that would not be attained during the proposed clinical exposure or 
presenting evidence of a mechanism not expected to be operative in vivo.  Positive responses that 
are satisfactorily explained by an MOA may allow clinical studies in normal volunteers or in 
patients to proceed without additional studies. 
 
C.  Additional Supportive Studies   
 
On occasion, results from in vitro studies demonstrate a reproducible positive dose-response.  
Results from bone marrow cytogenetic studies are frequently negative, even for those 
compounds giving positive results in in vitro genetic toxicology assays.  This discrepancy can 
result from a number of differences between cultured cells and intact animals: differing 
metabolic pathways occurring in vitro and in vivo, metabolic inactivation in the intact animal, 
failure of the parent compound or active metabolite to reach the target cell, or simply, an 
inability to achieve plasma levels in vivo comparable to concentrations that generated positive 
responses in the in vitro assays.   
 
Additional in vivo assays can be useful in clarifying in vitro positive results.  For example, 
peripheral blood smears from repeat-dose toxicity studies in mice can be evaluated for 
  3 Contains Nonbinding Recommendations 
 
micronucleus induction, and peripheral blood lymphocytes from repeat-dose studies in rats or 
monkeys can be cultured and assessed for chromosome damage in metaphase spreads.  DNA 
damage can be assessed in potential target tissues (e.g., DNA adducts or DNA strand breakage 
using the Comet or alkaline elution assay), or transgenic rats or mice can be used to assess 
mutagenicity in potential target tissues.6  
 
The Syrian hamster embryo cell (SHE) transformation assay has been suggested as a follow-up 
assay in the face of positive in vitro genotoxicity results.  Data in the literature suggest that the 
SHE assay correlates well with rodent carcinogenicity results for chemicals in general (Isfort et 
al. 1996).  Results from an International Life Sciences Institute (ILSI) validation effort on human 
pharmaceuticals, although smaller in scope, suggest that the SHE assay is less predictive for 
human carcinogenic risk (Mauthe et al. 2001).  With respect to human pharmaceuticals, the ILSI 
study found that the SHE assay had high sensitivity (83 percent) for detection of human 
carcinogens.  However, its low specificity (15 percent) for prediction of putative human 
noncarcinogens led to a poor overall concordance of 37 percent.  Although transformation assays 
measure endpoints more akin to the health effect of concern (cancer) and can be useful in making 
a WOE judgment, they also have inherent limitations.  Many pharmaceuticals that give positive 
responses in 2-year rodent carcinogenicity studies do so through exaggerated pharmacological 
effects, immune suppression, or hormonal disequilibrium.  It is unclear how an in vitro assay 
could be responsive to these mechanisms.  
 
In the last several years, a number of transgenic mouse strains have become available for use in 
short-term carcinogenicity studies.  The p53 haplo insufficient mouse has been found to be 
useful in the identification of mutagenic carcinogens (MacDonald et al. 2004).  Negative results 
in a p53 carcinogenicity study are considered evidence that a genotoxic agent does not present a 
carcinogenic hazard to humans through a p53-mediated mechanism.   
 
Supportive studies contribute to the WOE determination as to whether a drug giving a positive 
response in one of the ICH-specified assays presents a risk of genetic damage to subjects 
involved in clinical trials.  The decision as to whether early assessment of oncogenic potential 
will be needed will, out of necessity, be on a case-by-case basis.  Factors influencing the decision 
include target population, disease indication, duration of exposure, and safety profile of other 
drugs in the class or other drugs serving the same medical need. 
 
 
                                                 
6 ICH guidance for industry S2B Genotoxicity:  A Standard Battery for Genotoxicity Testing of Pharmaceuticals.  
(http://www.fda.gov/cder/guidance/index.htm) 
  4 Contains Nonbinding Recommendations 
 
REFERENCES 
 
ICH guidance for industry S1A The Need for Long-Term Rodent Carcinogenicity Studies of 
Pharmaceuticals.  (http://www.fda.gov/cder/guidance/index.htm) 
 
Isfort, RJ, GA Kerckaert, and RA LeBoeuf, 1996, Comparison of the Standard and Reduced pH 
Syrian Hamster Embryo (SHE) Cell Transformation Assays in Predicting the 
Carcinogenic Potential of Chemicals, Mutat. Res. 356:11-63. 
 
MacDonald, J, JE French, RJ Gerson, J Goodman, T Inoue et al., 2004, The Utility of Transgenic 
Mouse Assays for Identifying Human Carcinogens  A Basic Understanding and Path 
Forward, Toxicol. Sci. 77(2):188-194. 
 
Mauthe, RJ, DP Gibson, RT Bunch, and L Custer, 2001, The Syrian Hamster Embryo (SHE) 
Cell Transformation Assay:  Review of Methods and Results, Toxicologic Pathology 29 
(Supplement): 138-146. 
 
  5