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 

Guidance for Industry and Review Staff1

Recommended Approaches to Integration of 

Genetic Toxicology Study Results

This guidance represents the Food and Drug Administration’s (FDA’s) 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. 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the Agency’s 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 shorterterm 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 investigator’s 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 

5

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.