U.S. flag An official website of the United States government

On Oct. 1, 2024, the FDA began implementing a reorganization impacting many parts of the agency. We are in the process of updating FDA.gov content to reflect these changes.

U.S. Food and Drug Administration
  1. Home
  2. Regulatory Information
  3. Search for FDA Guidance Documents
  4. Redbook 2000: IV.C.1. Short-Term Tests for Genetic Toxicity
  1. Search for FDA Guidance Documents

GUIDANCE DOCUMENT

Redbook 2000: IV.C.1. Short-Term Tests for Genetic Toxicity July 2007

Final
Issued by:
Guidance Issuing Office
Human Foods Program

Toxicological Principles for the Safety Assessment of Food Ingredients

Return to Redbook 2000 table of contents

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.

Genetic changes known to be associated with adverse human health effects include gene mutations, chromosomal rearrangements or deletions, and loss or gain of whole chromosomes (aneuploidy) or chromosomal segments. Genotoxicity tests are in vitro and in vivo tests designed to detect compounds that induce genetic damage. Such tests include: (1) tests that directly assess the key types of genetic alterations (gene mutations and chromosomal effects) and (2) indirect genotoxicity tests that respond to types of DNA damage known to lead to these alterations. The latter category of tests may assess either DNA damage (e.g., DNA adducts or DNA strand breakage) or cellular responses to DNA damage (e.g., unscheduled DNA synthesis).

FDA recommends the use of a battery of short-term genetic toxicity tests for all when the cumulative estimated dietary intake exceeds 1.5 µg per person per day, corresponding to 0.5 parts per billion (ppb) in the total diet. The recommended tests directly measure gene mutations and/or chromosomal effects. The Agency uses such data, in the absence of long-term animal feeding studies, to determine whether or not a chemical should be considered to be a possible carcinogen. Such data may also indicate whether a chemical may have adverse heritable effects. When long-term animal feeding studies are available for the evaluation of carcinogenicity, genetic toxicity data may assist in the interpretation of the results of such studies.

Genetic Toxicity Test Battery

We consider it essential that chemicals be evaluated for their ability to induce both gene mutations and chromosomal aberrations. The most widely used test for gene mutations is performed using bacteria as the target cells. Tests for chemicals that induce gene mutations can also be performed in mammalian cells grown in vitro. Tests that detect the induction of chromosomal aberrations are performed using cells exposed to chemicals in vitro or in vivo. While the recommended battery consists of specific genetic toxicity tests, data from other systems that measure gene mutations, chromosomal effects, DNA damage, or cellular responses to DNA damage may be relevant to the overall genotoxicity evaluation of a chemical. Therefore, all available data relating to such endpoints in any test system should be submitted.

Recommended Tests

The recommended genetic toxicity test battery for food ingredients whose cumulative estimated daily intake exceeds 50 ppb in the diet (150 µg per person per day) generally includes:

  1. a test for gene mutations in bacteria

    and

  2. an in vitro test with cytogenetic evaluation of chromosomal damage using mammalian cells

    or

    an in vitro mouse lymphoma thymidine kinase+/- gene mutation assay

    (the mouse lymphoma assay is preferred)

    and

  3. an in vivo test for chromosomal damage using mammalian hematopoietic cells.

The Agency prefers the mouse lymphoma tk+/- assay in item "b" because this assay measures heritable genetic damage arising by several mechanisms in living cells and is capable of detecting chemicals that induce either gene mutations or heritable chromosomal events, including genetic events associated with carcinogenesis. In performing the mouse lymphoma tk+/- assay, either the soft agar or the microwell method is acceptable.

When the cumulative estimated daily intake of a food ingredient is 50 ppb or less but greater than 0.5 ppb, then the recommended genetic toxicity test battery generally includes items "a" and "b" in the above list since there are few chemicals that are uniquely genotoxic when tested in vivo for chromosomal damage.

Guidance for performing a test for gene mutations in bacteria and an in vivo micronucleus assay (which is an acceptable in vivo test for chromosomal damage using mammalian hematopoietic cells) is presented here. This guidance is based directly on the guidelines published by the Organization for Economic Cooperation and Development (OECD) or those published by the United States Environmental Protection Agency (US EPA), which are virtually identical to each other. The reports of the International Conference on Ha rmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) were used as the basis for the general recommendations for genetic toxicity testing and were also referred to in drafting the guidelines on specific test systems. The guidance for an in vivo test for chromosomal damage using mammalian hematopoietic cells (micronucleus assay) was also based on the report of the 1998 International Workshop on Genotoxicity Test Procedures (Hayashi, et al., Environmental and Molecular Mutagenesis, 2000, in press). The FDA has modified these previous guidelines as appropriate. For specific guidance on the conduct of genotoxicity tests for which guidance is not included here, the above referenced ICH, OECD, or US EPA guidelines should be consulted.

Types of Genetic Damage

The mutation assays specified in the standard battery are capable of detecting different spectra of genetic damage. The tests for gene mutations in bacteria detect point mutations which involve substitution, addition, or deletion of one or a few DNA base pairs. Point mutations may also be detected in in vitro mammalian cell mutagenicity assays. The mouse lymphoma tk+/- assay specified in the battery also detects large deletions, translocations, mitotic recombination/gene conversion and aneuploidy in addition to point mutations, making this the assay with the broadest spectrum of detectable genetic damage in the battery. The in vitro test with cytogenetic evaluation of chromosomal damage using mammalian cells detects structural chromosomal aberrations. The in vivo tests for chromosomal damage using mammalian hematopoietic cells detect structural chromosomal aberrations in the case of the mammalian bone marrow chromosome aberration test and structural damage to chromosomes or damage to the mitotic apparatus in the case of the mammalian erythrocyte micronucleus test.

It should also be noted that, although the mouse lymphoma tk+/- assay and the in vitro test with cytogenetic evaluation of chromosomal damage using mammalian cells both detect structural chromosomal damage, the mouse lymphoma tk+/- assay detects damage that is heritable but not lethal to the cells, while the in vitro test with cytogenetic evaluation of chromosomal damage detects additional types of damage that are lethal to the cells. Additionally, the mouse lymphoma tk+/- assay is the only assay in the battery capable of detecting gene conversion/mitotic recombination.

Thus, the tests included in the battery were chosen to complement each other in terms of the specific types of genetic damage detected. It is not expected that chemicals will always give uniformly positive or negative results in the various assays. A chemical may, for example, be positive only in the bacterial mutagenicity assay. Such a result may, nevertheless, be relevant to potential human health effects, including carcinogenicity. Conversely, it has been shown that some chemicals are negative in the bacterial assay but positive in other assays. These chemicals are likely operating by mechanisms that cause chromosomal mutations (large deletions, translocations, gene conversion/mitotic recombination and/or aneuploidy), while the bacterial assay detects only point and other very small-scale gene mutations.

In addition to the mouse lymphoma tk+/- assay, other in vitro mammalian gene mutation assays, which employ other cell lines including the CHO, AS52 and V79 lines of Chinese hamster cells and the TK6 human lymphoblastoid cells, are sometimes used. In these cell lines the most commonly used genetic endpoints measure mutation at either hypoxanthine-guanine phosphoribosyl transferase (hprt), a transgene of xanthine-guanine phosphoribosyl transferase (xprt), or thymidine kinase (tk). The tk, hprt, and xprt mutation tests detect different spectra of genetic events. Differences in sequences in the genes will lead to different spectra of point mutations that can be detected. In addition, the autosomal location of tk and xprt genes appears to allow for the detection of genetic events (e.g., chromosomal exchange events) that are not detected at the hprt locus on the X-chromosome. This is because genetic damage that involves essential genes adjacent to the hprt locus on the X-chromosome will likely be lethal to the cell, while damage to essential genes on an autosome will be compensated for by intact genes on the homologous chromosome (which lacks functional tk or xprt). Also, the lack of a homologous chromosome in the case of the hprt gene may preclude mutations that arise via homologous recombination. The tk locus in mouse lymphoma cells (the mouse lymphoma tk+/- assay) is the preferred target for mammalian cell genotoxicity assays because of the wealth of data which exists to support this locus and assay and because of the broad spectrum of damage detected at this locus.

Modifications of the Test Battery 

a. Limited Effectiveness of Bacterial Tests

There are circumstances where the performance of the bacterial reverse mutation test does not provide sufficient information for the assessment of genotoxicity. This may be the case for compounds that are highly toxic to bacteria (e.g., some antibiotics) and compounds thought or known to interfere with mammalian cell-specific systems (e.g., topoisomerase inhibitors, nucleoside analogues, or certain inhibitors of DNA metabolism). In these cases, usually two in vitro mammalian cell tests should be performed using two different cell types and two different endpoints, i.e., gene mutation and chromosomal damage. Test approaches currently accepted for the assessment of mammalian cell gene mutation include tests for mutation: 1) at the tk locus using mouse lymphoma L5178Y cells or human lymphoblastoid TK6 cells; 2) at the hprt locus using CHO cells, V79 cells, or L5178Y cells; or 3) at the gpt locus using AS52 cells. When such additional tests are performed because of the high level of toxicity of the test chemical to bacteria, it is still important to perform the bacterial reverse mutation test because some antibacterial agents, albeit highly toxic to the tester strains, are genotoxic at very low, sub-lethal concentrations in the bacterial reverse mutation test (e.g., nitrofuran antibiotics).

b. Compounds bearing structural alerts for genotoxic activity

Structurally alerting compounds are usually detectable in the standard test battery. However, compounds bearing structural alerts that have given negative results in the standard test battery may necessitate limited additional testing. The choice of additional test(s) or protocol modification(s) depends on the chemical nature, the known reactivity and metabolism data on the structurally alerting compound under question. For some classes of compounds with specific structural alerts, it is established that protocol modifications/additional tests are necessary for optimum detection of genotoxicity (e.g., molecules containing an azo-group requiring testing of azo reduction products, glycosides requiring testing of hydrolysis products, compounds such as nitroimidazoles requiring nitroreduction for activation, compounds such as phenacetin requiring another rodent S9 for metabolic activation). When the standard test battery gives negative results with a chemical that falls within a class known to require special test conditions, then additional testing with appropriate test modifications should be performed.

c. Limitations to the use of standard in vivo tests

There are compounds for which standard in vivo tests do not provide additional useful information. This includes compounds for which data from studies on toxicokinetics or pharmacokinetics indicate that they are not systemically absorbed and therefore are not available for the target tissues in standard in vivo genotoxicity tests. In cases where sufficient target tissue exposure cannot be achieved, it may be appropriate to base the evaluation only on in vitro testing.

d. Evidence for tumor response

Additional genotoxicity testing in appropriate models may be conducted for food ingredients that were negative in the standard test battery but which have shown effects in carcinogenicity bioassay(s) with no clear evidence for a non-genotoxic mechanism. To help understand the mechanism of action, additional testing can include modified conditions for metabolic activation in in vitro tests or can include in vivo tests measuring genetic damage in target organs of tumor induction (e.g., liver UDS test, 32P-postlabelling, mutation induction in transgenes, or molecular characterization of genetic changes in tumor-related genes).

e. Structurally unique chemical classes

On rare occasions, a completely novel compound in a unique structural chemical class will be introduced. When such a compound will not be tested in chronic rodent carcinogenicity bioassays, more extensive genotoxicity evaluation may be indicated.

Submit Comments

Submit comments on this guidance document electronically via docket ID: FDA-2013-S-0610 - Specific Electronic Submissions Intended For FDA's Dockets Management Staff (i.e., Citizen Petitions, Draft Proposed Guidance Documents, Variances, and other administrative record submissions)

If unable to submit comments online, please mail written comments to:

Dockets Management
Food and Drug Administration
5630 Fishers Lane, Rm 1061
Rockville, MD 20852

All comments should be identified with the title of the guidance.

Back to Top