Guidance for Industry: S2(R1) Genotoxicity Testing
and Data Interpretation for Pharmaceuticals
Intended for Human Use

(PDF version of this document)

This document reached step 2 of the ICH Process on March 6, 2008.

For questions regarding this draft document contact (CDER) David Jacobson-Kram 301-796-0175.

DRAFT
Guidance on Genotoxicity Testing and Data Interpretation
for Pharmaceuticals Intended for Human Use

1.         INTRODUCTION

            1.1  Objectives of the Guideline
This guidance replaces and combines the ICH S2A and S2B guidelines.  The purpose of the revision is to optimize the standard genetic toxicology battery for prediction of potential human risks, and to provide guidance on interpretation of results, with the ultimate goal of improving risk characterization for carcinogenic effects that have their basis in changes in the genetic material.  The revised guidance describes internationally agreed upon standards for follow-up testing and interpretation of positive results in vitro and in vivo in the standard genetic toxicology battery, including assessment of non-relevant findings.

          1.2  Background

Unless otherwise noted in this guidance, the recommendations from the latest OECD guidelines and the reports from the International Workshops on Genotoxicity Testing (IWGT) have been considered where relevant.  The following notes for guidance should be applied in conjunction with other ICH guidances.

1.3  Scope of the Guideline
The primary focus of this guidance is testing of “small molecule” drug substances, and not biologics as defined in the ICH S6 guidance.

1.4  General Principles
Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage by various mechanisms.  These tests enable hazard identification with respect to damage to DNA and its fixation.  Fixation of damage to DNA in the form of gene mutations, larger scale chromosomal damage or recombination is generally considered to be essential for heritable effects and in the multi-step process of malignancy, a complex process in which genetic changes may play only a part.  Numerical chromosome changes have also been associated with tumorigenesis and can indicate a potential for aneuploidy in germ cells.  Compounds that are positive in tests that detect such kinds of damage have the potential to be human carcinogens and/or mutagens.  Because the relationship between exposure to particular chemicals and carcinogenesis is established for humans, whilst a similar relationship has been difficult to prove for heritable diseases, genotoxicity tests have been used mainly for the prediction of carcinogenicity.  Nevertheless, because germ line mutations are clearly associated with human disease, the suspicion that a compound might induce heritable effects is considered to be just as serious as the suspicion that a compound might induce cancer.  In addition, the outcome of genotoxicity tests can be valuable for the interpretation of carcinogenicity studies.

 

2.         THE STANDARD TEST BATTERY FOR GENOTOXICITY

2.1       Rationale
Registration of pharmaceuticals requires a comprehensive assessment of their genotoxic potential.  Extensive reviews have shown that many compounds that are mutagenic in the bacterial reverse mutation (Ames) test are rodent carcinogens.  Addition of in vitro mammalian tests increases sensitivity and broadens the spectrum of genetic events detected, but also decreases the specificity of prediction; i.e., increases the incidence of positive results that do not correlate with rodent carcinogenicity.  Nevertheless, a battery approach is still reasonable because no single test is capable of detecting all genotoxic mechanisms relevant in tumorigenesis.

The general features of a standard test battery are as follows:

1. Assessment of mutagenicity in a bacterial reverse mutation test.  This test has been shown to detect relevant genetic changes and the majority of genotoxic rodent and human carcinogens.
2. Genotoxicity should also be evaluated in mammalian cells in vitro and/or in vivo.

Several in vitro mammalian cell systems are widely used and can be considered sufficiently validated:  The in vitro metaphase chromosome aberration assay, the in vitro micronucleus assay (note 1) and the mouse lymphoma L5178Y cell tk gene mutation assay.  These three assays are currently considered equally appropriate and therefore interchangeable when used together with other genotoxicity tests in a standard battery for testing of pharmaceuticals, if the test protocols recommended in this guideline are used.
In vivo test(s) for genetic damage should usually be a part of the test battery to provide additional relevant factors (absorption, distribution metabolism, excretion) that can influence the genotoxic activity of a compound and permit the detection of some additional genotoxic agents (note 2).  An in vivo test for chromosomal damage in rodent cells largely fulfills this need, either an analysis of micronuclei in erythrocytes in blood or bone marrow, or of chromosomal aberrations at metaphase in bone marrow cells (note 3).  Lymphocytes cultured from treated animals can also be used for cytogenetic analysis, although experience with such analyses is less widespread.

In vitro and in vivo tests that measure chromosomal aberrations in metaphase cells can detect a wide spectrum of changes in chromosomal integrity.  Breakage of chromatids or chromosomes can result in micronucleus formation if an acentric fragment is produced; therefore assays that detect either chromosomal aberrations or micronuclei are appropriate for detecting clastogens.  Micronuclei can also result from lagging of one or more whole chromosome(s) at anaphase and thus micronucleus tests have the potential to detect some aneuploidy inducers.  The mouse lymphoma cell mutation assay detects mutations in the tk gene that result from both gene mutations and changes in chromosome integrity.  There is some evidence that the mouse lymphoma assay can also detect chromosome loss.

There are several additional in vivo assays that can be used in the battery or as follow-up tests to develop weight of evidence in assessing results of in vitro or in vivo assays (see below).  Negative results in appropriate in vivo assays (usually two), with adequate justification for the endpoints measured, and demonstration of exposure (see section 4.8) is sufficient to demonstrate absence of genotoxic activity.

2.2       Description of the two options for the standard battery
The following two options for the standard battery are considered equally suitable:

Option 1

1. A test for gene mutation in bacteria.
2. A cytogenetic test for chromosomal damage (the in vitro metaphase chromosome aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma tk gene mutation assay.
3. An in vivo test for genotoxicity, generally a test for chromosomal damage using rodent hematopoietic cells, either for micronuclei or for chromosomal aberrations in metaphase cells.

Option 2

1. A test for gene mutation in bacteria.
2. An in vivo assessment of genotoxicity with two tissues, usually an assay for micronuclei using rodent hematopoietic cells and a second in vivo assay.

            Under both standard battery options, the in vivo genotoxicity assays can often be integrated into repeat-dose toxicity studies when the doses are sufficient (see section 4.7).  Under Option 2, if dose/exposure is not appropriate, an acute in vivo study (incorporating two genotoxicity assays in one study where possible) should be performed to optimize dose selection based on exposure/toxicity (see sections 4.7.2 and 4.7.3), or Option 1, including an in vitro mammalian cell assay, should be followed.

            For compounds that give negative results, the completion of either test battery, performed and evaluated in accordance with current recommendations, will usually provide sufficient assurance of the absence of genotoxic activity and no additional tests will be needed.  Compounds that give positive results in the standard test battery may, depending on their therapeutic use, need to be tested more extensively (see Section 5).

            The standard battery does not include a required independent test designed specifically to test for aneuploidy.  However, information on numerical changes can be derived from the mammalian cell assays in vitro and from the micronucleus assays.  Elements of the standard protocols that provide such information are elevations in the mitotic index, polyploidy induction and micronucleus evaluation.  There is also experimental evidence that spindle poisons can be detected in the mouse lymphoma tk assay.  The preferred in vivo cytogenetic test under Option 2 is the micronucleus assay, not a chromosome aberration assay, to include more direct capability for detection of chromosome loss (potential for aneuploidy).

            There are several in vivo assays (note 4) that may be used as the second part of the in vivo assessment under option 2 (see section 4.3).  The liver is typically the preferred tissue because of exposure and metabolizing capacity, but choice of in vivo tissue and assay should be based on factors such as any knowledge of the potential mechanism, of the metabolism in vivo, and of the exposed tissues thought to be relevant.  The in vivo genotoxicity assays may be integrated into existing (repeat dose) toxicity studies when the dose levels are justifiable (see section 4.7) and the protocols are compatible.

            The suggested standard set of tests does not imply that other genotoxicity tests are generally considered inadequate or inappropriate.  Additional tests can be used for further investigation of genotoxicity test results obtained in the standard battery (see sections 4.3 and 5).  Alternative species, including non-rodents, can also be used if indicated, and if sufficiently validated.
Under extreme conditions in which one or more tests in the standard battery cannot be employed for technical reasons, alternative validated tests can serve as substitutes provided sufficient scientific justification is given to support the argument that a given standard battery test is not appropriate.

2.3       Modifications to the test battery
            The following sections give situations where modification of the standard test battery may be advisable.

2.3.1    Compounds from well characterized classes
            For compounds from well characterized classes where genotoxicity is expected, e.g., some quinolone antibiotics and some nucleoside analogues, the battery may be modified to characterize these appropriately in the tests/protocols known to respond to them.  (See also note 8).

2.3.2    Testing compounds that are toxic to bacteria
            In cases where compounds are highly toxic to bacteria (e.g., some antibiotics), the bacterial reverse mutation (Ames) test should still be carried out, because mutagenicity can occur at lower, less toxic concentrations.  In such cases, any one of the in vitro mammalian cell assays should be done, i.e., Option 1 is followed.

2.3.3    Compounds bearing structural alerts for genotoxic activity
            Structurally alerting compounds (Note 5) are usually detectable in the standard test battery since the majority of “structural alerts” are defined in relation to bacterial mutagenicity.  A few chemical classes are known to be more easily detected in mammalian cell chromosome damage assays than bacterial mutation assays.  Thus negative results in either test battery with a compound that has a structural alert is usually sufficient assurance of a lack of genotoxicity.  However, for compounds bearing certain specific structural alerts modification to standard protocols can be appropriate (Note 5).  The choice of additional test(s) or protocol modification(s) depends on the chemical nature, the known reactivity and any metabolism data on the structurally alerting compound in question.

2.3.4   Limitations to the use of in vivo tests
            There are compounds for which many in vivo tests (typically in bone marrow, blood or liver) do not provide additional useful information.  These include compounds for which data on toxicokinetics or pharmacokinetics indicate that they are not systemically absorbed and therefore are not available to the target tissues.  Examples of such compounds are some radioimaging agents, aluminum based antacids, some compounds given by inhalation, and some dermally or other topically applied pharmaceuticals.  In cases where a modification of the route of administration does not provide sufficient target tissue exposure, and no suitable genotoxicity assay is available in the most exposed tissue, it may be appropriate to base the evaluation only on in vitro testing.  In some cases evaluation of genotoxic effects at the site of contact may be warranted, although such assays have not yet been widely used (note 6).

2.4       Detection of germ cell mutagens
            Results of comparative studies have shown that, in a qualitative sense, most germ cell mutagens are likely to be detected as genotoxic in somatic cell tests so that negative results of in vivo somatic cell genotoxicity tests generally indicate the absence of germ cell effects.

3.         RECOMMENDATIONS FOR IN VITRO TESTS

3.1       Test repetition and interpretation
            Reproducibility of experimental results is an essential component of research involving novel methods or unexpected findings; however, the routine testing of drugs with standard, widely used genotoxicity tests often does not need replication.  These tests are sufficiently well characterized and have sufficient internal controls that repetition of a clearly positive or negative assay is not usually needed.  Ideally it should be possible to declare test results clearly negative or clearly positive.  However, test results sometimes do not fit the predetermined criteria for a positive or negative call and therefore are declared “equivocal”.  The application of statistical methods can aid in data interpretation; however, adequate biological interpretation is of critical importance.  An equivocal test that is repeated may result in (i) a clearly positive outcome, and thus an overall positive result; (ii) a negative outcome, so that the result is not reproducible and overall negative, or (iii) another equivocal result, with a final conclusion that remains equivocal.

3.2       Recommended protocol for the bacterial mutation assays
           Advice on the protocols is given in the OECD guideline (1997) and the IWGT report (Gatehouse et al, 1994).

3.2.1    Selection of top dose level

Maximum dose level
            The maximum dose level recommended is 5000 µg/plate when not limited by solubility or cytotoxicity.

Limit of solubility
            For bacterial cultures, precipitating doses are scored provided precipitate does not interfere with scoring, toxicity is not limiting, and the top concentration does not exceed 5000µg/plate.  There is some evidence that dose-related genotoxic activity can be detected when testing certain compounds in the insoluble range in bacterial genotoxicity tests.  On the other hand, heavy precipitates can interfere with scoring colonies or render the test compound unavailable to enter cells and interact with DNA. If no cytotoxicity is observed, then the lowest precipitating dose should be used as the top dose scored.  If dose related cytotoxicity or mutagenicity is noted, irrespective of solubility, the top dose scored is based on cytotoxicity as described below.

Limit of cytotoxicity:
            In the bacterial reverse mutation test, the doses scored should show evidence of significant toxicity, but without exceeding a top dose of 5000 µg/plate.  Toxicity may be detected by a reduction in the number of revertants, and/or clearing or diminution of the background lawn.

3.2.2    Study design/Test protocol
            The recommended set of bacterial strains (OECD) includes those that detect base substitution and frameshift mutations as follows:  Salmonella typhimurium TA98; TA100; TA1535; either TA1537 or TA97 or TA97a; and either TA102 or Escherichia coli WP2 uvrA or Escherichia coli WP2 uvrA (pKM101).
            One difference from the OECD and IWGT recommendations is that, based on experience with testing pharmaceuticals, a single bacterial mutation (Ames) test is sufficient when it is clearly negative or positive, and carried out with a fully adequate protocol including all strains with and without metabolic activation, a suitable dose range that fulfills criteria for top dose selection, and appropriate positive and negative controls.  Also, for testing pharmaceuticals, either the plate incorporation or the pre-incubation method is appropriate for this single experiment (note 7).  Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified protocol such as appropriate spacing of dose levels.

3.3       Recommended protocols for the mammalian cell assays
            Advice on the protocols is given in the OECD guidelines (1997) and the IWGT publications (Kirsch-Volders et al 2003; Moore et al 2006).  Several differences from these recommendations are noted here for testing pharmaceuticals, notably for selection of the top concentration, related to the maximum concentration, cytotoxicity and solubility (see details below).

3.3.1    Selection of top concentration

Maximum concentration
            The maximum top concentration recommended is 1 mM or 0.5 mg/ml, whichever is lower, when not limited by solubility or cytotoxicity (note 8).

Limit of solubility
            When solubility is limiting, the maximum concentration if not limited by cytotoxicity, should be the lowest concentration at which minimal precipitate is visible in cultures, provided there is no interference with scoring.  Evaluation of precipitation should be done by methods such as light microscopy, noting precipitate that persists, or appears during culture (by the end of treatment).

Cytotoxicity
            It is not necessary to exceed a reduction of about 50% in cell growth (notes 9 and 10) for in vitro cytogenetic assays for metaphase chromosome aberrations or for micronuclei, or a reduction of about 80% in RTG (relative total growth) for the mouse lymphoma tk mutation assay (note 9).

3.3.2    Study design/Test protocols

            For the cytogenetic evaluation of chromosomal damage in metaphase cells in vitro, the test protocol includes the conduct of tests with and without metabolic activation, with appropriate positive and negative controls.  Treatment with the test articles is for 3 to 6 hours with a sampling time approximately 1.5 normal cell cycles from the beginning of the treatment.  A continuous treatment without metabolic activation up to the sampling time of approximately 1.5 normal cell cycles is needed in case of negative or equivocal results for both short treatments, with and without metabolic activation.  The same principles apply to the in vitro micronucleus assay, except that the sampling time is typically 1.5 to 2 normal cell cycles from the beginning of treatment to allow cells to complete mitosis and enter the next interphase.  For both in vitro cytogenetic assays, certain chemicals may be more readily detected by longer treatment, delayed sampling times or recovery periods, e.g., some nucleoside analogues and some nitrosamines.  In the metaphase aberration assay, information on the ploidy status should be obtained by recording the incidence of polyploid (including endoreduplicated) metaphases as a percentage of the number of metaphase cells.  An elevated mitotic index (MI) or an increased incidence of polyploid cells may give an indication of the potential of a compound to induce aneuploidy.  For the mouse lymphoma tk assay, the test protocol includes the conduct of tests with and without metabolic activation, with appropriate positive and negative controls, where the treatment with the test article is for 3 to 4 hours.  A continuous treatment without metabolic activation for approximately 24 hours is needed in case of a negative or equivocal result for both short treatments, with and without metabolic activation.  An appropriate mouse lymphoma tk assay includes (i) the incorporation of positive controls that induce mainly small colonies, and (ii) colony sizing for positive controls, solvent controls and at least one positive test compound concentration (should any exist), including the culture that gave the greatest mutant frequency.
            For mammalian cell assays in vitro, built-in confirmatory elements, such as those outlined above (e.g., different treatment lengths, tests with and without metabolic activation), are used.  Following such testing, further confirmatory testing in the case of clearly negative or positive test results is not usually needed.  Equivocal or weak positive results may require repeating tests, possibly with a modified protocol such as appropriate spacing of the test concentrations.

3.3.3    Positive controls
            Concurrent positive controls are important, but in vitro mammalian cell tests for genetic toxicity are sufficiently standardized that use of positive controls for chromosome aberration and MLA assays can be confined to a positive control with metabolic activation (provided it is done concurrently with the non-activated test) to demonstrate the activity of the metabolic activation system and the responsiveness of the test system.

4.         RECOMMENDATIONS FOR IN VIVO TESTS

4.1       Tests for the detection of chromosome damage in vivo
            Either the analysis of chromosomal aberrations or the measurement of micronucleated polychromatic erythrocytes in bone marrow cells in vivo is appropriate for the detection of clastogens.  Both rats and mice are appropriate for use in the bone marrow micronucleus test.  Micronuclei may also be measured in immature (e.g., polychromatic) erythrocytes in peripheral blood in the mouse, or in the newly formed reticulocytes in rat blood (note 3).  Likewise, immature erythrocytes can be used from any other species which has shown an adequate sensitivity to detect clastogens/aneuploidy inducers in bone marrow or peripheral blood (note 3).  Chromosomal aberrations can also be analyzed in peripheral lymphocytes cultured from treated rodents (note 11).
            Note that when no in vitro mammalian cell assay is conducted, (Option 2), the micronucleus test in vivo is recommended, not the metaphase chromosome aberration assay, to include more direct capability for detection of chromosome loss (potential for aneuploidy).

4.2       Automated analysis of micronuclei
              Systems for automated analysis (image analysis and flow cytometry) can be used if appropriately validated (OECD, 1997; Hayashi et al 2000; 2007).

4.3       Other in vivo genotoxicity tests
The same in vivo tests described as the second test in the standard battery (option 2) can be used as follow-up tests to develop weight of evidence in assessing results of in vitro or in vivo assays (notes 4 and 11).  While the type of effect seen in vitro and any knowledge of the mechanism can help guide the choice of in vivo assay, investigation of chromosomal aberrations or of gene mutations in endogenous genes is not feasible with standard methods in most tissues; while mutation can be measured in transgenes in rodents this entails prolonged treatment (e.g., 28 days) to allow for mutation expression/fixation, especially in tissues with little cell division.  Thus the second in vivo assay will often evaluate a surrogate (DNA damage) endpoint.  Assays with the most published experience and advice on protocols include the DNA strand break assays such as the single cell gel electrophoresis (“Comet”) assay and alkaline elution assay, the in vivo transgenic mouse mutation assays and DNA covalent binding assays, (all of which may be applied in many tissues, note 4), in addition to the liver unscheduled DNA synthesis (UDS) assay.

4.4       Use of male/female rodents in in vivo genotoxicity tests
            If sex-specific drugs are to be tested, then the assay can be done in the appropriate sex.  In vivo tests by the acute protocol may generally be carried out in only one sex (note 12).  For acute tests both sexes should be considered only if any existing toxicity/metabolism data indicate a substantial sex difference in the species being used.  Otherwise, males alone are appropriate for acute genotoxicity tests.  When the genotoxicity test is integrated into a repeat-dose toxicology study in two sexes, samples can be collected from both sexes, but a single sex can be scored if there is no substantial sex difference evident in toxicity/metabolism.  The dose levels for the sex(es) scored should meet the criteria for appropriate dose levels (sections 4.7.2 and 4.7.3).
Similar principles can be applied for other established in vivo genotoxicitytests.

4.5       Use of multiple administrations in genotoxicity assays in vivo and integration into toxicology studies

4.5.1    Sampling times
            When micronucleus analysis is integrated into multi-week studies, sampling of blood or bone marrow can be done the day after the final administration (see recommendation for additional blood sampling time below).
            When blood or bone marrow is used for micronucleus measurement in a multiweek study (e.g., 28 days), marked hematotoxicity may affect the ability to detect micronuclei, i.e., a dose that induces detectable increases in micronuclei after acute treatment may be too toxic to analyze after multiple treatments.  It can be useful to obtain an additional sample blood on day 2 to 4 of dosing (Hamada et al, 2001); see section 4.7.3).  The early sample can be used if needed to provide assurance that clastogens and potential aneugens are detected (but see notes 13 and 17).
            For other genotoxicity assays, sampling time is selected as appropriate for the endpoint measured; for example DNA damage/strand break measurements are usually made a few (e.g., 2-6) hours after the last administration.
            In principle, studies of any length may be appropriate provided the top dose/exposure is adequate.

4.5.2    Number of animals analyzed
The number of animals analyzed is determined by current recommendations for the micronucleus assay (OECD) or other genotoxicity assays and generally does not include all the animals treated for a toxicology study.  (Animals used for genotoxicity analyses should be randomly selected).

4.6       Route of administration
            The route of administration is generally the expected clinical route, e.g., oral, intravenous or subcutaneous, but can be modified if needed to obtain systemic exposure, e.g., for topically applied compounds (see section 2.3.4).

4.7      Dose selection for in vivo assays
Typically three dose levels are used (Hayashi et al, 2005).

4.7.1    Short-term studies
            For short term (usually 1 to 2 administrations) protocols, the top dose recommended for genotoxicity assays is a limit dose of 2000 mg/kg if this is tolerated, or maximum tolerated dose defined, for example for the micronucleus assay (OECD 474) as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality.  (Similar recommendations have been made for the Comet assay [Hartmann et al, 2003] and transgenic mutation assay [Heddle et al, 2000]).  Suppression of bone marrow red blood cell production may also be taken into account in dose selection.  Lower doses are generally spaced at approximately two to three fold intervals below this. 

4.7.2    Multiple administration studies
            In the Option 1 battery, when the in vitro mammalian cell assay is negative (or “non-relevant positive” (see section 5), if the in vivo genotoxicity test is integrated into a multiple administration toxicology study, the doses are generally considered appropriate when the toxicology study meets the criteria for an adequate study to support human clinical trials.  However, when carrying out follow-up studies to address any indication of genotoxicity, or when using Option 2 with no in vitro mammalian cell assay, several factors should be evaluated to demonstrate that the top dose is appropriate for genotoxicity evaluation, as follows:
Recommendations for determining whether the top dose in a toxicology study (typically in rats) is appropriate for micronucleus analysis and for other genotoxicity evaluation (any one of the following):

1. Maximum feasible dose (MFD) based on physico-chemical properties of the drug in the vehicle (provided the MFD in that vehicle is similar to that achievable with acute administration; note 14).
2. Limit dose of 1000 mg/kg for studies of 14 days or longer, if this is tolerated
3. Exposure: 
   1. Plateau/saturation in exposure
   2. Accumulation 

Substantial reduction in exposure to parent drug with time (e.g., ³ 50% reduction from initial exposure) would usually disqualify the study.  If this is seen in one sex, generally the sex with reduced exposure would not be scored, unless there is enhanced exposure to a metabolite of interest.
iv           Top dose is ³ 50% of the top dose that would be used for acute administration, i.e., close to the minimum lethal dose, if such acute data are available for other reasons.  (The top dose for acute administration micronucleus test is currently described in OECD guidance as the dose above which lethality would be expected; similar guidance is given [e.g. Hartmann et al, 2003] for other in vivo assays.)
            Selection of a top dose based only on an exposure margin (multiple over clinical exposure) without toxicity is not considered sufficient justification.
           If dose levels/exposure are not appropriate, acute in vivo assays should be performed to maximize exposure or obtain the appropriate toxicity range, (preferably conducting two genotoxicity assays in the same animals), or an in vitro mammalian cell assay should be done if not already completed.

4.7.3    Additional guidance on dose selection for multiple administration studies
            Compounds that induce aneuploidy, such as spindle poisons, are typically detectable in in vivo micronucleus assays in bone marrow or blood only within a narrow range of doses approaching toxic doses.  This is also true for some clastogens.  If toxicological data indicate severe toxicity to red blood cell lineage (e.g., marked suppression of PCEs or reticulocytes), doses scored should be spaced not more than about 2 fold below the top, cytotoxic dose.  If suitable doses are not included in a multi-week study, additional data may be required to ensure detection of aneugens and some toxic clastogens; these could be derived from any one of the following:

1. 2 -4 day blood sampling from the multiweek study before substantial hematotoxicity developed
2. an in vitro mammalian cell micronucleus assay
3. An acute bone marrow micronucleus assay

4.8       Demonstration of target tissue exposure for negative in vivo test results
            In vivo tests have an important role in genotoxicity test strategies.  The value of in vivo results is directly related to the demonstration of adequate exposure of the target tissue to the test compound.  This is especially true for negative in vivo test results when in vitro test(s) have shown convincing evidence of genotoxicity, or when no in vitro mammalian cell assay is used.  Evidence of adequate exposure could include toxicity in the tissue in question, or toxicokinetic data.

4.8.1When an in vitro genotoxicity test is positive (or not done)
            Assessments of in vivo exposure should be made at the top dose or other relevant doses using the same species, strain and dosing route used in the genotoxicity assay.  When genotoxicity is measured in toxicology assays, exposure information is generally available as part of the toxicology assessment.
            Demonstration of in vivo exposure should be made by any of the following measurements:

1. Cytotoxicity
   1. For cytogenetic assays: By obtaining a significant change in the proportion of immature erythrocytes among total erythrocytes in the tissue used (bone marrow or blood), at the doses and sampling times used in the micronucleus test or by measuring a significant reduction in mitotic index for the chromosomal aberration assay.
   2. For other in vivo genotoxicity assays:  Toxicity in the liver or tissue being assessed, e.g., by histopathological evaluation or blood biochemistry toxicity indicators.
2. Bioavailability
   1. Measurement of drug related material either in blood or plasma.  The bone marrow is a well perfused tissue and levels of drug related materials in blood or plasma are generally similar to those observed in bone marrow.  Liver is expected to be exposed for drugs with systemic exposure regardless of the route of administration.
   2. Direct measurement of drug-related material in target tissue, or autoradiographic assessment of tissue exposure.

            If systemic exposure is similar to or lower than expected clinical exposure, alternative strategies may be needed such as (i) use of a different route of administration; (ii) use of a different species with higher exposure; (iii) use of a different tissue or assay (see section 2.3.4, “Limitations to the use of standard in vivo tests”.
            If adequate exposure cannot be achieved e.g., with compounds showing very poor target tissue availability, conventional in vivo genotoxicity tests may have little value.

4.8.2    When in vitro genotoxicity tests are negative
            If in vitro tests do not show genotoxic potential, in vivo (systemic) exposure can be assessed by any of the methods above, or can be assumed from the results of standard absorption, distribution, metabolism and excretion (ADME) studies in rodents done for other purposes.

4.9       Use of positive controls for in vivo studies
            For in vivo studies, it is not necessary to include concurrent treatments with positive controls in every study, after a laboratory has established competence in the use of the assay (note 15).

5.           GUIDANCE ON EVALUATION OF TEST RESULTS AND ON FOLLOW-UP TEST STRATEGIES

            Comparative trials have shown conclusively that each in vitro test system generates both false negative and false positive results in relation to predicting rodent carcinogenicity.  Genotoxicity test batteries (of in vitro and in vivo tests) detect carcinogens that are thought to act primarily via a mechanism involving direct genetic damage, such as the majority of known human carcinogens.  Therefore, these batteries are not expected to detect non-genotoxic carcinogens.  Experimental conditions, such as the limited capability of the in vitro metabolic activation systems, can lead to false negative results in in vitro tests.  The test battery approach is designed to reduce the risk of false negative results for compounds with genotoxic potential, whereas a positive result in any assay for genotoxicity does not necessarily mean that the test compound poses a genotoxic/carcinogenic hazard to humans.
            Although positive in vitro data may indicate intrinsic genotoxic properties of a drug, appropriate in vivo data determine the biological significance of these in vitro signals in most cases.  Also, because there are several indirect mechanisms of genotoxicity that operate only above certain concentrations, it is possible to establish a safe level (threshold) for classes of drugs with evidence for such mechanisms (see 5.2. below, Müller and Kasper, 2000; Scott et al, 1991; Thybaud et al 2007).

5.1       Assessment of biological relevance
            The recommendations below assume that the test has been conducted using appropriate spacing of doses, levels of toxicity etc.
            Small increases in apparent genotoxicity in vitro or in vivo should first be assessed for reproducibility and biological significance.  Examples of results that are not considered biologically meaningful include:

1. Small increases that are statistically significant compared with the negative or solvent control values but are within the historical control range for the testing facility
2. Weak/equivocal responses that are not reproducible
               If any of the above conditions apply the weight of evidence indicates a lack of genotoxic potential, the test is considered negative or the findings not biologically relevant, and no further testing is required.
   
   5.2       Evaluation of results obtained in in vitro tests
   In evaluating positive results, especially for the microbial mutagenicity test, the purity of the test compound should be considered, to determine whether the positive result may be attributable to a contaminant.
   
   5.2.1    Evaluation of positive results obtained in vitro in a bacterial mutation assay
               There are some well characterized examples of artefactual increases in colonies that are not truly revertants.  These may occur due to contamination with amino acids, (providing histidine for Salmonella strains or tryptophan for Escherichia Coli strains), so that the bacterial reversion assay is not suitable for testing a peptide that is likely to degrade.  Certain cases exist where positive results in bacterial mutation assays may be shown not to indicate genotoxic potential in vivo in humans, for example when bacterial-specific metabolism occurs, such as activation by bacterial nitroreductases.
   
   5.2.2    Evaluation of positive results obtained in vitro in mammalian cell assays
               Recommendations for assessing weight of evidence and follow-up testing for positive genotoxicity results are discussed in IWGT reports (e.g., Thybaud et al 2007).  In addition, the scientific literature gives a number of conditions that may lead to a positive in vitro result of questionable relevance.  Therefore, any in vitro positive test result should be evaluated based on an assessment of the weight of evidence as indicated below.  This list is not exhaustive, but is given as an aid to decision-making.

1. Conditions that do not occur in vivo (pH; osmolality; precipitates)

Note that the 1 mM limit avoids increases in osmolality, and that if the test compound alters pH it is advisable to adjust pH to the normal pH of untreated cultures at the time of treatment.

2. The effect occurs only at the most toxic concentrations.

          In the MLA increases at ≥80% reduction in RTG
          For in vitro cytogenetics assays when growth is suppressed by ≥50%
      If any of the above conditions apply the weight of evidence indicates a lack of genotoxic potential and no additional testing beyond the standard battery (option 1) with one negative in vivo test would be needed.

5.2.3    Evaluation of in vitro negative results
            For in vitro negative results further testing should be considered in special cases, such as (the examples given are not exhaustive, but are given as an aid to decision-making): The structure or known metabolism of the compound indicates that standard techniques for in vitro metabolic activation (e.g., rodent liver S9) may be inadequate; the structure or known activity of the compound indicates that the use of other test methods/systems may be appropriate.

5.3       Evaluation of results obtained from in vivo tests

            In vivo tests have the advantage of taking into account absorption, distribution and excretion, which are not factors in in vitro tests, but are potentially relevant to human use.  In addition metabolism is likely to be more relevant in vivo compared to the systems normally used in vitro.  If the in vivo and in vitro results do not agree, then the difference should be considered/explained on a case-by-case basis, e.g., difference in metabolism; rapid and efficient excretion of a compound may occur in vivo, etc.
            In vivo genotoxicity tests also have the potential to give misleading positive results that do not indicate true genotoxicity.  For example, increases in micronuclei can occur without administration of any genotoxic agent, due to disturbance in erythropoeisis (Tweats et al, 2007 I), DNA adduct data should be interpreted in the light of the known background level of endogenous adducts, and indirect, toxicity-related effects can influence the results of the DNA strand break assays (e.g., alkaline elution and Comet assays).  Thus it is important to take into account all the toxicological and hematological findings when evaluating the genotoxicity data (note 17).  Indirect effects related to toxicological changes may have a safety margin and may not to be clinically relevant.

5.4       Follow-up strategies for positive results

5.4.1    Follow-up to findings in vitro in mammalian cell tests
The following discussion assumes negative results in the Ames bacterial mutation assay.

5.4.1.1 Mechanistic/in vivo follow-up
            To evaluate in vitro mammalian cell assay positive results for which there is insufficient weight of evidence to indicate lack of relevance, recommended follow-up for mammalian cell assays would be to provide experimental evidence, either by additional in vitro studies or by carrying out two appropriate in vivo assays, as follows:

i.Mechanistic information that contributes to a weight of evidence for a lack of relevant genotoxicity is often generated in vitro, for example evidence that a test compound that induces chromosome aberrations, or mutations in the MLA is not a DNA damaging agent (e.g., other negative mutation/DNA damage tests in addition to the Ames test; structural considerations), or evidence for an indirect/threshold mechanism not relevant in vivo (e.g., inhibition of DNA synthesis, reactive oxygen species produced only at high concentrations, etc, (Galloway et al, 1998; Scott et al, 1991; Muller and Kasper, 2000).  Similar studies can be used to follow up a positive result in the in vitro micronucleus assay, or in this case evidence can include a known mechanism that indicates chromosome loss/aneuploidy, or centromere staining experiments (note 18) that indicate chromosome loss.
If the above mechanistic information and weight of evidence supports the lack of relevant genotoxicity, only a single in vivo test is needed, with appropriate evidence of exposure, to establish the lack of genotoxic activity.  This is typically a cytogenetic assay, and the micronucleus assay in vivo is needed when following up potential for chromosome loss.

Polyploidy is a common finding in chromosome aberration assays in vitro.  While aneugens can induce polyploidy, polyploidy alone does not indicate aneugenic potential and may simply indicate cell cycle perturbation; it is also commonly associated with increasing cytotoxicity.  If polyploidy, but no structural chromosome breakage, is seen in an in vitro assay, generally a negative in vivo micronucleus assay with assurance of appropriate exposure would provide sufficient assurance of lack of potential for aneuploidy induction.

Or
ii.     Two appropriate in vivo assays are done, usually with different tissues, and with supporting demonstration of exposure.

            In summary, if the results of the in vitro mammalian cell assay are positive and there is not sufficient weight of evidence or mechanistic information to rule out relevant genotoxic potential, two in vivo tests are required, with appropriate endpoints and in appropriate tissues (usually two different tissues), and with an emphasis on obtaining sufficient exposure in the in vivo models.
            Negative results in appropriate in vivo assays, with adequate justification for the endpoints measured, and demonstration of exposure (see section 4.8.1) is sufficient to demonstrate absence of genotoxic activity. 

            5.4.1.2 Follow-up to an in vitro positive result that is dependent upon S-9 activation
            When positive results are seen only in the presence of the S-9 activation system, it should first be verified that metabolic activation is responsible and not some other difference in conditions (e.g., low or no serum in the S-9 mix, compared with ³10% serum in the non-activated incubations).  The follow-up strategy is then aimed at determining the relevance of any reactive metabolites produced in vitro to conditions in vivo, and will generally focus on in vivo studies in liver (note 16).

5.4.2    Follow-up to a positive in vivo micronucleus assay
            If there is an increase in micronuclei in vivo, all the toxicological data should be evaluated to determine whether a non-genotoxic effect may be the cause or a contributing factor (note 17).  If non-specific effects of disturbed erythropoeisis or physiology (such as hypo/hyperthermia) are suspected, an in vivo assay for chromosome aberrations may be more appropriate.  If a “real’ increase is suspected, strategies would be needed to demonstrate whether the increase is due to chromosome loss or chromosome breakage (note 18).  There is evidence that aneuploidy induction, e.g., with spindle poisons, follows a non-linear dose response.  Thus, it may be possible to determine that there is a threshold exposure below which chromosome loss is not expected and to determine whether an appropriate safety margin exists compared with clinical exposure.
            In conclusion, the assessment of the genotoxic potential of a compound should take into account the totality of the findings and acknowledge the intrinsic values and limitations of both in vitro and in vivo tests.

5.5       Follow-up genotoxicity testing in relation to tumor findings in a carcinogenicity bioassay
            Additional genotoxicity testing in appropriate models may be conducted for compounds that were negative in the standard test battery but which have shown increases in tumors in carcinogenicity bioassay(s) with insufficient evidence to establish a non-genotoxic mechanism.  To help understand the mode 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 tumour induction, such as DNA strand break assays (e.g., comet or alkaline elution assays), liver UDS test, DNA covalent binding (e.g., by 32P-postlabelling), mutation induction in transgenes, or molecular characterization of genetic changes in tumor-related genes (Kasper et al, 2007).

6.         NOTES

1.         The in vitro micronucleus assay has been widely evaluated in international collaborative studies (Kirsch-Volders et all, 2003), is considered validated by ECVAM (Corvi et al, 2008), and an OECD guideline is in preparation.

2.         There is a small but significant number of genotoxic carcinogens that are reliably detected by the bone marrow tests for chromosomal damage but have yielded negative/weak/conflicting results in the in vitro tests outlined in the standard battery options.  Carcinogens such as procarbazine, hydroquinone, urethane and benzene fall into this category.  Some other examples from a survey of companies are described by Tweats et al, 2007, II.

3.         In principle, micronuclei in hematopoeitic cells may be evaluated in bone marrow from any species, and in blood from species that do not filter out circulating micronucleated erythrocytes in the spleen.  In laboratory mice, micronuclei can be measured in polychromatic erythrocytes in blood, and mature (normochromatic) erythrocytes can be used when mice are treated continuously for about 4 weeks or more.  Although rats rapidly remove micronucleated erythrocytes from the circulation, it has been established that micronucleus induction by a range of clastogens and aneugens can be detected in rat blood reticulocytes (Wakata et al, 1998; Hamada et al 2001).  Rat blood may be used for micronucleus analysis provided methods are used to ensure analysis of the newly formed reticulocytes (Hayashi et al, 2007; MacGregor et al, 2006), and the sample size is sufficiently large to provide appropriate statistical sensitivity given the lower micronucleus levels in rat blood than in bone marrow (Kissling et al, 2007).  Whichever method is chosen, bone marrow or blood, automated or manual analysis, each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation.
     Some experience is now available for micronucleus induction in the dog.  One example where such alternative species might be useful would be in evaluation of a human metabolite that was not sufficiently represented in rodents but was formed in the dog.

4.         The inclusion of a second in vivo assay in the battery is to provide assurance of lack of genotoxicity by use of a tissue that is well exposed to a drug and/or its metabolites; a small number of carcinogens that are considered genotoxic gave positive results in a test in liver but were negative in a cytogenetics assay in vivo in bone marrow.  These examples likely reflect a lack of appropriate metabolic activity or lack of reactive intermediates delivered to the hematopoietic cells of the bone marrow.
Assays for DNA strand breaks, DNA adducts, and mutation in transgenes have the advantage that they can be applied in many tissues.  Internationally agreed protocols are not yet in place for all the in vivo assays, although considerable experience and published data exist for DNA strand break assays (Comet and alkaline elution assays) DNA adduct (covalent binding) measurements and transgenic rodent mutation assays, in addition to the UDS assay.  Because cytotoxicity induces DNA strand breakage, careful cytotoxicity assessment is needed to avoid confounding the results of DNA strand break assays.  This has been well characterized for the alkaline elution assay (Storer et al, 1996) but not yet fully validated for the Comet assay.  In principle the DNA strand break assays may be used in repeat-dose toxicology assays with appropriate dose levels and sampling times.
            Since liver of mature animals is not a highly mitotic tissue, often a non-cytogenetic endpoint is used for the second assay, but with special protocols, or in young rats (Suzuki et al 2005), micronucleus analysis in liver is possible, and detects known genotoxic compounds.

5.         Certain structurally alerting molecular entities are recognized as being causally related to the carcinogenic and/or mutagenic potential of chemicals.  Examples of structural alerts include alkylating electrophilic centers, unstable epoxides, aromatic amines, azo-structures, N-nitroso groups, and aromatic nitro-groups (Ashby and Paton 1994).  For some classes of compounds with specific structural alerts, it is established that specific protocol modifications/additional tests are important for optimum detection of genotoxicity (e.g., molecules containing an azo-group, glycosides, compounds such as nitroimidazoles requiring nitroreduction for activation, compounds such as phenacetin requiring a different rodent S9 for metabolic activation).

6.         There is some experience with in vivo assays for micronucleus induction in skin, liver and colon (Hayashi et al 2007) and DNA damage assays in these tissues can also be an appropriate substitute.

7.         A few chemicals are more easily detectable either with plate-incorporation or with pre-incubation methods though differences are typically quantitative rather than qualitative (Gatehouse et al, IWGT, 1994).  Experience in the pharmaceutical industry where drugs have been tested in both protocols has not resulted in different results for the two methods and in the IWGT report the examples of chemical classes listed as more easily detectable in the pre-incubation protocol are generally not pharmaceuticals and are positive in in vivo genotoxicity tests in liver.  These include short chain aliphatic nitrosamines; divalent metals; aldehydes (e.g., formaldehyde, crotonaldehyde); azo dyes (e.g., butter yellow); pyrrolizidine alkaloids; allyl compounds (Allylisothiocyanate, allyl chloride), and nitro (aromatic, aliphatic) compounds.

8.         The rationale for a maximum concentration of 1 mM for in vitro mammalian cell assays includes the following:  The test battery includes the Ames test and an in vivo assay.  Viewing the battery as a whole means that it is not necessary to detect in the mammalian cell assay every compound considered to be a genotoxic carcinogen.  There is a low likelihood of such compounds of concern (DNA damaging carcinogens) that are not detected in Ames test or in vivo genotoxicity assay, but are detectable in an in vitro mammalian assay only above 1 mM.  Second, a limit of 1 mM maintains the element of hazard identification, being higher than clinical exposures to known pharmaceuticals, including those that concentrate in tissues (Goodman & Gilman's, 2001), and is also higher than the levels generally achievable in preclinical studies in vivo.  Certain drugs are known to require quite high clinical exposures, e.g., nucleoside analogs and some antibiotics.  While comparison of potency with existing drugs may be of interest to sponsors, perhaps even above the 1 mM limit, it is ultimately the in vivo tests that determine relevance for human safety.

9.         Although some genotoxic carcinogens are not detectable in in vitro genotoxicity assays unless the concentrations tested induce some degree of cytotoxicity, particularly when measured by colony forming assays, DNA damaging agents are generally detectable with only moderate levels of toxicity (e.g., 30% reduction in growth measured at the time of sampling in the chromosome aberration assay, Greenwood et al, 2004).  As cytotoxicity increases, mechanisms other than direct DNA damage by a compound or its metabolitescan lead to ‘positive’ results that are related to cytotoxicity and not genotoxicity.  Such indirect induction of DNA damage secondary to damage to non-DNA targets are more likely to occur above a certain concentration threshold.  The disruption of cellular processes is not expected to occur at lower, pharmacologically relevant concentrations.
In cytogenetic assays, even weak clastogens that are known to be carcinogens are positive without exceeding a 50% reduction in cell counts.  On the other hand, compounds that are not DNA damaging, mutagenic or carcinogenic can induce chromosome breakage at toxic concentrations.  For both in vitro cytogenetic assays, the chromosome aberration assay and the in vitro micronucleus assay, a limit of about 50% growth reduction is appropriate.
For cytogenetic assays in cell lines, measurement of cell population growth over time (by measuring the change in cell number during culture relative to control, e.g., by the method referred to as population doubling (PD; note 10), has been shown to be a useful measure of cytotoxicity, as it is known that cell numbers can underestimate toxicity.  For lymphocyte cultures, an inhibition of proliferation not exceeding about 50% is considered sufficient; this can be measured by mitotic index (MI) for metaphase aberration assays and by an index based on cytokinesis block for in vitro micronucleus assays.  In addition, for the in vitro micronucleus assay, since micronuclei are scored in the interphase subsequent to a mitotic division, it is important to verify that cells have progressed through the cell cycle.  This can be done by use of cytochalasin B to allow nuclear division but not cell division, so that micronuclei can be scored in binucleate cells (the preferred method for lymphocytes).  For cell lines other methods to demonstrate cell proliferation, including cell population growth over time (PD) as described above, may be used (Kirsch-Volders et al 2003).
For the mouse lymphoma assay, appropriate sensitivity is achieved by limiting the top concentration to one with close to 20% Relative Total Growth (RTG) both for soft agar and for microwell methods (IWGT).  Reviews of published data using the current criteria described by Moore et al (2006) found very few chemicals that were positive in MLA only at concentrations with less than 20% RTG and that were rodent carcinogens, and convincing evidence of genotoxic carcinogenesis for this category is lacking.  The consensus (Moore et al, 2006) is that caution is needed in interpreting results when increases in mutation are seen only below 20% RTG, and a result would not be considered positive if the increase in mutant fraction occurred only at £ 10% RTG.
In conclusion, caution is appropriate in interpreting positive results obtained as reduction in growth/survival approaches or exceeds 50% for cytogenetics assays or 80% for the mouse lymphoma assay.  It is acknowledged that the evaluation of cells treated at these levels of cytotoxicity/clonal survival may result in greater sensitivity, but bears an increased risk of non-relevant positive results.  The battery approach for genotoxicity is designed to ensure appropriate sensitivity without the need to rely on single in vitro mammalian cell tests at high cytotoxicity.
To obtain an appropriate toxicity range, a preliminary range-finding assay over a broad range of concentrations is useful, but in the genotoxicity assay it is often critical to use multiple concentrations that are spaced quite closely (less than two–fold dilutions).  Extra concentrations may be tested but not all need be evaluated for genotoxicity.  It is not intended that multiple experiments be carried out to reach exactly 50% reduction in growth, for example, or exactly 80% reduction in RTG.

10.       For in vitro cytogenetic assays it is appropriate to use a measure of relative cell growth to assess toxicity, because cell counts can underestimate toxicity (Greenwood et al, 2004).  Using calculated population doublings to estimate the 50% growth reduction level it was demonstrated that the frequency of positive results with compounds that are not mutagenic or carcinogenic is reduced, while true DNA damaging agents are reliably positive.

11.       In certain cases it may be useful to examine chromosome aberrations at metaphase in lymphocytes cultured from test animals after one or more administrations of test compound, just as bone marrow metaphase cells may be used.  Because some lymphocytes are relatively long-lived, in principle there is the potential for accumulation of un-repaired DNA damage in vivo, that would give rise to aberrations when the cells are stimulated to divide in vitro.  The in vivo lymphocyte assay may be useful in following up indications of clastogenicity, but in general another tissue such as liver is a more informative supplement to the micronucleus assay in hematopoeitic cells because exposure to drug and metabolite(s) is often higher in liver.

12.       Extensive studies of the activity of known clastogens in the acute mouse bone marrow micronucleus test have shown that in general male mice are more sensitive than female mice for micronucleus induction.  Quantitative differences in micronucleus induction have been identified between the sexes, but no qualitative differences have been described.  Where marked quantitative differences exist, there is invariably a difference in toxicity between the sexes.  Thus males alone can be appropriate for acute in vivo micronucleus tests.

13.       Caution is required if the toxicological study design includes additional blood sampling, e.g., for measurement of exposure.  Such bleeding could perturb the results of micronucleus analysis since erythropoeisis stimulated by bleeding can lead to increases in micronucleated erythrocytes.

14.       For common vehicles like aqueous methyl cellulose this would usually be appropriate, but for vehicles such as Tween 80, the volume that can be administered could be as much as 30 fold lower than that given acutely.

15.       For micronucleus (and other cytogenetic) assays, the purpose of the positive control is to verify that the individuals scoring the slides can reliably detect increases in micronuclei.  This can be accomplished by use of samples from periodic studies of small groups of positive control animals (one sex).  For manual scoring such slides can be included in coded slides scored from each study, or used for periodic demonstration of ability of readers to recognize positive responses.  Positive control slides should not be obvious to readers based on their staining properties or micronucleus frequency.  For automated scoring, appropriate quality control samples should be used with each assay.
            For other in vivo genotoxicity assays, the purpose of positive controls is to demonstrate reliable detection of an increase in DNA damage/mutagenicity using the assay in the chosen species, tissue and protocol.  After a laboratory has demonstrated that it can consistently detect appropriate positive control compounds in multiple independent experiments, it is no longer necessary to carry out concurrent controls with every assay using that protocol, but controls can be tested periodically.

16.       Standard induced S-9 mix has higher activation capacity than human S-9, and lacks phase two detoxification capability unless specific cofactors are supplied.  Also, non-specific activation can occur in vitro with high test substrate concentrations (see Kirkland et al, 2007).  Genotoxicity testing with human S-9 or other human-relevant activation systems can be helpful.  Analysis of the metabolite profile in the genotoxicity test incubations for comparison with known metabolite profiles in preclinical species, (in uninduced microsomes or hepatocytes, or in vivo), or in preparations from humans, canalso help determine the relevance of test results (Ku et al, 2007), and follow-up studies will usually focus on in vivo testing in liver.  A compound that gives positive results in vitro with S-9 may not induce genotoxicity in vivo because the metabolite is not formed, is formed in very small quantities, or is metabolically detoxified or rapidly excreted, indicating a lack of risk in vivo.

17.       Increases in micronuclei can occur without administration of any genotoxic agent, due to disturbance in erythropoeisis (such as regenerative anemia; extramedullary hematopoeisis), stress, hypo- and hyperthermia (reviewed by Tweats et al 2007I, IWGT).  In blood, changes in spleen function that affect clearance of micronucleated cells from the blood are expected to lead to increases in circulating micronucleated red blood cells.

18.       Determination of whether micronucleus induction is due primarily to chromosome loss or to chromosome breakage could include staining micronuclei in vitro or in vivo to determine whether centromeres are present. e.g., using fluorescent in situ hybridization (FISH) with probes for DNA sequences in the centromeric region, or a labeled antibody to kinetochore proteins.  If the majority of induced micronuclei are centromere positive, this suggests chromosome loss.  (Note that even potent tubule poisons like colchicine and vinblastine do not produce 100% kinetochore positive micronuclei, but more typically 70 to 80%, but are accepted as primarily aneugens for assessing risk).  An alternative approach is to carry out an in vitro or in vivo assay for metaphase structural aberrations; if negative this would infer that micronucleus induction is related to chromosome loss.

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