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THE SCIENCE CORNER

GENOTOXICITY TESTING of CANDIDATE DRUGSUPDATE ON ICH S2 (R1)

Safety and Health Research Laboratories

Safety and Health Research Laboratories

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Safety and Health Research Laboratories

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GLOBAL EXPERTISE, LOCAL RESPONSE

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The ICH S2(R1) «Guidance on genotoxicity testing and data

interpretation for pharmaceuticals intended for human use»

was approved by the ICH Steering Committee on

9 November 2011 and has since been adopted by major

regulatory authorities around the world. It replaces the

previous ICH S2A and S2B documents. The guidance has

been updated extensively to reflect scientific developments

relating to study selection and design, and also to provide

a useful expanded section on supplementary testing

when genotoxicity is detected in the initial test battery.

In this brief overview, we have outlined some of the changes,

weighed the merits of the two battery options and explained

some of the possible testing strategies.

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Experience shows that false positive results may be obtained in genotoxicity testing. There had been some concern that in vitro mammalian cell tests were too sensitive for some compounds, detecting genotoxic effects not thought to be relevant to humans. These «false positive» results were often attributed to non-specific effects (e.g. excessive toxicity or other marked changes in cell physiology) which are only observed at in vitro concentrations significantly above those expected in the intended human use.

This concern has been largely addressed for the in vitro mouse lymphoma assay over recent years by an expert working group of the International Workshop on Genotoxicity Testing, using data-based evaluations to establish appropriate criteria for test performance and data interpretation. S2(R1) refers to these criteria and adds that the maximum test concentration required for soluble compounds of low toxicity is reduced to 0.5 mg/mL, or 1 mM in all in vitro mammalian cell studies. Testing at higher concentrations should only be considered for pharmaceuticals with unusually low molecular weight (e.g. less than 200 g/mol).

In vitro studies previously included at least two tests to investigate the reproducibility of results, except when a test item was clearly genotoxic in the first test. It is now accepted that a single test including appropriate treatment conditions and replicate cultures is adequate, except when the data is equivocal or weakly positive.

Most in vitro mammalian cell studies have included positive control treatments both with and without metabolic activation (e.g. S9 mix), but it is now accepted that a positive control treatment with metabolic activation is sufficient when the test without metabolic activation is made concurrently.

S2(R1) includes several changes that improve animal welfare (e.g. the assessment of genotoxicity endpoints in general toxicology studies and the incorporation of multiple endpoints into a single study are encouraged, which avoids the use of additional animals in one or more separate specific genotoxicity studies).

Detailed advice is provided, concerning the design of combination general toxicology/genotoxicity studies, including considerations for dose selection, target tissues, sampling times, numbers of animals, use of one or both sexes, route of administration, etc. (e.g. dose levels for a multi-administration toxicology study that meet ICH criteria to support human clinical trials will also generally be considered acceptable for genotoxicity determination in that study, provided the compound is non-genotoxic in the in vitro mammalian cell study). Dose levels chosen using the S2(R1) guidance can differ from those chosen according to OECD criteria for an in vivo micronucleus study (e.g. OECD 474).

SUMMARY OF CHANGES

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Guidance is also provided on dose level selection for an in vivo combination study when the compound has not been tested in an in vitro mammalian cell genotoxicity study, or has given positive results in that study.

In S2(R1), it is recognised that positive in vivo control treatments are included in studies to demonstrate the

reliability of scoring or the study system. Experienced laboratories can now make positive control treatments periodically. Reference slides or other samples generated can then be used for scoring in multiple studies. This reduces animal usage.

OPTION 1 OPTION 2

A test for gene mutation in bacteria A test for gene mutation in bacteria

A cytogenetic test for chromosomal damage (in vitro metaphase chromosome aberration test or in vitro micronucleus test)

or

an in vitro mouse lymphoma Tk gene mutation assay

An in vivo assessment of genotoxicity with two different tissues.

Acceptable tests include:

• Chromosome aberrations and micronucleus assays

• DNA strand break assays, such as: a single cell gel electrophoresis (“Comet”) assay and an alkaline elution assay

• In vivo transgenic mouse mutation assay and DNA covalent binding assay

• Liver unscheduled DNA synthesis (UDS) assay

An in vivo genotoxicity test (generally a chromosomal damage test using rodent hematopoietic cells, either for micronuclei or chromosome aberrations in metaphase cells)

5OPTION 1 VERSUS OPTION 2

There are two testing pathways, each with a standard battery of tests.

The major advantage of Option 1 is that a smaller amount of test item is usually required and, as only one in vivo test should be included, it also has an advantage over Option 2 from an animal welfare point of view (except for multi-endpoint studies). Also, the recommended maximum concentration has been lowered for in vitro genotoxicity tests, so the occurrence of false positive results will be reduced. However, a positive in vitro result should be followed by two in vivo studies (as in Option 2) to provide sufficient evidence of a lack of genotoxic potential in vivo.

In Option 2, some of the tests are laborious and costly, but the overall cost of the package could be lowered by integration of the two in vivo tests or an in vivo genotoxicity test into other regulatory toxicology assays (without impacting scientific value or human risk evaluation).

Assessment for DNA strand breaks and DNA adducts have the advantage that they can be applied in many tissues; thus they could be easily incorporated into routine repeat-dose toxicology assays with appropriate dose levels and sampling times.

The liver is typically the preferred tissue because of exposure and metabolizing capacity, but the choice of an in vivo tissue and assay should be based on factors such as mechanism of action, route of administration and target tissue. A popular combination is an in vivo micronucleus test (from blood or bone marrow) and an in vivo Comet assay (from liver or colon).

Internationally agreed protocols are not currently available for all study types that can be selected as the second in vivo study in Option 2.

Experience with the study types may vary between different regulatory authorities, so their preferences for specific study types may vary. It is advisable to check that the relevant regulatory authorities consider that the proposed selection and design of genotoxicity studies are acceptable and scientifically appropriate.

In some cases, modification of the standard test battery might be advisable. There are compounds for which many in vivo tests (typically in bone marrow, blood or liver) do not provide additional useful information, as the compounds are not systemically absorbed, and thus not available to target tissues (some radio-imaging agents, aluminium based antacids, some compounds given by inhalation, some ocular drugs, dietary polymeric drugs and some dermally or other topically applied pharmaceuticals are included in this category). In cases where a modification of the administration route does not provide sufficient target tissue exposure, and no suitable genotoxicity assay is available in the most exposed tissue, it might be appropriate to base the evaluation on in vitro testing only.

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Though Option 1 and 2 strategies identify most intrinsically genotoxic compounds with a high degree of specificity, a number of false positives are still generated. Follow-up strategies exist to qualify the human biological significance of a positive result and may, in some cases, allow the further development of a drug candidate even though it has tested positive in in vitro tests.

Low test item purity can cause a bacterial mutation positive result. As the Ames test is placed earlier in the drug development process, batches may have lower purity, and the likelihood of a test item containing genotoxic contaminants leading to false positives is higher.

If contaminants are not an issue, the current guideline suggests extensive in vivo follow up studies to assess potential risk to humans. For both bacterial mutation and in vitro mammalian positive results that only occur in the presence of metabolizing enzymes, it would be relevant to review the species-related differences in metabolite formation and, if necessary, repeat the study with human metabolic activation as opposed to the more typical rat liver S9 fraction.

An important consideration for mammalian cell tests is the choice of cell type, as recent evidence suggests misleading

(false) positive results with certain cell lines. Other general considerations are the in vivo relevance of in vitro testing conditions (e.g. pH, osmolality and precipitation exclusively observed in vitro).

The appearance of cytotoxicity concurrently with genotoxicity may be indicative of specific mechanisms, so knowledge of the mechanism by which DNA is damaged can help to make a weight of evidence argument.

If DNA damage is the result of reactive oxygen species (ROS), causing a general decline in cell health and the appearance of genotoxicity, in vitro studies to demonstrate ROS production and the detection of oxidized DNA bases may allow the weight of evidence to suggest unlikely in vivo relevance. Similarly, compounds that interfere with cell cycle progression, chromosome separation and spindle formation are often found positive in the in vitro micronucleus assay through a mechanism of chromosome loss. Distinguishing between clastogens and aneugens through fluorescent in situ hybridization of centromeres can help with risk assessment, as aneugens are considered to have a threshold of no effect at low dose levels, whilst clastogens are not.

Ultimately, the revised ICH guideline recommends two in vivo studies for test items with in vitro mammalian positive results for which the mechanism is not understood or the weight of evidence insufficient. The in vivo studies should then be performed as suggested earlier, with justified endpoints in appropriate tissues.

An in vivo micronucleus positive result may also be evaluated from a weight of evidence perspective. Compound-induced effects on erythropoiesis and body temperature can increase the formation of micronuclei, leading to a false positive result, so an alternative end point for the study (chromosome aberrations) might be more appropriate.

POSITIVE RESULTS, WHAT NEXT?

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The ICH S2(R1) guideline is a far-reaching revision that introduces many

refreshing new elements into genetic toxicology testing of candidate drugs,

rendering test procedures more efficient, logical and 3R’s friendly, and assisting

the investigator faced with apparently positive results.

Numerous changes in study design are described in S2(R1); these changes

have already been implemented at CiToxLAB, and our study plans and the

testing procedures in use at CiToxLAB are now fully ICH S2(R1) compliant.

For the Sponsor, the major issue is the choice of testing strategy – whether

to select testing Option 1 or 2 – and we have provided some comments

on approaching this choice. If you are unlucky enough to obtain positive

genotoxicity results for your candidate drug, the ICH document describes

possible pathways to resolution of the problem.

The CiToxLAB Genetic Toxicology team looks forward to discussing your testing needs with you in further detail !

Genetic Toxicologists at CiToxLABCiToxLAB France

•Nick Pearson•Guillaume Sire•Séverine Sarlang•Alizée Brient

CiToxLAB Scantox

•Nick Edwards

CiToxLAB Hungary

•Judit Hargitai

GLOBAL EXPERTISE, LOCAL RESPONSESafety and Health Research Laboratories

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