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Implications of the revised ICH guideline on genotoxicity testing S2(R1)

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Implications of the revised ICH guideline on genotoxicity testing S2(R1)

he International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has approved a revision to its genotoxicity testing and data interpretation guidelines. The revision, designated S2(R1), was approved for publication in the three ICH regions (Europe, USA and Japan) on 9th November 2011, and replaces and combines the previous S2A and S2B guidelines, issued in 1995 and 1997 respectively[1,2,3]. The purpose of the revision is to optimise 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 characterisation for carcinogenic effects that have their basis in changes in the genetic material. The revised guidance describes internationally agreed 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. The primary focus of this guidance is testing of small molecule drug substances, and not biologics. Significant benefits of the revision are that it incorporates accumulated knowledge specific to pharmaceuticals, takes advantage of new technologies, includes more options in the test battery, reduces delays caused by non-relevant in vitro positive results, reduces animal use and enables more efficient use of resources. Table 1: Test battery: comparison with original S2A/B guidelines S2A/B
Bacterial reverse mutation test Chromosome aberration test or mouse lymphoma assay In vivo chromosome aberration test or In vivo micronucleus test

It was recognised that the two in vitro mammalian genotoxicity tests described in the previous guideline, the chromosome aberration assay and the mouse lymphoma tk mutation assay, had a tendency towards generating a high level of positive results that may not be relevant to human risk[4]. The revised guideline therefore outlines changes to the methods to reduce the incidence of so-called false positives, and includes an additional third option, the in vitro micronucleus test. Some of the changes have caused some concern with regard to animal usage and the 3Rs (replacement, refinement and reduction) for in vivo testing. However, it is clear that these concerns have been addressed with the option of running dual micronucleus and comet assay endpoints in the same animal. These are discussed later in the document.

Background

S2(R1) Option 1:
Bacterial reverse mutation test Chromosome aberration test or Mouse lymphoma assay or In vitro micronucleus test In vivo chromosome aberration test or In vivo micronucleus test

For bacterial reverse mutation (Ames) tests, the previous guideline required that two independent assays be conducted; if a negative or equivocal result was obtained in the first assay, the test conditions should be modified, e.g. by varying the test concentrations or including a pre-incubation stage. There is no requirement in the revised guideline for a second assay when the result is clearly either negative or positive. Experience with testing pharmaceuticals has shown that there is rarely any advantage in the pre-incubation method[5]. For the single test, therefore, either the plate-incorporation or pre-incubation options will be equally accepted. For mammalian cell assays, the maximum test concentration of a soluble, non-cytotoxic substance has been lowered from 10 mM to 1mM (or 0.5 mg/mL, whichever is the lower). The previous guideline required that testing continue into the insoluble (i.e. precipitating) range if cytotoxicity continued to increase. The maximum test concentration will now be the lowest precipitating concentration, provided that there is no interference with scoring. The new guideline also clarifies cytotoxicity limits. For in vitro assays which detect metaphase chromosome aberrations or micronuclei, it will not be necessary to exceed a reduction of approximately 50% in cell growth. For the

In vitro tests

Option 2:
Bacterial reverse mutation test In vivo micronucleus test In vivo comet assay/UDS

mouse lymphoma tk mutation assay, there should be 80-90% cytotoxicity as measured by a reduction in RTG (relative total growth) to 10-20%. In addition, it will no longer be necessary to include positive controls without metabolic activation provided that the non-activated test is conducted concurrently with an activated test which includes a positive control with metabolic activation. Table 2: In vitro tests: comparison with original S2A/B guidelines S2A/B
Two replicate bacterial tests required Maximum concentration for mammalian cell assays 10 mM or 0.5 mg/mL Analysis of precipitating concentrations required for mammalian cell assays

50% of the top dose that would be used for acute administration, if such data are available. In addition, it will be necessary to score only male animals if there is no meaningful sex difference.

Table 3: In vivo tests: comparison with original S2A/B guidelines S2A/B

Positive controls required in every test) Integration with repeat-dose toxicity studies not considered

S2(R1)
Positive controls not required in every test Guidance provided for integration with repeat-dose toxicity studies

S2(R1)
Only one bacterial test required Maximum concentration for mammalian cell assays 1 mM or 0.5 mg/mL Analysis of only one precipitating concentration required

In vivo tests
Either the analysis of chromosome aberrations or the rodent bone marrow micronucleus test remain the recommended tests, with an option of analysing micronucleus induction in peripheral blood, provided that methods are used to ensure analysis of newlyformed reticulocytes. The micronucleus test is thus readily analysable using flow cytometry[6]. The requirement for inclusion of concurrent positive controls in every in vivo micronucleus test has been removed for laboratories that have demonstrated competence in the use of the assay, but remains advisable for the comet assay. It should also be noted that, although there is no OECD guideline for the comet assay, readily acceptable protocols are available which conform to the IWGT or JaCVAM recommendations[7,8]. There is now an option of integrating in vivo genotoxicity tests, such as the rodent bone marrow micronucleus test or the comet assay, into general toxicology tests, e.g. a 28-day repeat dose test[9,10], and guidance is given on ensuring that the highest dose is appropriate for genotoxicity evaluation. For a stand-alone shortterm in vivo genotoxicity study, the recommended top dose remains at 2000 mg/kg or the maximum tolerated dose (MTD). The revised guidelines state that any one of the following criteria is sufficient to demonstrate that the top dose is adequate for micronucleus or other genotoxicity evaluation: Maximum Feasible Dose (MFD), based on physicochemical properties of the drug; limit dose of 1000 mg/ kg for studies of 14 days or longer, if tolerated: Demonstration of exposure, plateau/saturation or accumulation, provided that exposure is not reduced with time.

The new guideline provides two options for a genotoxicity test battery. The test battery approach is designed to reduce the risk of false negative results for substances with genotoxic potential as well as reducing false positives. The first option is broadly similar to the battery described in the original guideline, i.e. a bacterial reverse mutation test, an in vitro mammalian cell assay (chromosome aberration, micronucleus or mouse lymphoma tk) and an in vivo test (chromosome aberration or micronucleus). The second option is to conduct a bacterial reverse mutation test and two in vivo tests, one being a micronucleus test and the second selected from the comet assay, transgenic mouse mutation assay, alkaline elution assay, DNA covalent binding assay, or the liver unscheduled DNA synthesis (UDS) assay (although this assay is viewed with some concern because of its apparent insensitivity). The selection of the second assay requires suitable justification and, as in the previous guideline, evidence of exposure to the selected tissue should be obtained.

Overall testing strategy

The new guideline provides advice on the evaluation of test results and on follow-up strategies. Key to evaluation of in vitro positive results is the assessment of biological relevance, e.g. small but statistically significant increases in genotoxicity that do not fall outside historical data ranges or are not reproducible, or where the conditions under which the positive result was obtained do not occur in vivo. If there is insufficient weight of evidence, the guideline recommends follow-up in vitro mechanistic studies or two in vivo assays, usually with different tissues. The guideline recommends that a positive in vivo micronucleus assay be followed up by first examining all the toxicology data to determine whether or not a non-genotoxic event could be the cause or a contributing factor. If not, a strategy to determine whether the effect is due to chromosome loss or chromosome breakage, e.g. using fluorescence in situ hybridization (FISH) staining techniques should be applied.

Guidance on interpretation of data

Aneuploidy induction may be non-linear and it may also 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[11].

9. Hayashi, M. et al, In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing and automated scoring. Environmental and Molecular Mutagenesis 35, 234-252. 2000. 10. Rothfuss, A. Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 732 (2), 108-120. 2011. 11. Mller, L. and Kasper, P. Human biological relevance and the use of threshold arguments in regulatory genotoxicity assessment: Experience with pharmaceuticals. Mutation Research 464, 9-34. 2000.

References:
1.ICH Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. 1995.
2. ICH Guideline S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. 1997. 3. ICH Guideline S2(R1). Genotoxicity testing and data interpretation for pharmaceuticals intended for human use. EMA/CHMP/ ICH/126642/2008. November 2011. 4. Kirkland, D. et al, How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop. Mutation Research 628, 31-55. 2007. 5. Gatehouse, D.G. et al, Report from the working group on bacterial mutation assays: International Workshop on Standardisation of Genotoxicity Test Procedures. Mutation Research 312, 2170-233. 1994. 6. MacGregor, J.T. et al, Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal changes in the rat. Toxicological Sciences 94, 92107. 2006. 7. Burlinson, B. The In Vitro and In Vivo Comet Assays. Methods in Molecular Biology, 817, 143-163, DOI: 10.1007/978-1-61779-4216_8). 2012. 8. Burlinson, B. et al, Fourth International Workgroup on Genotoxicity testing: Results of the in vivo Comet assay workgroup. Mutation Research, 607, 31-35. 2007.

Key Points

Original S2A and S2B guidelines merged. Additional options provided for test battery. Reduction of irrelevant positives in in Integration

vitro mammalian cell assays by reduction of maximum test concentration. of in vivo assays into repeat dose toxicology studies. every in vivo assay. as standard.

Concurrent positive controls no longer required in Duplicate bacterial mutation test no longer required