Guidance on Genotoxicity testing and data interpretation for pharamaceutials intended for human use S2(R1)

        INTRODUCTION

1. Objectives of the Guideline

The ICH S2A and S2B Guidelines are combined and replaced by this guidance. With the ultimate goal of improving risk characterisation for carcinogenic effects that have their basis in genetic material, the revision aims to optimise the standard genetic toxicology battery for prediction of potential human risks and to offer guidance on result interpretation. The updated guidelines outline globally accepted criteria for in vitro and in vivo follow-up testing and interpretation of positive results in the standard genetic toxicology battery, including assessment of non-relevant data. Only products being developed as human pharmaceuticals are meant to be covered by this guidance.

1.2.       Scope of the Guideline

          This guidance does not apply to biologics; instead, it focuses on testing novel “small molecule” drugs. The ICH M3 (R2) guidance offers recommendations regarding the scheduling of the studies in relation to clinical development.

1. General Principles

          Genotoxicity tests, conducted both in vitro and in vivo, are used to identify compounds that cause genetic damage through various mechanisms, crucial for hazard identification related to DNA damage and its fixation. This fixation leads to gene mutations and chromosomal damage, important in hereditary effects and tumorigenesis. Compounds that exhibit positive results in these tests may be carcinogenic or mutagenic. While establishing a direct link between chemical exposure and heritable diseases is challenging, these tests primarily predict carcinogenicity, with germ line mutations also posing significant health concerns. The results from genotoxicity tests are instrumental in interpreting carcinogenicity studies.

• THE STANDARD TEST BATTERY FOR GENOTOXICITY
  • Rationale

Registration of pharmaceuticals necessitates a thorough evaluation of their genotoxic potential. Reviews indicate that many compounds identified as mutagenic in the Ames test are carcinogenic in rodents. While incorporating in vitro mammalian tests enhances sensitivity for identifying rodent carcinogens and expands the range of genetic events detected, it also reduces predictive specificity, leading to more positive results that may not align with rodent carcinogenicity. Despite this limitation, a battery approach remains sensible as no single test can detect all genotoxic mechanisms involved in tumorigenesis.

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

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

There are several popular and well validated in vitro mammalian cell systems: The mouse lymphoma L5178Y cell Tk (thymidine kinase) gene mutation test (MLA), the in vitro metaphase chromosome aberration test, and the in vitro micronucleus test (Note 1). If the test procedures suggested in this Guideline are followed, these three assays are currently regarded as equally suitable and hence interchangeable for measuring chromosomal damage when used in conjunction with other genotoxicity tests in a standard battery for pharmaceutical testing.

Tests that quantify chromosomal abnormalities in metaphase cells both in vitro and in vivo can identify a broad range of chromosomal integrity alterations. Assays that identify either chromosomal abnormalities or micronuclei are thought to be suitable for identifying clastogens since chromatid or chromosome breakage can lead to micronucleus development if an acentric fragment is created. Micronucleus tests have the ability to identify some aneuploidy inducers because micronuclei can also arise from lagging of one or more entire chromosomes during anaphase. The MLA finds Tk gene mutations caused by chromosome damage as well as gene mutations. There is considerable indication that chromosomal loss can also be detected by MLA.

In order to develop the weight of evidence when evaluating the results of in vitro or in vivo assays, a number of additional in vivo tests can be employed in the battery or as follow-up tests (see below). It is generally accepted that the lack of a major genotoxic risk can be demonstrated by negative results in suitable in vivo assays (often two), sufficient justification for the endpoints assessed, and evidence of exposure (see Section 4.4).

• Description of the Two Options for the Standard Battery

The following two options for the standard battery are considered equally suitable (Note 4):

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 different tissues, usually an assay for micronuclei using rodent hematopoietic cells and a second in vivo assay. Typically this would be a DNA strand breakage assay in liver, unless otherwise justified (see below; also Section 4.2 and Note 12).

There is a greater historical basis for Option 1 due to its alignment with S2A and B. Both Options 1 and 2 are considered equally acceptable because a positive outcome in an in vitro mammalian cell assay, accompanied by negative results from two well-conducted in vivo assays with appropriate tissue and exposure evidence, constitutes sufficient proof of the absence of genotoxic potential in vivo. Thus, the testing strategy involving two in vivo assays reflects the approach taken to validate a positive in vitro result.

It is possible to apply either acute or repeat dose study designs in vivo under both standard battery options. If scientifically supported, efforts should be made to include the genotoxicity endpoints in toxicity studies for repeated administrations.
It is best to combine many endpoints that are assessed in vivo into a single research. Before the study starts, there is frequently enough information about the expected acceptability of the doses for the repeat-dose toxicological study. This information can be used to decide whether an acute or integrated test will be appropriate.

When a compound yields negative results, completing any of the standard test battery’s options and evaluating them in line with current guidelines will typically provide enough assurance that there is no genotoxic activity, negating the need for further testing. Depending on their therapeutic usage, compounds that score well on the conventional test battery may require additional testing (see Section 5).

A number of in vivo tests, some of which can be incorporated into repeat-dose toxicity studies, can be utilised as the second phase of the in vivo assessment under Option 2 (see Section 4.2).  Because of its exposure and metabolising ability, the liver is usually the preferred tissue. However, the choice of in vivo tissue and assay should be based on criteria like any understanding of the potential mechanism, of the metabolism in vivo, or of the exposed tissues assumed to be relevant. 

Information on numerical changes can be obtained from mammalian cell assays and micronucleus assays, both in vitro and in vivo. Key indicators include elevations in the mitotic index, polyploidy induction, and micronucleus evaluation. Additionally, spindle poisons can be detected in MLA. The micronucleus assay is the preferred in vivo cytogenetic test as it offers a more direct assessment of chromosome loss, indicating potential for aneuploidy, compared to chromosome aberration assays.

The standard set of genotoxicity tests does not exclude the use of additional tests or alternative species, including non-rodents, for further investigations if validated.

Alternative validated tests can be used as substitutes when one or more of the standard battery’s tests cannot be used for technical reasons, as long as adequate scientific rationale is supplied.

• Modifications to the Test Battery

The following sections outline circumstances in which it could be advisable to alter the standard test battery.

• Exploratory Clinical Studies

For certain exploratory clinical studies, fewer genotoxicity assays or different criteria for justification of the maximum dose in vivo might apply (see ICH M3(R2) guidance).

• 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, just as cytotoxic compounds are tested in mammalian cells, because mutagenicity can occur at lower, less toxic concentrations. In such cases, any one of the in vitro mammalian cell assays should also be done, i.e., Option 1 should be followed.

• Compounds Bearing Structural Alerts for Genotoxic Activity

Structurally alerting compounds are typically detectable in standard test batteries, as most structural alerts relate to bacterial mutagenicity. Some chemical classes are more effectively identified in mammalian cell chromosome damage assays than in bacterial mutation assays. Consequently, negative results in either test with structurally alerting compounds provide assurance of a lack of genotoxicity. However, for compounds with specific structural alerts, modifications to standard protocols may be warranted. The choice of additional tests or protocol modifications is guided by the compound’s chemical nature, known reactivity, and metabolism data.

• Limitations to the Use of In Vivo Tests

Many in vivo experiments (usually in bone marrow, blood, or liver) do not yield extra valuable information for certain substances.  These include substances for which pharmacokinetic or toxicokinetic data show that the target tissues cannot access them because they are not systemically absorbed.

In vivo tests often provide limited information for certain compounds that are not systemically absorbed, such as specific radioimaging agents, aluminum-based antacids, inhaled substances, and topical pharmaceuticals. When a change in administration route fails to enhance exposure to target tissues and no suitable genotoxicity assays are available, it may be appropriate to rely solely on in vitro testing. Additionally, evaluating genotoxic effects at the contact site may be justified, though such assays remain underutilized.

• Detection of Germ Cell Mutagens

Comparative studies indicate that most germ cell mutagens are likely to be detected as genotoxic in somatic cell tests, suggesting that negative results in in vivo somatic cell genotoxicity tests typically imply no germ cell effects.

The entire text of ICH S2 (R1) could not be presented in this article due to space constraints. To properly grasp, GUIDANCE ON GENOTOXICITY TESTING AND DATA INTERPRETATION FOR PHARMACEUTICALS INTENDED FOR HUMAN USE,it is advised that you read the entire guideline. The link is provided below:

Reference: https://database.ich.org/sites/default/files/S2%28R1%29%20Guideline.pdf

                                                                                  Dr Subramanian S Iyer