A Standard Battery for Genotoxicity Testing of Pharmaceuticals

GENERAL PURPOSE OF GENOTOXICITY TESTING

Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds which induce genetic damage directly or indirectly by various mechanisms. These tests should enable a 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, recombination and numerical chromosome changes 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. Compounds which are positive in tests that detect such kinds of damage have the potential to be human carcinogens and/or mutagens, i.e. may induce cancer and/or heritable defects. Because the relationship between exposure to particular chemicals and carcinogenesis is established for man, 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 may induce heritable effects is considered to be just as serious as the suspicion that a compound may induce cancer. In addition, the outcome of such tests may be valuable for the interpretation of carcinogenicity studies.

THE STANDARD TEST BATTERY FOR GENOTOXICITY

Registration of pharmaceuticals requires a comprehensive assessment of their genotoxic potential. It is clear that no single test is capable of detecting all relevant genotoxic agents. Therefore, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. Such tests are complementary rather than representing different levels of hierarchy.

The general features of a standard test battery can be outlined as follows:

i) It is appropriate to assess genotoxicity in a bacterial reverse mutation test. This test has been shown to detect relevant genetic changes and the majority of genotoxic rodent carcinogens.
ii) DNA damage considered to be relevant for mammalian cells and not adequately measured in bacteria should be evaluated in mammalian cells. Several mammalian cell systems are in use: systems that detect gross chromosomal damage systems that detect primarily gene mutations, and a system that detects gene mutations and clastogenic effects . The various in vitro tests for chromosomal damage and the mouse lymphoma assay yield results with a high level of congruence for compounds that are regarded as genotoxic but yield negative results in the bacterial reverse mutation assay. Therefore, these systems are currently considered interchangeable when used together with other genotoxicity tests in a standard battery for genotoxicity testing of pharmaceuticals, if these test protocols are used.
iii) An in vivo test for genetic damage should usually be a part of the test battery to provide a test model in which additional relevant factors that may influence the genotoxic activity of a compound are included. As a result, in vivo tests permit the detection of some additional genotoxic agents . An in vivo test for chromosomal damage in rodent hematopoietic cells fulfills this need. This in vivo test for chromosomal damage in rodents could be either an analysis of chromosomal aberrations in bone marrow cells or an analysis of micronuclei in bone marrow or peripheral blood erythrocytes.

The following standard test battery is recommended based upon the considerations mentioned above:

1. A test for gene mutation in bacteria.
2. An in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay.
3. An in vivo test for chromosomal damage using rodent hematopoietic cells.

For compounds giving negative results, the completion of this 3-test battery, performed and evaluated in accordance with current recommendations, will usually provide a sufficient level of safety to demonstrate the absence of genotoxic activity . Compounds giving positive results in the standard test battery may, depending on their therapeutic use, need to be tested more extensively .

The suggested standard set of tests does not imply that other genotoxicity tests are generally considered as inadequate or inappropriate . Such tests serve as options in addition to the standard battery for further investigation of genotoxicity test results obtained in the standard battery. Furthermore, molecular techniques to study mechanisms of genotoxicity in the standard battery systems may be useful for risk assessment. Only under extreme conditions in which one or more tests comprising the standard battery cannot be employed for technical reasons, alternative validated tests can serve as substitutes. For this to occur, sufficient scientific justification should be provided to support the argument that a given standard battery test is not appropriate.

The standard battery does not include an independent test designed specifically to test for aneuploidy. However, information on this type of damage may be derived from the tests for chromosomal damage in vitro and in vivo. Elements of the standard protocols that provide such information are elevations in the mitotic index, polyploidy induction and micronucleus evaluation. There is also limited experimental evidence that aneuploidy inducers can be detected in the mouse lymphoma tk assay . In such cases, further testing may be needed.

MODIFICATIONS OF THE 3-TEST BATTERY

The following sections give situations where the standard 3-test battery may need modification.

Limitations to the use of bacterial test organisms
There are circumstances where the performance of the bacterial reverse mutation test does not provide appropriate or sufficient information for the assessment of genotoxicity. This may be the case for compounds that are excessively toxic to bacteria and compounds thought or known to interfere with the mammalian cell replication system. For these cases, usually two in vitro mammalian cell tests should be performed using two different cell types and of two different endpoints. Nevertheless, it is still important to perform the bacterial reverse mutation test .

Compounds bearing structural alerts for genotoxic activity
Structurally alerting compounds are usually detectable in the standard 3-test battery. However, compounds bearing structural alerts that have given negative results in the standard 3-test battery may necessitate limited additional testing. The choice of additional test or protocol modification depend on the chemical nature, the known reactivity and metabolism data on the structurally alerting compound under question.
Limitations to the use of standard in vivo tests.

There are compounds for which standard in vivo tests do not provide additional useful information. This includes compounds for which data from studies on toxicokinetics or pharmacokinetics indicate that they are not systemically absorbed and therefore are not available for the target tissues in standard in vivo genotoxicity tests. Examples of such compounds are some radioimaging agents, aluminum based antacids, and some dermally applied pharmaceuticals. In cases where a modification of the route of administration does not provide sufficient target tissue exposure, it may be appropriate to base the evaluation only on in vitro testing.

Additional genotoxicity testing in relation to the carcinogenicity bioassay
Evidence for tumor response

Additional genotoxicity testing in appropriate models may be conducted for compounds that were negative in the standard 3-test battery but which have shown effects in carcinogenicity bioassay(s) with no clear evidence for a non-genotoxic mechanism.To help understand the mechanism of action, additional testing can include modified conditions for metabolic activation in in vitro tests or can include in vivo tests measuring genetic damage in target organs of tumour induction.

Structurally unique chemical classes

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

STANDARD PROCEDURES FOR IN VITRO TESTS

Reproducibility of experimental results is an essential component of research involving novel methods or unexpected findings; however, the routine testing of chemicals with standard, widely used genotoxicity tests need not always be completely replicated. These tests are sufficiently well characterized and have sufficient internal controls that repetition can usually be avoided if protocols with built-in confirmatory elements, such as those outlined below, are used.

For both bacterial and mammalian cell gene mutation tests, the results of a range-finding test can be used to guide the selection of concentrations to be used in the definitive mutagenicity test. By these means, a range-finding test may supply sufficient data to provide reassurance that the reported result is the correct one. In bacterial mutagenicity tests, preliminary range-finding tests performed on all bacterial strains, with and without metabolic activation, with appropriate positive and negative controls, and with quantification of mutants, may be considered a sufficient replication of a subsequent complete test. Similarly, a range-finding test may also be a satisfactory substitute for a complete repeat of a test in gene mutation tests with mammalian cells other than the mouse lymphoma tk assay if the range-finding test is performed with and without metabolic activation, with appropriate positive and negative controls, and with quantification of mutants .

For the cytogenetic evaluation of chromosomal damage in vitro, the test protocol includes the conduct of tests with and without metabolic activation, with appropriate positive and negative controls, where the exposure to the test articles is 3 to 6 hours and a sampling time of 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 a negative result for the short treatment period without metabolic activation. Certain chemicals may be more readily detected by longer treatment or delayed sampling times, e.g. some nucleoside analogues or some nitrosamines. Negative results in the presence of a metabolic activation system may need confirmation on a case by case basis. In any case information on the ploidy status should be obtained by recording the incidence of polyploid cells as a percentage of the number of metaphase cells. An elevated mitotic index or an increased incidence of polyploid cells may give an indication of the potential of a compound to induce aneuploidy. In such cases, further testing may be needed.

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 exposure to the test articles is 3 to 4 hours. A continuous treatment without metabolic activation for approximately 24 hours is needed in case of a negative result for the short treatment without metabolic activation . Negative results in the presence of a metabolic activation system may need confirmation on a case by case basis. In any case, an acceptable mouse lymphoma tk assay includes (i) the incorporation of positive controls which induces mainly small colonies, (ii) colony sizing for positive controls, solvent controls and at least one positive test compound dose (should any exist), including the culture that gave the greatest mutant frequency.

Following such testing, further confirmatory testing in the case of clearly negative or positive test results is not usually needed.

Ideally it should be possible to declare test results as 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 aids in data interpretation, however, adequate biological interpretation is of critical importance. Nonetheless, further testing is usually indicated for equivocal results.

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