Regulatory aspects of genotoxicity testing: from hazard identification to risk assessment.

This symposium focused on the use of tests for chromosomal damage, and other genotoxicity measures, for detection of potentially harmful chemicals. The speakers discussed the information that has been gained over the last three decades about the use of "short-term tests" for genotoxicity in cultured cells and in animals (mainly rodents), and the ongoing debates about the rational use of data from such experimental systems in trying to extrapolate to an understanding of potential human risk. The overall theme was that the field of regulatory toxicology currently is over-reliant on qualitative outcomes of in vitro hazard-screening tests, generally conducted at the maximum achievable exposures, and needs a more realistic approach that incorporates in vivo exposure levels and dose-response information.

In vitro approaches to develop weight of evidence (WoE) and mode of action (MoA) discussions with positive in vitro genotoxicity results.

A recent analysis by Kirkland et al. [Kirkland, D., Aardema, M., Henderson, L. and Müller, L. (2005) Evaluation of the ability of a battery of 3 in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat. Res. 584, 1-256] demonstrated an extremely high false positive rate for in vitro genotoxicity tests when compared with carcinogenicity in rodents. In many industries, decisions have to be made on the safety of new substances, and health risk to humans, without rodent carcinogenicity data being available. In such cases, the usual way to determine whether a positive in vitro genotoxicity result is relevant (i.e. indicates a hazard) for humans is to develop weight of evidence (WoE) or mode of action (MoA) arguments. These are based partly on further in vitro investigations, but usually rely heavily on tests for genotoxicity in one or more in vivo assays. However, for certain product types in the European Union, the use of animals for genotoxicity testing (as well as for other endpoints) will be prohibited within the next few years. Many different examples have been described that indicate DNA damage and genotoxic responses in vitro can arise through non-relevant in vitro events that are a result of the test systems and conditions used. The majority of these non-relevant in vitro events can be grouped under a category of 'overload of normal physiology' that would not be expected to occur in exposed humans. However, obtaining evidence in support of such MoAs is not easy, particularly for those industries prohibited from carrying out in vivo testing. It will become necessary to focus on in vitro studies to provide evidence of non-DNA, threshold or in vitro-specific processes and to discuss the potential for such genotoxic effects to occur in exposed humans. Toward this end, we surveyed the published literature for in vitro approaches that may be followed to determine whether a genotoxic effect observed in vitro will occur in humans. Unfortunately, many of the approaches we found are based on only a few published examples and validated approaches with consensus recommendations often do not exist. This analysis highlights the urgent need for developing consensus approaches that do not rely on animal studies for dealing with in vitro genotoxins.

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes.

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds.

Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.

Recommendations for spacing of test chemical concentrations in the mouse lymphoma tk mutation assay (MLA)

Recent test guidelines for the mouse lymphoma tk mutation assay (MLA) have highlighted the need to achieve 80-90% reduction in cell survival for a valid, robust assay with toxic chemicals. For many pharmaceuticals, under new ICH recommendations, this may be the only in vitro mammalian cell test that is performed. It was important to discover, therefore, how critical it is to achieve 80-90% toxicity, and how best to select the number and spacing of test concentrations to fulfil this requirement. We analysed data from 121 positive chemicals, provided by nine industrial and commercial laboratories, and found that for 17 chemicals (14%), the response profiles were so steep that using a conventional 2-fold dilution series of test concentrations would have failed to identify the active range (> 90% toxicity at one concentration, and no significant mutation at 50% of this dose), and positive responses would have been missed. Analysis of genotoxicity results in other test systems with these 17 chemicals revealed no differences in overall response profiles from the 104 chemicals that exhibited less steep MLA responses. The MLA results were therefore deemed to be equally biologically relevant. From this analysis, it is recommended that concentration spacing in the MLA needs to be closer than that obtained with a 2-fold dilution series, and a dilution factor where each concentration is 0.75 or 0.8 of the one above is recommended to identify the active range of positive mutagens.

Fluorescence in situ hybridisation with chromosome-specific centromeric probes: a sensitive method to detect aneuploidy.

Cytochalasin B-blocked binucleate human lymphocytes from female donors have been used to measure micronucleus induction and other aneuploidy events after treatment with colchicine, vinblastine or carbendazim. For the aneuploidy events, centromeric probes for 6 selected chromosomes (1, 8, X, 11, 17, 18) were used to measure chromosome loss, addition and non-disjunction in the interphase nuclei of these binucleate cells. The chromosomes were probed in pairs using Cy-3 (red) and FITC (green) labels for the 2 different centromeric regions. For colchicine, the total non-disjunction frequencies for chromosomes 1 and 8 were similar to the total micronucleus frequencies, but were detected as significant at lower concentrations. For vinblastine (chromosomes 1 and 8) and carbendazim (all 6 chromosomes) the frequencies of non-disjunction far exceeded (7 and > 2-fold, respectively) the peak frequencies of micronucleus induction. Although most chromosomes exhibited similar sensitivity in all the aneuploidy events measured, there was an indication that chromosome X was more than susceptible to non-disjunction than the other chromosomes. We believe that measurement of non-disjunction in binucleate human lymphocytes using chromosome specific centromeric probes offers a sensitive method for detection of aneuploidy and is particularly appropriate for the establishment of thresholds.

Special considerations for conducting genotoxicity tests with protein materials.

Standard genotoxicity tests are often inappropriate for testing new biological entities, in particular for recombinant proteins which are nature-identical. Arguments that these may contain mutagenic impurities are not substantiated; however, we have produced evidence that such impurities would be detected amidst a vast excess of protein. Concerns that human patients receiving therapy may be at risk from higher-than-physiological levels of proteins are also somewhat theoretical. However, it is apparent that genotoxicity testing will be required for these products for the time being, even if pragmatic approaches reduce the battery of in vitro tests to Ames and chromosomal aberrations only, and reduce the top dose in vivo to 1000x the human therapeutic dose. There is a number of physical and chemical properties of proteins that demand special approaches to methodology if the tests are to produce accurate results. The potential for adsorption to certain forms of glass and plastic means special care must be taken in dissolving and diluting test solutions; adherence to filters means special low protein binding, non-pyrogenic filters should be used for sterilisation of test solutions, where this is necessary; freeze-dried powders aliquotted in multiple vials should be dissolved in minimal solvent and cascaded from vial to vial rather than trying to empty the solid contents for bulk weighing. As proteins are often supplied in solution, in order to achieve sufficiently high test concentrations, it may be necessary to resuspend test bacteria/cells in the test solutions for short periods of time before centrifuging and resuspending in selective or growth media.(ABSTRACT TRUNCATED AT 250 WORDS)

On the need for confirmation of negative genotoxicity results in vitro and on the usefulness of mammalian cell mutation tests in a core battery: experiences of a contract research laboratory.

There are currently unresolved discussions on two important topics in regulatory genetic toxicology, namely whether or not it is necessary to confirm clearly negative results from in vitro assays in independent experiments, and whether or not the mammalian cell gene mutation test should be part of a core battery of tests. Analysis of in-house data, using full regulatory protocols, suggests that for bacterial mutation tests (144 compounds reviewed) it is impractical to design a single experiment to incorporate all relevant variables and, therefore, confirmation of negative results using modified methodology is desirable. On the other hand, data from TK mutation assays (65 compounds reviewed) and chromosomal aberration tests (94 compounds reviewed) suggest that confirmation of negative results in well-designed mammalian cell studies is not necessary. Analysis of 32 chemicals, each tested in Ames, TK mutation and chromosomal aberration tests, revealed two positives unique to the TK assay and one unique to the chromosomal aberration test. As the TK assay did not show increased susceptibility to false positives (frequency of positives is similar to other in vitro assays) and these two unique positives were clearly observed (> 2-fold increase in mutation frequency at 60-70% relative survival in both cases), they do appear to be 'real' results. Both compounds induced small colony mutants (one also induced 'large'), and yet in vitro chromosomal aberration and in vivo micronucleus tests were negative. The single unique chromosomal aberration positive may be an artefact of high cytotoxicity, and certainly the substance was negative for micronuclei and UDS in vivo, so it might be argued that the chromosomal aberration test is surplus to requirements. Overall, however, it would seem premature to reject either assay at this time, and experience suggests the extra information provided by two mammalian cell tests instead of one is extremely valuable in assessing risk and deciding upon appropriate follow-up tests in vivo.

Report from working group on in vitro tests for chromosomal aberrations.

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Statistical evaluation of mutagenicity test data: recommendations of the U.K. Environmental Mutagen Society.

Most of the many published guidelines on how to conduct mutagenicity tests do not give advice or references on statistical analysis of data. The U.K. Environmental Mutagen Society decided to address this omission, and in 1985 established 8 working groups comprising genetic toxicologists from all sectors of the science, plus at least 2 statisticians per group, to produce statistics guidelines on 10 different test systems. Each group gave advice on how to determine the suitability of data for distribution fitting, when data are unsuitable, when and how data should be transformed, which statistical tests are suitable for a given set of data, which factors govern the choice of statistical test, an order of preference, and some worked examples using real data. In addition, groups gave advice on statistical issues in the design of experiments. Strong recommendations were made that for in vitro tests, sufficient cells be treated and sampled to provide meaningful values of spontaneous mutant/aberration frequencies, for genuine, independent replicate treatments to be used, and that the acceptability of an experiment should be based on homogeneity between replicates as well as comparison of negative and positive control responses with historical ranges. It was recommended that most in vitro studies should include independent repeat experiments, and advice was given on how to check for consistency between experiments and then combine data for further significance testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenic and clastogenic properties of (3-[3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl) benzoyl]-1-ethoxymethyl-5-fluorouracil) (BOF-A2), a new 5-fluorouracil derivative with anti-tumour activity.

As part of the safety assessment of (3-[3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl)benzoyl]-1-ethoxymethyl-5-fluorouracil) (BOF-A2), a new 5-fluorouracil (5-FU) derivative with anti-tumour activity, its potential genotoxicity was studied in 3 different tests. BOF-A2 was negative in a reverse mutation (Ames) test in strains of S. typhimurium and E. coli. BOF-A2 induced chromosomal aberrations in Chinese hamster cells in vitro especially in the presence of exogenous metabolic activation, and was clastogenic in vivo, inducing micronuclei in mouse bone marrow. The clastogenic activity of BOF-A2 was similar to that of 5-FU.

Genetic toxicology testing requirements: official and unofficial views from Europe.

This work presents genetic toxicity testing requirements in three industry sectors; pharmaceuticals, pesticides, and industrial chemicals.