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Introduction

1. The advice of the COM previously has been that compounds that are clearly positive in any in-vivo mutagenicity assay should be regarded as mammalian mutagens (and hence potential genotoxic carcinogens).(1) (http://www.doh.gov.uk/com/guidance.pdf). Furthermore the COM consider that for such compounds it is prudent to assume that there is no threshold for their mutagenic activity, unless appropriate mechanistic data can be provided to identify a threshold related mechanism eg induction of aneuploidy where the site of initial action is not DNA but the spindle apparatus.(2) (http://www.doh.gov.uk/comivm.htm). Such mechanistic data are rarely available.

2. This approach has major implications with regard to risk assessment of such chemicals, with the assumption that there is no 'safe' level and that any exposure results in some increased health detriment, albeit this may be very small.

3. In practice this means that much weight is placed on the in-vivo genotoxicity assays recommended by the COM in their strategy of testing, in particular on one of the bone marrow assays for clastogenicity (usually the micronucleus test) which is usually the initial assay investigated. A positive in one of these assays results in the compound being considered as an in-vivo mutagen. It is thus especially important that such decisions are based on sound data, and that the positive results truly reflect in-vivo mutagenic activity. In some cases such positive results in the bone marrow assays have only been seen at high dose levels associated with severe toxicity including lethality, giving rise to concern at the relevance of the results. This is particularly true if the overall weight of evidence from the in-vivo data, including the results of carcinogenicity bioassays, is not supportive of the compound being an in-vivo genotoxin.

4. Furthermore it is now recognised that in the case of the in-vivo micronucleus test, micronuclei can be induced by mechanisms such as hypothermia, hyperthermia, stimulation of erythropoiesis and possibly indirectly due to severe cytotoxicity in the bone marrow. In such cases the positive results, if not supported by other in-vivo data, may be regarded as artefacts, which do not reflect mutagenicity of the compound itself in animals.

5. Draft COM guidance on the interpretation of in-vivo bone marrow mutagenicity data that may give rise to concern with regard to being a 'false positive' is provided in this document.

Guidance on 'false positive' in-vivo mutagenicity data

6. The COM guidance on a strategy for testing chemicals for mutagenicity is based on the identification of compounds with mutagenic potential on the basis of a small number of well conducted in-vitro tests. This is followed by establishing whether activity seen in-vitro can be expressed in-vivo in somatic cells, to establish whether the compound can be regarded as an in-vivo somatic cell mutagen and hence a potential carcinogen. In the initial in-vitro testing it is recognised in the guidelines that artifactual positives may be obtained in the mammalian cell assays which do not reflect intrinsic mutagenic activity, and that this uncertainty may be resolved by in-vivo assays, and specifically the bone marrow assays for clastogenicity. Until recently there has been little consideration as to whether artifactual positive results can also be obtained in the in-vivo bone marrow assays.

7. However it is now known that effects such as hyperthermia (3,4) or hypothermia (5,6), which can be produced as a secondary toxic effect at high dose levels with certain compounds, can produce micronuclei in bone marrow cells in-vivo. Another possible mechanism is the induction of erythropoiesis. This was demonstrated following unexpected positive results in the bone marrow micronucleus test with a series of recombinant human erythropoietin products; similar effects were later shown with native erythropoietin(7,8,9) The induction of micronuclei by such non-DNA reactive compounds has been shown to be due to the accelerated proliferation and differentiation of erythrocytes and promotion of early release of PCEs resulting in the micronucleated PCEs.

8. Another possible mechanism is excessive toxicity at high dose levels, associated with severe toxicity including lethality, ie higher levels than recommended in the current OECD guidelines. Such 'high dose' studies may possibly give positive results due to the severe toxicity seen in the bone marrow. Thus there are a number of reasons why the in-vivo data may not indicate that the compound is an in-vivo mutagen but further information would be needed to draw any definite conclusions.

9. In considering whether positive results in in-vivo bone marrow mutagenicity data may be caused by factors other than mutagenicity of the compound itself, or its metabolites, it is important to consider all the available data regarding the mutagenic profile of the chemical and whether the hypothesis that it is not an in-vivo mutagen is biologically plausible. This will include information on structure-activity relationships, data from in-vitro mutagenicity assays, other in-vivo mutagenicity data, information on toxicokinetics of the substances and any available data from carcinogenicity bioassays. Each compound needs to be considered on a case-by-case basis.

10. An important consideration with regard to data from bone marrow assays for clastogenicity (metaphase analysis or micronuclei) is the highest dose level used. The guidance in the current OECD guidelines is that the top dose should produce signs of toxicity such that higher dose levels, based on the same dosing regime, would be expected to produce lethality (or for non-toxic compounds it should be set at a limit dose of 2 grams/kg for single doses and 1 gram/kg for multiple dosing regimes)(10). In the past considerably higher dose levels have been used, and on occasions the relevance of such data needs to be assessed. In the most extreme case, when positive results have been obtained only at highly toxic or lethal dose levels, the Committee believe that the results obtained are confounded by toxicity and cannot be interpreted with any certainty in these circumstances; it is thus necessary to repeat the test using lower, more appropriate dose levels. It may, however, be possible to utilise the available data from the lower dose levels, together with other relevant information, to enable the isolated high dose in-vivo positive data to be discounted.

Consideration of carcinogenicity data (if available)

11. The implications of mutagenicity data with regard to carcinogenicity was considered in some detail in the 1989 COM guidelines for the testing of chemicals for mutagenicity and also in the 1991 COC guidelines on the evaluation of chemicals for carcinogenicity.(11, 12) At that time both committees concluded that it was reasonable and prudent to regard compounds that were mutagens in-vivo in the somatic cell assays recommended by the COM to also have carcinogenic potential even though such potential had not been examined or had not been demonstrated in formal carcinogenicity tests.

12. The updated COM guidelines on a strategy for testing chemicals for mutagenicity published in December 2000 endorsed the view that in-vivo somatic cell mutagens should be regarded as potential carcinogens.(1) These conclusions have implications with regard to the testing of chemicals for carcinogenicity. The need to use large numbers of animals in carcinogenicity bioassays on compounds that were clear in-vivo somatic cell mutagens is unlikely to be justified; this is recognised in the 1991 COC guidelines. The COC is currently updating its guidance on risk assessment of carcinogens.

13. These conclusions also have implications regarding how carcinogenicity bioassay data that are not supportive of a compound being a genotoxic carcinogen may be used to assist in the interpretation of in-vivo mutation data when there is concern about the relevance of such data.

14. On occasions in the past the COM have used negative data from carcinogenicity bioassays in this way. An example was the chemical incapacitant CS (2-chlorobenzylidene malonitrile). This compound is mutagenic in-vitro inducing both clastogenic and aneugenic effects in mammalian cells. Although there were negative results from in-vivo bone marrow micronucleus assays it was noted that no data were available to indicate whether adequate amounts of CS, or short lived reactive metabolites, reached the target organ. Data from DNA binding studies in the liver and kidney did not help with regard to concerns in tissues of initial contact due to the direct acting mutagenic potential. However the COM took into account the negative carcinogenicity bioassay data from the NTP programme comprising inhalation studies in the rat and the mouse.(13) The advice from the COC was that these assays had been adequately conduced and were negative. The COM agreed these negative data provided reassurances that the in-vitro effects seen with CS do not occur in-vivo at the site of initial contact in animals. (Full information can be found at www.doh.gov.uk/cot/csgas/htm.)

15. Thus consideration of the results from carcinogenicity bioassays, if available, is important when assessing the overall weight of evidence as to whether the compound should be regarded as an in-vivo mutagen. In this regard an assessment needs to be made as to whether the carcinogenicity data are consistent with the compound being a genotoxic carcinogen. Alternatively the tumour profile may be suggestive of a non-genotoxic mechanism of chemical carcinogenesis, or that the chemical is not carcinogenic. Consideration may need to be taken of any difference in dose levels used but it must be recognised that the difference in duration of the mutagenicity and carcinogenicity bioassays will severely limit the utility of any comparison. However, consideration of the available information on the toxicokinetics of the compound will often be useful.

16. It is not possible to give more than generic guidance in this area, as each compound will need to be considered on a case-by-case basis. Expert judgement will be needed with particular consideration of a number of key points that are summarised in the conclusions.

Conclusions

17. The following points need to be considered when deciding whether positive in-vivo bone marrow clastogenicity data can be discounted with regard to considering a compound as an in-vivo mutagen.

i) The totality of the relevant data relating to the specific compound in question needs to be considered, including any information on its chemical reactivity, toxicokinetics, its mutagenic profile (in-vitro and in-vivo), and also any available data from carcinogenicity bioassays to enable an assessment to be made as to whether the compound appears to be a genotoxic carcinogen.

(ii) Positive bone marrow in-vivo mutagenicity data from dose levels that are associated with high levels of toxicity or lethality (ie above the maximum dose level recommended in the current OECD guidelines) cannot be interpreted with any certainty because of the confounding effects of toxicity. If adequate information is not available at lower, non-lethal dose levels then retesting is necessary.

(iii) It is also important to consider whether there is any evidence for a plausible mechanism to support the contention that the observed positive results in the bone marrow assays at high dose levels may be secondary to other non-genotoxic effects rather than being a mutagenic effect of the compound (or its metabolites). Examples of such mechanisms include (but are not limited to) hypothermia, hyperthermia, and erythropoiesis.

(iv) Data from adequately conducted carcinogenicity bioassays, if available, provide important information to help in the assessment of the significance of such high dose bone marrow mutagenicity results. Such data may indicate that the carcinogenic profile of the chemical is consistent with either a genotoxic or a non-genotoxic mechanism, or that the compound is not carcinogenic.

(v) Only generic advice can be given in this area and it should be emphasised that each compound needs to be considered in a case-by-case basis. However consideration of the above factors, with expert judgement, may provide sufficient evidence to conclude that the positive in-vivo bone marrow data at high dose levels was due to a non-genotoxic effect. A threshold based risk assessment may thus be appropriate.

November 2003

References

1. COM. Guidance on a strategy for testing chemicals for mutagenicity. December 2000. (http://www.doh.gov.uk/com/guidance.pdf).

2. COM. Statement on risk assessment of in-vivo mutagens (and genotoxic carcinogens). June 2001. (http://www.doh.gov.uk/comivm.htm).

3. Chrisman CL and Baungartner AP. Micronuclei in bone marrow cells of mice subjects to hypthermia. Mut. Res. 77 95-7 (1980).

4. King MT and Wild D. The mutagenic potential of hypthermia and fever in mice. Mut. Res. 111 219-26 (1983).

5. Asanami S and Shimono K. Hypothermia induces micronuclei in mouse bone marrow cells. Mut. Res. 393 91-8 (1997).

6. Asanami S, Shimono K and Kaneda S. Transient hypothermia induced micronuclei in mice. Mut. Res. 413 7-14 (1998).

7. Yajima N, Kurata Y, Sawai T and Takeshita Y. Induction of micronucleated erythrocytes by recombinant human erthropoietin. Mutagenesis 8 221-9 (1993).

8. Yajimi N, Kurata Y, Imai E, Sawai T and Takashita Y. Genotoxicity of genetic recombinant human erythropoietin in a novel test system. Mutagenesis 8 231-6 (1993).

9. Yajima N, Kurata Y, Sawait T and Takeshita Y. Comparative induction of micronuclei by 3 genetically recombinant and urinary human erthropoietins. Mutagenesis 8 237-41 (1993).

10. OECD (1997) OECD guidelines for the testing of chemicals. Mammalian Erythrocyte Micronucleus Test (Guideline No 474) and Mammalian Bone Marrow Chromosome Aberration Test (Guideline No 475). OECD Paris.

11. Department of Health (1989) Guidelines for the Testing of Chemicals for Mutagenicity. London. HMSO (Report on Health and Social Subjects No 35).

12. Department of Health (1991). Guidelines for the Evaluation of Chemicals for Carcinogenicity. London. HMSO (Report on Health and Social Subjects No 42).

13. COM (1998) Annual Report. 2-Chlorobenzylidene Malonitrile (CS) p. 32. (Full statement on www.doh.gov.uk/cot/csgas.htm).

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