Genetic toxicity assessment: employing the best science for human safety evaluation part IV: Recommendation of a working group of the Gesellschaft fuer Umwelt-Mutationsforschung (GUM) for a simple and straightforward approach to genotoxicity testing.

Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.

Photo-chemically induced DNA effects in the comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA.

UNLABELLED: Due to the need for in vivo photo-genotoxicity tests, the in vivo photo-comet assay was established in epidermal cells of the SKH-1 mouse. Groups of 10 male SKH-1 mice each were treated once orally with vehicle only, with three fluoroquinolones (25 mg/kg clinafloxacin, 20 mg/kg lomefloxacin, 200 mg/kg ciprofloxacin) or with 200mg/kg 8-methoxypsoralene (8-MOP). Thirty minutes after treatment half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were killed and their epidermal cells tested in the alkaline (pH >13) comet assay; at the same time after administration, compound-treated, non-irradiated mice were killed and analysed. A negative control group of ten male SKH-1 mice received the vehicle only; half of these animals were exposed to UVA, half were not. The comet tail lengths of epidermal cells of the mice were statistically significantly increased for all three fluoroquinolones (FQ) tested in combination with UV irradiation. Treatment with 8-methoxypsoralene+UV induced a significant reduction of comet tail length. Tail intensity and tail moment gave essentially the same results after combined exposure (compound+UV). Without irradiation, the tail lengths of controls and compound-treated mice were comparable under the conditions of this study. In contrast, tail intensity and tail moment were increased for all test compounds (including 8-MOP), without irradiation. Irradiated controls had a tail length comparable to non-irradiated controls, while tail intensity and tail moment were clearly increased in irradiated controls. IN CONCLUSION: under the present experimental conditions the in vivo photo-comet assay is able to detect photo-chemically induced DNA strand breaks as well as photo-chemically induced DNA cross-links.

Genotoxic activities of aniline and its metabolites and their relationship to the carcinogenicity of aniline in the spleen of rats.

Aniline (in the form of its hydrochloride) has been shown to induce a rather rare spectrum of tumors in the spleen of Fischer 344 rats. The dose levels necessary for this carcinogenic activity were in a range where also massive effects on the blood and non-neoplastic splenotoxicity as a consequence of methemoglobinemia were to be observed. This review aimed at clarifying if aniline itself or one of its metabolites has a genotoxic potential which would explain the occurrence of the spleen tumors in rats as a result of a primary genetic activity. The database for aniline and its metabolites is extremely heterogeneous. With validated assays it ranges from a few limited Ames tests (o- and m-hydroxyacetanilide, phenylhydroxylamine, nitrosobenzene) to a broad range of studies covering all genetic endpoints partly with several studies of the same or different test systems (aniline, p-aminophenol, p-hydroxyacetanilide). This makes a direct comparison rather difficult. In addition, a varying number of results with as yet not validated systems are available for aniline and its metabolites. Most results, especially those with validated and well performed/documented studies, did not indicate a potential of aniline to induce gene mutations. In five different mouse lymphoma tests, where colony sizing was performed only in one test, aniline was positive. If this indicates a peculiar feature of a point mutagenic potential or does represent a part of the clastogenic activity for which there is evidence in vitro as well as in vivo remains to be investigated. There is little evidence for a DNA damaging potential of aniline. The clastogenic activity in vivo is confined to dose levels, which are close to lethality essentially due to hematotoxic effects. The quantitatively most important metabolites for experimental animals as well as for humans (p-aminophenol, p-hydroxyacetanilide) seem to have a potential for inducing chromosomal damage in vitro and, at relatively high dose levels, also in vivo. This could be the explanation for the clastogenic effects that have been observed after high doses/concentrations with aniline. They do not induce gene mutations and there is little evidence for a DNA damaging potential. None of these metabolites revealed a splenotoxic potential comparable to that of aniline in studies with repeated or long-term administration to rats. The genotoxicity database on those metabolites with a demonstrated and marked splenotoxic potential, i.e. phenylhydroxylamine, nitrosobenzene, is unfortunately very limited and does not allow to exclude with certainty primary genotoxic events in the development of spleen tumors. But quite a number of considerations by analogy from other investigations support the conclusion that the effects in the spleen do not develop on a primary genotoxic basis. The weight of evidences suggests that the carcinogenic effects in the spleen of rats are the endstage of a chronic high-dose damage of the blood leading to a massive overload of the spleen with iron, which causes chronic oxidative stress. This conclusion, based essentially on pathomorphological observations, and analogy considerations thereof by previous authors, is herewith reconfirmed under consideration of the more recently reported studies on the genotoxicity of aniline and its metabolites, on biochemical measurements indicating oxidative stress, and on the metabolism of aniline. It is concluded that there is no relationship between the damage to the chromosomes at high, toxic doses of aniline and its major metabolites p-aminophenol/p-hydroxyacetanilide and the aniline-induced spleen tumors in the rat.

Dimethylhydrazine: a reliable positive control for the short sampling time in the UDS assay in vivo.

In the first international guideline addressing the unscheduled DNA synthesis (UDS) assay in vivo (OECD guideline no. 486, adopted July 1997) only the genotoxic liver carcinogen N-nitrosodimethylamine (NDMA) is proposed as positive control for the short sampling time. Since NDMA is extremely volatile, alternative positive controls should be identified to facilitate handling and reduce exposure risk during routine testing. At Bayer AG and at RCC-CCR GmbH, the genotoxic but non-volatile dimethylhydrazine (DMH; as dihydrochloride) was used instead as positive control in livers of Wistar rats and to a limited extent of NRMI mice after 2-4h exposure. As shown by the data presented in this paper DMH induced a positive result in a total of 21 UDS in vivo studies over a period of 7 years. A negative result was never seen for DMH. Due to these results DMH was proven to be a suitable and reliable positive control in the UDS assay in vivo. Consequently, DMH should be considered as positive control for the short sampling time in the next issue of OECD guideline no. 486.