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Mutagenic activity of o-anisidine was investigated in a reliable bacterial reverse mutation assay similar to guidelines which was selected as key study. This study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 as well as Escherichia coli WP2 uvrA with and without metabolic activation (induced rat liver S9-mix) at concentrations of 0.004, 0.02, 0.1, 0.5, 2.5 and 10 µl/plate. Under the conditions tested the test compound was not mutagenic because no significant increase in the number of revertant colonies and no dose dependent effects were observed.

A variety of studies described in the EU-Risk Assessment Report gave negative as well as positive results for o-anisidine genotoxicity in vitro and in vivo:

Negative as well as positive or ambiguous results were observed in bacterial reverse mutation assays under different conditions.

No unscheduled DNA synthesis could be observed in rat hepatocytes and only weak positive results were shown in an alkaline elution assay. Positive test results were obtained in a sister chromatide assay and two chromosome aberration assays, as well as in a mouse lymphoma assay (with or without metabolic activation).

o- Anisidine was tested in several in vivo micronucleus assays. In one assay the test compound was administered orally by gavage to male and female mice. The animals were treated once with 1000 mg/kg bw (including vehicle and positive control) and according to the test procedure the animals were killed 24, 48 or 72 hours after application. No induction of micronuclei was observed under these test conditions, but systemic toxicity was already detectable. Negative results were also obtained in several other in vivo micronucleus assays. Additionally, no formation of DNA-adducts, no DNA-single strand break and UDS were detectable in vivo. Negative results were also obtained in a sex linked recessive lethal assay.

Positive results in vivo were obtained in a host mediated assay, somatic mutation and recombination assay in Drosophila melanogaster and a transgenic mouse mutation assay (Big Blue Mouse TM), furthermore, inhibition of testicular DNA-synthesis was observed in male mice.

Short description of key information:
In a variety of reverse bacterial mutation assays the test material showed negative as well as positive results in the presence and absence of metabolic activation. Mutagenic effects in vitro were reported for chromosome aberration tests and mouse lymphoma assays with and without metabolic activation. Tests for unscheduled DNA-synthesis in vitro were consistently negative. In vivo assays gave mostly negative results (e.g. micronucleus assay in vivo), but also some positive results were obtained (e.g. transgenic mouse mutation assay).

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

In accordance with the evaluation in the EU-RAR it is concluded that there is sufficient evidence that o-anisidine is genotoxic. There are contradictory results in in vitro short-term tests as well as in in vivo assays. The evidence from the transgenic mouse mutation assay and supporting in vitro and in vivo evidence is considered to be sufficient for classification as Muta 2, H341 or Muta. Cat. 3; R68, which fits to current classification in Annex VI to Regulation (EC) No.1272/2008.