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Additional information

Genetic toxicity in vitro:

1,3-dioxepane was not mutagenic in an Ames test using S. typhimurium TA1535, 100, 1537 and 98 with test concentrations up to 5000 µg/plate (BASF AG, 1991). The test was performed in absence and presence of a metabolic activation system but according to the standard guideline at the date of test performance no E.coli strain was included in the test protocol.

 

In an in vitro chromosome aberration test Chinese hamster lung fibroblasts (V79) were incubated with dose levels of 1250, 2500 and 5000 µg/ml 1,3-dioxepane with and without the presence of a metabolic activation system (BASF AG, 1994). The test was performed according to OECD Guideline 473 under GLP. At the highest tested dose level (5000 µg/ml) and in presence of a metabolic activation system the number of structurally aberrant metaphases was significantly increased, with a high proportion of exchanges. In detail, at 5000 µg/ml the number of structurally aberrant metaphases including gaps was increased by a factor of 2.4 and the number of structurally aberrant metaphases excluding gaps by a factor of 4.4 compared to the vehicle controls, respectively.The detailed analysis of the aberrant metaphases excluding gaps revealed, that besides a slight increase in chromatid breaks predominantly the number of chromatidal/chromosomal exchanges were increased.

An increase in the frequency of cells incl. numerical aberrations (aneuploidy/polyploidy) was not demonstrated.

The observation of an increase in the number of structurally aberrant metaphases was restricted to the highest tested dose level and therefore no dose dependency could be established. Furthermore a slightly reduced attachment, a reduced amount of analyzable cells and a slight decrease in the number of mitotic indices were observed in the highest dose tested.

Summarized, a clastogenic effect at the limit dose of 5000µg/ml was observed in this in vitro assay, but based on the presented considerations the physiological relevance of this high dose effect can not be evaluated. Furthermore this observation at unphysiologically high concentrations of 1,3-dioxepane may, at least partly, result from formaldehyde formation in the presence of metabolic activation (S9-mix). The release of low amounts of formalehyde under cell culture conditions in the presence of a metabolic activation system was confirmed in a subsequent in vitro assay (BASF AG, 1998, see chapter toxicokinetics). The released formaldehyde was shown to be further metabolized by the S9 mix.

Chromosome mutagenic activity of formaldehyde is well documented from in vitro studies. From mechanistic studies is was possible to postulate a mechanism for this activity (DNA-protein cross-links as pre-mutagenic lesion) and to establish a correlated threshold for repair (>10 μM/0.3 μg/mL in cultured human lymphocytes).

 

In an in vitro gene mutation in mammalian cells (HPRT locus assay in CHO-cells) no indications for a mutagenic potential of 1,3-dioxepane were found (BASF AG, 1994). The assay was performed according to the OECD guideline 476 under GLP. The cells were incubated with test concentrations up to 5000 µg/ml with and without metabolic activation.

 

 

Genetic toxicity in vivo:

In a Miconucleus Assay, mice were injected twice intraperitoneally with dose levels of 437.5, 875.0 and 1750 mg/kg bw (BASF AG, 1999). The assay was performed according to the OECD guideline 474 under GLP. No increase in the number of polychromatic erythrocytes containing either small or large micronuclei was observed. A slight inhibition of erythropoiesis at the highest tested dose level indicated the validity of the test and therefore no clastogenic and/or aneugenic potential can be anticipated under the conditions of this assay.

 

In an unscheduled DNA synthesis assay rats were gavaged with 1000 and 2000mg/kg bw 1,3-dioxepane (BASF AG, 1999). The assay was performed according to the OECD guideline 486 under GLP. 3 and 16h treatment periods were chosen and subsequently the potential to induce DNA repair was assayed in primary hepatocyte cultures established from the treated animals. The viability of the hepatocytes was not affected by the treatment and no dose level revealed UDS induction. Therefore no DNA-damage resulting in increased repair synthesis was observed in hepatocytes of rats orally treated with 1,3-dioxepane.

 

Summarized no indication for a mutagenic potential was identified in in vitro and in vivo assays (Ames, HPRT, UDS).

No clear conclusions on the genotoxic potential of 1,3-dioxepane can be drawn from an in vitro chromosome aberration test in Chinese hamster lung fibroblasts (V79). A clastogenic effect was only reported at the limit dose of 5000µl/ml. Though reproducible, no dose response was observed in presence of slight cytotoxicity.

The physiological relevance of this observation is further questioned by the results of an in vivo miconucleus assay in mice. No clastogenic (and/or aneugenic) potential was identified under the harsh exposure conditions of this assay (2 intraperitoneal applications with up to 1750 mg/kg bw).

The observation with unphysiologically high concentrations of 1,3-dioxepane in the in vitro chromosome aberration testat may, at least partly, result from formaldehyde formation in the presence of metabolic activation (S9-mix).


Short description of key information:
Negative Ames assay (BASF AG, 1991).
Ambiguous in vitro Chromosomal aberration assay (BASF AG, 1994).
Negative in vitro HPRT assay (BASF AG, 1994).
Negative in vivo Micronucleus assay (BASF AG, 1998).
Negative UDS assay (BASF AG, 1998).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based in the results of the existing in vitro and in vivo assays no classification for genotoxicity is warranted according to the current EU C&L guidance.