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In vitro gene mutation assays in bacteria

TP-90B Rubber Chemical was not mutagenic in the Ames test in Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 and Escherichia coli strain WP2 urvA, both in the presence and in the absence of S-9 mix when tested up to cytotoxic doses (Scarcella, 2003). The study was conducted according to OECD TG 471 and GLP regulations, and comprised three individual experiments: one experiment using the standard plate test method, two experiments using the preincubation method. There was no increase in the number of revertants caused by the test substance in any of the experiments or tester strains.

 

In vitro gene mutation assays in mammalian cells

In a GLP conform study according to OECD guideline 476, the mutagenic activity of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane (purity 92 area-%) was evaluated in a gene mutation test with V79 Chinese hamster cells with independent repeat (NOTOX, 2010). 

The test was performed in two independent experiments in the absence and presence of S9-mix.  The test substance was dissolved in dimethyl sulfoxide.

In the first experiment, Bis-(2-(2-butoxyethoxy)-ethoxy)-methane was tested up to concentrations of 2500 µg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. Bis-(2-(2-butoxyethoxy)-ethoxy)-methane was tested up to cytotoxic levels of 70 and 61% in the absence and presence of S9-mix, respectively.

In the second experiment, Bis-(2-(2-butoxyethoxy)-ethoxy)-methane was tested up to concentrations of 1500 and 2700 µg/mL in the absence and presence of S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Bis-(2-(2-butoxyethoxy)-ethoxy)-methane was tested up to cytotoxic levels of 93% in the absence of S9-mix and up to 68% in the presence of S9-mix, respectively.

The spontaneous mutation frequencies in the solvent-treated control cultures were within the historical control data range and within the acceptability criteria of this assay. Except the response in the absence of S9-mix (second experiment), in which the mutation frequency was below the limit of the historical control data range. However since all observed mutation frequencies of the solvent control substances were within the acceptability criteria of this assay, the validity of the test was considered to be not affected.

Mutation frequencies in cultures treated with positive control chemicals were increased by 7.2-fold forin the absence of S9-mix, and by 5.2- and 43-fold for DMN in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In all treatments, Bis-(2-(2-butoxyethoxy)-ethoxy)-methane did not induce a significant increase in the mutation frequency. It is concluded that Bis-(2-(2-butoxyethoxy)-ethoxy)-methane is not mutagenic in the gene mutation test with V79 Chinese hamster cells under the given experimental conditions.

  

 

In vivo Studies

 In a CD-1 mouse bone marrow micronucleus assay according to GLP requirements and following OECD TG 474 (Getuli, 2003), the test animals were given a single oral dose (gavage) of the test substance in corn oil at doses of 0, 375, 750 and 1500 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours after treatment. The number of micronuclei in polychromatic erythrocytes was evaluated in 2000 polychromatic erythrocytes and the percentage of polychromatic erythrocytes was evaluated in 200 erythrocytes. Signs of toxicity and cytotoxicity were reported. There was no increase in the number of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. The positive control induced the appropriate response.

 


Short description of key information:
in vitro
Gene mutation in bacteria
Ames test; S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA, with and without metabolic activation: negative (GLP, OECD 471; Scarella 2003)

Gene mutation in mammalian cells
HPRT Test, V79 cells, with and without metabolic activation: negative (GLP, OECD 476; NOTOX 2010)

Cytogenicity in mammalian cells
no data available

in vivo
Micronucleus test; mouse, oral: negative (GLP, OECD 474; Getulli 2003)

Endpoint Conclusion:

Justification for classification or non-classification

Based on the available data on the genetic toxicity of hexaoxatricosane no classification is warranted according to the criteria of both, 67/548/EEC and CLP regulation (EC) 1272/2008.