O,O,O',O'-tetraoctyl-(branched and linear) S,S'-{methylenebis[imino(3-oxopropane-3,1-diyl)]} bis(phosphorothioate)

Endpoint summary

Additional information

In vitro

Ames (bacterial) Test

Three AMES test were performed and all reported results were negative. Therefore, the test material is conisdered non-mutagenic.

Chromosome aberration

The test material did not induce any statistically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times. The test material was therefore considered to be non-clastogenic to CHL and CHO cells in vitro.

Mouse lymmphoma assay

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose level used in the main test was limited by test item induced toxicity

A precipitate of test item was observed at and above 312.5 ug/mL. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

In vivo

Mouse micronucleus assay

The potential of this substance to cause germ cell mutagenicity was evaluated in an in vivo mouse micronucleus assay (OECD 474). Based on the results of a range finding study groups of mice (7/group) were administered single i.p. doses or 500, 1000, and 2000 mg/kg with a dose volume of 10 ml. The study included a concurrent vehicle (arachis oil) and positive (cyclophosphamide) control groups. Immediately following sacrifice (ie. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re­ suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium. A positive mutagenic response was considered demonstrated when a statistically significant dose-responsive increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was considered demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group. No evidence of a significant increase in the incidence of micronucleated PCEs with the test material compared to the vehicle control was observed. Also, no statistically significant decreases in the PCE/NCE ratios were found in the 24 or 48 hour test material groups. Evidence that absorption was demonstrated by the presence of clinical signs in one animal in the 48 hour, 2000 mg/kg group. The study was determined to be valid based on the marked increase in the frequency of micronucleated PCEs in the positive control group. Based on the results of this study this substance does not require classification under EU Regulation (EC) No. 1272/2008 for germ cell mutagenicity.

Conclusion

Overall, no adverse effects were observed and is considered negative for genotoxicity.

Short description of key information:
Three AMES test were performed and all reported results were negative. Therefore, the test material is considered negative for genotoxicity.

The test material did not induce any statistically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times. The test material was therefore considered to be non-clastogenic to CHL and CHO cells in vitro.

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

In an in vivo mouse micronucleus assay no evidence of a significant increase in the incidence of micronucleated PCEs with the test material compared to the vehicle control was observed. Also, no statistically significant decreases in the PCE/NCE ratios were found in the 24 or 48 hour test material groups.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Five in vitro and one in vivo genetic toxicity studies were conducted and all demonstrated negative results. Therefore, classification for mutatgenicity is not required.