Reaction Products of Dimethyl hydrogen phosphite and Alcohols C14-18, Even, Linear

Endpoint summary

Additional information

Bacterial reverse mutation

GUIDELINE

The test item was evaluated in the Ames/Salmonella-E.coli liquid pre-incubation assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) plus the tryptophan locus in on Escherichia coli tester strain (WP2 uvrA). Testing took place in the presence and absence of an exogenous metabolic activation system (S9).

METHOD

Toxicity of the test item was first evaluated in a pre-screen by treating duplicate cultures of strains TA1538, TA100 and WP2 uvrA with the test article at doses of 50.0, 167, 500, 1670 and 5000 µg/plate in the absence of S9. Results of the pre-screen indicated that the test item was not toxic to any strain at a dose of 50 µg/plate, or to WP2 uvrA at does of 167 and 500 µg/plate in the absence of S9. Inhibited growth, characterised by a reduced background lawn and/or the presence of pindot colonies, was observed in TA1358 and TA100 at doses ≥ 167 µg/plate and in WP2 uvrA at doses ≥ 1670 µg/plate. In addition, the test article was completely soluble at doses ≥ 500 µg/plate.

Based on these findings, the test item was evaluated in triplicate cultures in all five Salmonelle tester strains at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 µg/plate and in WP2 uvrA at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate. Investigation took place in the presence and absence of S9, which included 6 % (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors. The test article was again found to be incompletely soluble at doses ≥ 1670 µg/plate but normal growth was observed in all tester strains at all doses evaluated with and without S9. Revertant frequencies for all doses of the test item in all tester strains with and without S9 approximated, or were less than, those observed in the concurrent negative control cultures.

The test item was re-evaluated in the confirmatory assay in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with and without S9. The test article was again found to be incompletely soluble at doses ≥ 500 µg/plate. Inhibited growth was observed in tester strains TA1537 and TA1538 at doses of 1670 and/or 5000 µg/plate with and without S9. Revertant frequencies for all doses of the test item in all tester strains with and without S9 again approximated or were less than control values.

The test item was re-evaluated under identical conditions in all five Salmonella tester strains to confirm the results observed at the higher dose levels investigated in the second assay. The test article was again found to be incompletely soluble at doses ≥ 1670 µg/plate and inhibited growth was again observed in tester strains TA1535 and TA100 at a dose of 5000 µg/plate with or without S9. Revertant frequencies for all doses of test item in all tester strains with S9, and in tester strains TA1535, TA1537, TA98 and TA100 without S9, approximated or were less than control values. In contrast, statistically significant increases in revertant frequencies (to approximately 2.4 to 2.7 -fold control values) were observed in strain TA1538 at doses of 16.7 and 50.0 µg/plate without S9. However, these increases were not dose dependent.

The test item was therefore re-evaluated in strain TA1538 at doses of 1.67, 5.00, 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate wihtout S9. The test article was again found to be incompletely soluble at doses ≥ 1670 µg/plate and inhibited growth was observed at doses of 167, 500, 1670 and 5000 µg/plate. Revertant frequencies for all doses of test item again approximated or were less than control values. Thus, the slight increases observed in strain TA1538 in the previous evaluation were considered to be statistical aberrations due to random fluctuation of the spontaneous revertant frequency. The apparent discrepency in toxicity between the pre-screen and mutation assays was likely due to interference as a result of test item insolubility. All positive and negative control values in all assays were within acceptable limits.

CONCLUSION

Results for the test item were negative in the Ames/Salmonella-E. coli liquid pre-incubation assay.

Chromosome aberration test

GUIDELINE

The investigation was designed to be compatible with the procedures indicated by the OECD Guidelines for Testing of Chemicals No. 473 "In VitroMammalian Chromosome Aberration Test", adopted 26 September 2014, and The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI) and Ministry of the Environment (MOE) Guidelines of 31 March 2011.

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. The main experiment was performed using three exposure conditions; a 4 -hour exposure in the presence and absence of a standard metabolising system (S9 at a final concentration of 2 %) and a 24 -hour exposure in the absence of metabolic activation.

Dose levels in the main experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. Final concentration test item dose levels for the main test were 0, 5, 10, 20, 30, 40 and 80 µg/L [4(20)-hour without S9]; 0, 2.5, 5, 10, 20, 40 and 80 µg/L [4(20)-hour with S9 (2 %)]; 0, 1.25, 2.5, 5, 10, 20, 30 and 40 µg/L [24 -hour without S9].

RESULTS

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The test item did not demonstrate any obvious toxicity in the preliminary toxicity test and therefore the dose range for the main experiment was limited by precipitate. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in any of the three exposure conditions of the main experiment using a dose range that included a dose level that was the lowest precipitating dose level.

CONCLUSION

The test item was considered to be non-clastogenic to human lymphocytesin vitro.

Mammalian Cell Gene Mutation Assay

GUIDELINE

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.

METHODS

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 six 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 six or 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 or solubility in the vehicle.

RESULTS

A precipitate of test item was observed at and above 156.25 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.

CONCLUSION

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.

Justification for selection of genetic toxicity endpoint
GLP quality studies performed according to the latest OECD test guidelines.

Short description of key information:
An Ames/Salmonella-E.coli liquid pre-incubation assay similar to OECD 471. A GLP chromosome aberration test in human lymphocytes according to OECD 473. A GLP gene mutation assay in mouse lymphoma L5178Y cells TK locus according to OECD 476.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification for mutagenicity is proposed according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).