Amines, C12-14-tert-alkyl, C14-18 and C18-unsatd.-alkyl phosphates

Endpoint summary

Additional information

In order to evaluate the potential for genetic toxicity of the substance “reaction products of alcohols, C14 - 18, C18 unsat., esterified with phosphorus pentoxide and salted with amines, C12 -14,-tert-alkyl", there are three key studies available which investigate chromosomal aberrations in mamallian cells, the genetic toxicity by Ames (1973) and the mutagenicity in mouse lymphoma cells.

Genotoxicity in bacterial strains

In a key study, the substance was tested according to OECD Guideline 471, EU Method B.13/14 and the USA, EPA (TSCA) OPPTS harmonised guidelines in order to determine its genetic toxicity in bacterial strains (Bowles, 2012). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day using an amended dose range ranging from 1.5 to 5000 µg/plate, depending on strain type and exposure. Fresh cultures of the bacterial strains and fresh test item formulations were prepared also.Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level either with or without metabolic activation. However, several of the bacterial strains (particularly TA98 and TA1537) exhibited lowered revertant counts at the upper test item dose levels although these responses were not fully reproducible. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests. Therefore, the test item (reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxid) was considered to be non-mutagenic under the conditions of this test.

Chromosomal aberration Test

An in vitro test for the detection of structural chromosomal aberrations in cultured mammalian cells according to OECD 473 and EU Method B.10 was performed (Morris, 2012). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period took place. In Experiment 2, a 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were 40, 80, 160, 320, 480 and 640 µg/mL in the 4(20)-hour group without S9 and the 4(20)-hour groups with 1% and 2% S9, respectively. Dose levels of 5, 10, 20, 40, 50, 60, 80 and 120 µg/mL were used for the 24-hour group without S9. All vehicle (solvent) control groups 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 indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. In conclusion, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Genotoxicity in mammalian cells

The evaluation of the mutagenic potential of the substance “reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxide” was performed 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 (Brown, 2012). 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 B.17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW 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 was selected following the results of a preliminary toxicity test, and was 20 to 960 µg/mL for Experiment 1 in both the absence and presence of metabolic activation. In Experiment 2, the dose range was 2.5 to 160 µg/mL in the absence of metabolic activation, and 40 to 720 µg/mL in the presence of metabolic activation. The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was observed at and above 80 µg/mL in the Mutagenicity Test. 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 metabolising system. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. In conclusion, the test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for selection of genetic toxicity endpoint
No study is selected since all three studies were negative.

Short description of key information:
Clearly negative in vitro studies - both with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the classification criteria outlined in section 3.5.2.2 (Guidance on the Application of CLP criteria, 2012), the test item need to be classified if the substance induces heritable mutations in the germ cells of humans (positive evidence) or the substance causes concern for humans owing to the possibility that they may induce such heritable mutations in humans. No positive results for the test item were found in all in vitro studies. Therefore, this substance does not meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification as a mutagen.