Phosphoric acid, mono- and di-C6-10-alkyl esters

Endpoint summary

Additional information

Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli

Method.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered), using both the Ames plate incorporation and pre-incubation methods 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 ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using a dose range of 5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations.

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.

Results.

The vehicle (dimethyl sulphoxide) 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.

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (pre-incubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. 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 (globular in appearance) was observed at 5000 µg/plate.

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 or exposure method.

Conclusion. The test item,Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered), was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration Test

Methods.

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, i.e. 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. In Experiment 2, the 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 as follows.

Group	Final concentration of Test Item (µg/ml)
4(20)-hour without S9	0, 6.25, 12.5, 25, 50, 75, 100, 150 and 200
4(20)-hour with S9 (2%)	0, 6.25, 12.5, 25, 50, 75, 100, 150 and 200
24-hour without S9	0, 12.5, 25, 50, 75, 100 and 200
4(20)-hour with S9 (1%)	0, 12.5, 25, 50, 75, 100 and 200

Results.

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 that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition.

Conclusion.

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

CHO HPRT forward mutation assay

Methods.

Chinese hamster ovary (CHO) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 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 ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

 

Exposure Group	Final concentration oftest item(µg/ml)
4-hour without S9	0, 0.5, 1, 2, 4, 6, 8, 12
4-hour with S9 (2%)	0, 8, 16, 32, 40, 44, 48
24-hour without S9	0, 1, 2, 4, 6, 8, 10, 12
4-hour with S9 (1%)	0, 2, 4, 8, 16, 32, 40, 48

 

 

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion.  The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Justification for selection of genetic toxicity endpoint
Three separate in vitro genetic toxicity studies have been conducted on the test item as follows:

Bowles (2012)/ Genetic toxicity in vitro: OECD Guideline 473 Genetic Toxicology: Chromosome Aberration Test
Bowles & Thompson (2012)/ Genetic toxicity in vitro: OECD Guideline 471 Bacterial Reverse Mutation Test
Morris (2012)/ Genetic toxicity in vitro: OECD 476 In Vitro Mammalian Cell Gene Mutation Tests

All 3 studies have been conducted according to OECD Guidelines and GLP and are adequately reported. All studies have been assigned a reliability 1.

Short description of key information:
OECD Guideline 471 Bacterial Reverse Mutation Test
The potential mutagenicity of the test item to cause gene mutation in bacteria was assessed according to OECD Guideline 471. The test item was considered to be non-mutagenic under the conditions of this test.

OECD Guideline 473 Chromosome Aberration Test
The potential mutagenicity of the test item to cause chromosomal aberrations was assessed according to OECD Guideline 473. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

OECD 476 In Vitro Mammalian Cell Gene Mutation Tests
The potenial mutagenicity of the test item to cause gene mutation in Chinese Hamster Ovary cells was assessed according to OECD Guideline 476. The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in the three following in-vitro studies, the substance is not classified for mutagencity.

 

- Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli:

The test item was considered to be non-mutagenic under the conditions of this test.

- Chromosome aberration test in human lymphocytes:

The test item is considered to be non-clastogenic in this chromosome aberration test.

- CHO HPRT forward mutation assay:

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.