Reaction mass of orthophosphoric acid and potassium dihexadecyl phosphate and dipotassium hexadecyl phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available weight of evidence information from the in vitro studies on the test substance as well as the main constituents, the test substance, ‘mono- C16 PSE and C16-OH’ is considered to be non-genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test and chromosomal aberration test are available for the test substance. However, in absence of a mammalian mutagenicity assay with the test substance, this endpoint been assessed based on studies available on substances representative of the two main constituents, which can be categorised as phosphate esters (PSE) and phosphoric acid. The results are presented below:

Ames test

Anin vitrostudy was conducted to determine the genotoxic potential of test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ (purity: 100%), using Ames test, according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test substance using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), in triplicate, both with and without metabolic activation (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and ranged from 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test substance concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally 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. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and second mutation test (pre-incubation method). A test substance precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and 2 (pre-incubation method). Under the study conditions, the test substance was determined to be non-genotoxic in the Ames test, with and without metabolic activation (Envigo, 2017).

Chromosomal aberration test

Anin vitrostudy was conducted to determine the clastogenicity of the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ (purity: 100%) using human lymphocytes, according to the OECD Guideline 473 (Chromosome Aberration test) and Japanese Guidelines, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at doses 0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL, together with vehicle and positive controls. In this study, three exposure conditions were investigated: 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-h expression period, 4 h exposure in the absence of metabolic activation (S9) with a 20-h expression period and a 24-h exposure in the absence of metabolic activation. The dose levels used 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. All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances (Mitomycin C and Cyclophosphamide) 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 substance was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (Envigo, 2017).

Mammalian mutagenicity assay:

Constituent 1: PSE - read across study

Anin vitrostudy was conducted to determine the genotoxic potential of the read across substance, ‘mono- C16 PSE and C16-OH’ (purity: 100%), using the thymidine kinase gene according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the read across substance to induce mutations at the thymidine kinase locus (TK1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y TK+/- cells. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the read across substance at 8 dose levels (ranging from 0.61 to 78 μg/mL) in duplicate, together with vehicle (DMSO), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 h exposure group in the absence of metabolic activation. The dose range of read across substance used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies in the preliminary test ranged from 2.44 to 625 μg/mL in the three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). A precipitate of the read across substance was observed at and above 39 μg/mL in all three exposure groups. Therefore, the maximum dose level used for the main test was limited by precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. There was evidence of moderate toxicity following exposure to the read across substance in the 4 h and 24 h exposure in the absence of metabolic, as indicated by the % RSG (relative suspension growth) and RTG (relative total growth) values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. The dose levels of 0.61 and 78 μg/mL in both the 4 and 24 h exposure –S9 were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 39 and 78 μg/mL in the 4 h +S9 exposure again were not plated out for 5-TFT resistance and viability due to excessive toxicity. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The read across substance did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the three exposure groups. The GEF (Global Evaluation Factor) value of the read across substance dose levels were not exceeded in any of the three exposure groups, including dose levels beyond the acceptable level of toxicity in the 24 h exposure (Envigo, 2017). Based on the results of the read across study, the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ is considered to be not-mutagenic in mouse lymphoma assay, with and without metabolic activation.

Constituent 2: Phosphoric acid: No mammalian mutagenicity study was available. An vivo mouse translocation assay conducted with disodium pyrophosphate was found to be negative when tested in male mice at doses up to 1400 mg/kg bw (CIR, 2016).

Overall, based on the available weight of evidence from the studies on test substance as well as main constituents, the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’, is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the available weight of evidence information from in vitro studies on the test substance as well as the main constituents, the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ does not warrant a classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​​