Amines, C12-14-alkyl, C6-10-alkyl phosphates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial mutagenicity:

Four valid experiments were performed. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item Deophos 228 was tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535). The test was performed in four experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.  

Experiment 1a:

In this experiment,the test item (dissolved in acetone) was tested up to concentrations of 5  µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

At the three highest concentrations (5, 1.5 and 0.5 µL/plate) no bacteria growth and no bacterial lawn was observed. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.

At the lower two concentrations, the bacterial background lawn was not reduced and no decrease of the spontaneous revertants was observed.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 1b:

Based on the toxicity results of the experiment 1a,the test item was tested up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

At the highest concentration (0.5 µL/plate), no bacteria growth was observed in the following bacteria strains: TA97a, TA98 and TA100. Towards the bacteria strains TA102 and TA1535 a clear decrease in the spontaneous revertants was observed.

The bacterial background lawn was observed in all concentrations.

 

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Experiment 2a:

In this experiment, the test item (dissolved in acetone) was tested up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

At the three highest concentrations (0.5, 0.25 and 0.125 µL/plate) no bacteria growth and no bacterial lawn was visible. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.

Towards the bacteria strains TA97a and TA100, signs of toxicity were observed in the concentrations 0.063, 0.031 and 0.016 µL/plate, too.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2b:

In this experiment, the test item was tested up to concentrations of 0.125 µL/plate in the absence and presence of S9-mix in the bacteria strains TA98, TA102 and TA1535 resp. up to concentrations of 0.016 µL/plate in the absence and presence of S9-mix in the bacteria strains TA97a and TA100.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed in the following concentrations towards the respective bacteria strains:

·        TA97a: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA98: 0.125 µL/plate (in the treatment without S9 mix no bacteria growth was observed, in the treatment with S9 mix a decrease in the spontaneous revertants was observed)

·        TA100: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA102: 0.125 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA1535: 0.125 µL/plate (no bacteria growth was observed in the treatment with and without S9 mix)

 

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the results of this study it is concluded that Deophos 228 is not mutagenic in theSalmonella typhimuriumstrains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

in vitro mammalian cell mutagenicity assay:

A study was conducted to determine the mutagenic potential of the read-across substance amines, C11 -14 -branched alkyl, monohexyl and dihexyl phosphatesaccording to OECD Guideline 476 and EU Method B.17. The study was conducted to investigate the induced gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed and a treatment period of 4 h at 0, 2.5, 5, 10, 20, 40, 60 and 80 μg/mL without metabolic activation and at 0, 1.3, 2.5, 5, 10, 20, 30 and 40 μg/mL with metabolic activation. The second experiment was performed with a treatment time of 4 h with metabolic activation at 0, 1.3, 2.5, 5, 10, 20, 30 and 40 μg/mL and 24 h without metabolic activation at 0.31, 0.63, 1.3, 2.5, 5, 7.5 and 10 μg/mL. The maximum concentration of the pre-experiments was 5000 μg/mL. The test substance was suspended (pre-experiment) or dissolved (main experiments) in culture medium. The concentration range of the main experiments was limited by cytotoxicity of the test substance. No substantial and reproducible dose-dependent increase in the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The reference mutagens (ethylmethane sulphonate and 7, 12 -dimethylbenzanthracene), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Under the study conditions, the test substance did not induce gene mutations at the HPRT locus in V79 cells and therefore, is considered to be non-mutagenic in this HPRT assay (Wollny, 2013).

in vitro mammalian cell cytogenetics assay:

As study was conducted to determine the in vitro cytogenicity potential of the read-across substance amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

according to OECD Guideline 473 and EU Method B.10, under GLP conditions. The test substance dissolved in culture medium, was assessed for its potential to induce structural chromosome aberrations inV79cells of the Chinese hamsterin vitroin two independent experiments. In each experimental group two parallel cultures were set up. Experiment I was conducted with 18 h exposure period at 0, 12.5, 25 and 50 μg/mL without metabolic activation and with 4 h exposure period at 0, 25, 50 and 100 μg/mL with metabolic activation. Experiment II was conducted with 18 h exposure period at 0, 0.6, 1.2 and 2.3 μg/mL and with 28 h exposure period at 0, 1.2, 2.3 and 4.7 μg/mL without metabolic activation and with 4 h exposure period at 0, 6.3, 12.5 and 25 μg/mL with metabolic activation. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without S9 mix and 18 h treatment, where only 50 metaphases were evaluated. Dose selection for the cytogenetic experiments was performed considering the toxicity data. Appropriate mutagens (ethylmethane sulphonate and cyclophosphamide) were used as positive controls. In Experiment I in the absence of S9 mix and in Experiment II in the absence of S9 mix with 18 hours continuous treatment concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I and II in the presence of S9 mix and in Experiment II in the absence of S9 mix with 28 h continuous treatment cytotoxicity indicated as reduced mitotic index was observed at the highest evaluated concentration. No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Positive controls induced statistically significant increases in cells with structural chromosome aberrations. Under the study conditions, the test substance was not found to induce structural chromosomal aberrations in V79 cells (Chinese hamster cell line)in vitro(Bohnberger, 2013).

Justification for classification or non-classification

Based on the results of the in vitro mutagenicity and cytogenicity assays with the substance and the read-across substance, no classification for genotoxicity is warranted according to EU CLP (EC 1272/2008) criteria.