Reaction product of 9-Octadecenoic acid, (Z)-, sulfonated, potassium salts, hydrogen peroxide and sulfuric acid

Endpoint summary

Additional information

IN VITRO

- Bacterial reverse mutation

An Ames test (OECD 471) was performed to investigate the potential of PSOA to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without metabolic activation. The plates incubated with the test item showed reduced background growth in nearly all strains. Toxic effects, evident as a reduction in the number of revertants, occurred in nearly all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with PSOA at any dose level, either in the presence or absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Therefore, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, PSOA is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

- Chromosome aberration test in vitro

The test item PSOA, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in five independent experiments. The following study design was performed:

 

	Without S9 mix		With S9 mix
	Exp.IA & IB	Exp. IIA	Exp.IA, IIA & IIB
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The highest applied concentration in the pre-test of toxicity (5000 µg/mL of the test item).

In all experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. However, in Experiment IA without S9 mix, although the mitotic index was in a normal range the number of metaphases was markedly reduced at the two highest evaluated concentrations. In Experiment IA with S9 mix, the mitotic index was reduced to approx. 60 % of control. In Experiment IB without S9 mix, with narrow concentration spacing, in Experiment IIA without S9 mix and in Experiment IIB with S9 mix the mitotic index was reduced to a range of 60 - 70 % of control at least at the highest evaluable concentration.

In Experiments IA and IB, no clastogenicity was observed at the concentrations evaluated. In Experiment IIA, in the absence of S9 mix, statistically significant and dose-dependent increases in chromosomal aberrations above the range of the historical control data (0.0 - 3.0 % aberrant cells, excluding gaps) were observed after continuous treatment with 163.3 and 285.7 µg/mL (4.0 and 12.0 % aberrant cells, excluding gaps). In Experiment IIA in the presence of S9 mix, one single statistically significant increase was observed after treatment with 400.0 µg/mL (2.5 % aberrant cells, excluding gaps). The value is in the range of the historical control data (0.0 - 3.0 % aberrant cells, excluding gaps). In Experiment IIB in the presence of S9 mix, statistically significant and dose-dependent increases were observed after treatment with 750.0 and 800.0 µg/mL (4.3 and 5.5 % aberrant cells, excluding gaps) exceeding the range of the historical control data (0.0 - 3.0 % aberrant cells, excluding gaps).

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Therefore, under the conditions of the study, it can be stated that PSOA induced structural chromosomal aberrations in human lymphocyte cells in vitro. Therefore, PSO is considered to be clastogenic in this chromosome aberration test.

-Gene mutation in mammalian cells
The study was performed to investigate the potential of PSOA to induce 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 with and without metabolic activation and had a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre- experiment (2500 Pg/mL) was based on available toxicity data. The concentration of the main experiments was limited by the cytotoxic potential of the test item. Appropriate reference mutagens, 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.

During the study no substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, PSOA is considered to be non-mutagenic in this HPRT assay.

 

 

IN VIVO

-Micronucleus Assay
Since the in vitro chromosome aberration test gave a positive result, an in vivo micronucleus assay was performed to deduce whether the positive result reflected a true mutagenic hazard of the test material or, alternatively to identify it as a false positive result.

During the study, the genetic toxicity of the test material was investigated in vivo in accordance with the standardised guideline OECD 474. The test material was dissolved in water, which was also used as the vehicle control. The pH was adjusted to 5 with NaOH. The volume administered orally was 10 mL/kg bw. 24 and 48 hours after a single administration of test material the bone marrow cells were collected for micronuclei analysis. Seven males per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes were scored for micronuclei per animal.

After treatment with the test material the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test material did not exert cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there were no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test material with any dose level. The mean values of micronuclei observed after treatment with test material were below or identical to the value of the vehicle control group and within the historical vehicle control range. The positive control showed a significant increase in micronucleus frequency.

In conclusion, it can be stated that under the conditions of the study, the test material did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat. Therefore, the test material is considered to be non-mutagenic in this micronucleus assay and identifies the finding of the in-vitro chromosome aberration test as a false positive result.

Justification for selection of genetic toxicity endpoint
No study selected because four key studies are available.

Short description of key information:
IN VITRO DATA
Gene mutation:
- negative in Salmonella typhimurium and Escherichia coli reverse mutation assay (Ames test, OECD 471).
- negative in mammalian cell gene mutation assay with Chinese hamster lung fibroblasts (V79) (HPRT) with and without metabolic activation (OECD 476)

Chromosome aberration:
- PSOA induced structural chromosomal aberrations in human lymphocytes in vitro (OECD 473). PSOA is considered to be clastogenic in the chromosome aberration test, when tested up to the highest evaluable concentration

IN VIVO DATA
Chromosome aberration:
- PSOA did not induce micronuclei during the micronucleus test which was conducted in the bone marrow cells of the rat (OECD 474)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance produced negative results in the Ames test and in the mammalian cell gene mutation test but a positive result in the in vitro chromosome aberration test. This result is mitigated by the negative result in the in vivo test investigating the same endpoint. Therefore, the substance is not considered to exhibit a potential for genetic toxicity.

In line with Directive 67/548/EEC and Regulation 1272/2008, the test substance is considered to be unclassified for genetic toxicity.