Fatty acids, tall-oil, reaction products with ethanolamine, ethoxylated

Endpoint summary

Additional information

Ames test

The test item was tested in the Salmonella typhimurium Reverse Mutation Assay for the induction of reverse mutations in a bacterial test system. The assay was performed with the Salmonella typhimurium strains TA98, TA 100, TA 1535, TA 1537 and TA 1538 in independent experiments, with and without metabolic activation by S9-mix. The second test without S9-mix was repeated, due to strong toxic effects in all strains in the solution control and all concentrations of the test substance.

Solutions of the test substance were prepared in ethanol and diluted with the same solvent just before use. The following concentrations were tested:

1st test: 8, 40, 200, 1000 and 5000 µg/plate

2nd test and repetition:

without S9-mix: 1.6, 8, 40, 200 and 1000 µg/plate

with S9-mix 1, 3, 10, 30 and 90 µg/plate

The direct plate incorporation assay was utilized. The following results were obtained:

Toxic effects were noted at the concentration of 40 µg/plate or higher. Precipitations were not noted.

No enhanced revertant rates compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation.

Therefore the test substance did not induce reverse mutations in the tested strains of Salmonella typhimurium in this bacterial mutagenicity test.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce gene mutations in Salmonella typhimurium strains. Subsequently, the test item is considered not to be mutagenic in this bacterial mutagenicity test in vitro.

 

Chromosome aberration in vitro

To investigate the potential of the test item to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out. The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

with metabolic activation: 100, 110, 120 and 140 µg/mL

without metabolic activation: 7.5, 15 and 30 µg/mL

Experiment Il:

with metabolic activation: 50, 70 and 80 µg/mL

without metabolic activation: 15, 30 and 60 µg/mL

The test item was suspended in cell culture medium. Precipitation of the test item was noted at a concentration of 30 µg/mL and higher. In experiment I without metabolic activation, toxic effects of the test item were noted at a concentration of 30 µg/mL, with metabolic activation at concentrations of 110 µg/mL and higher. In experiment Il without metabolic activation, toxic effects of the test item were observed at a concentration of 60 µg/mL. With metabolic activation, toxic effects of the test item were noted at concentrations of 70 µg/mL and higher.

In both experiments without metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In experiment I with metabolic activation an increase of aberrant cells was noted at a concentration of 140 µg/mL. The aberration rate found was above the historical control data of the negative control. In experiment II with metabolic activation an increase of aberrant cells was observed as well. The aberration rate found for the highest dose group (80 µg/mL) was clearly above the historical control data of the negative control. The clear clastogenic effect in both experiments with metabolic activation was correlated with high toxicity effects. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item induced structural chromosomal aberrations in the V79 Chinese hamster cell line in presence of metabolic activation. Therefore, the test item is considered to be clastogenic in vitro.

HPRT assay:

In a mammalian cell gene mutation assay for the HPRT locus (Harlan CCR, 2013; 1487004 ) V79 cells cultured in vitro were exposed to the test item in DMSO at concentrations of 0; 5; 10; 20; 40, 60 or 80.0 µg/mL in the presence and absence of mammalian metabolic activation (S9 -mix) in two independent experiments. The first main experiment was performed with and without liver microsomal activation and 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 concentration range of the main experiments was limited by cytotoxic effects and phase separation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus demonstration appropriate sensitivity of the test system. 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.

 

Chromosome aberration in vivo

The study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

 

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

 

The highest dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with the bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

 

Conclusion:

The test substance did not induce reverse mutations in Salmonella typhimurium strains. The test item did not induce gene mutations at the HPRT locus in V79 cellswith or without metabolic activation. The test item induced structural chromosomal aberrations in the V79 Chinese hamster cell line in presence of metabolic activation. However, no induction of micronuclei as determined by the micronucleus test with bone marrow cells of the mouse was observed. Therefore, the substance is considered to be not mutagenic.

Justification for selection of genetic toxicity endpoint
In vivo study according to OECD/EU guideline and the prnciples of GLP. Reliable without restrictions.

Short description of key information:
The test substance did not induce reverse mutations in Salmonella typhimurium strains. The test item induced structural chromosomal aberrations in the V79 Chinese hamster cell line in presence of metabolic activation. The test item did not induce gene mutations at the HPRT locus in V79 cellswith or without metabolic activation. No induction of micronuclei as determined by the micronucleus test with bone marrow cells of the mouse was observed. Thus, the substance is not classified as mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the experimental data available the test item is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 and Directive 67/548/EEC.