Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Endpoint summary

Additional information

Valid experimental data are available to assess the genetic toxicity of Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

GENETIC TOXICITY IN VITRO

In vitro:

Gene mutation in bacteria:

OECD conform studies:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) was tested in the Salmonella typhimurium Reverse Mutation Assay for the induction of reverse mutations in a bacterial test system according to OECD guideline 471 under GLP condition (Henkel, BASF PCN, 1991). The assay was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in two independent experiments, both with and without metabolic activation by S9-mix. Suspensions of the test substance were prepared in Tween80/bidist. water and diluted with bidist. water just before use. The following concentrations, related to the active content of the pure substance, were tested: 1st test: 8, 40, 200, 1000 and 5000 µg/plate (and repetition); 2nd test: 4, 20, 100, 500 and 2500 µg/plate. Toxic effects were noted at the concentration of 1000 µg/plate or higher. Precipitations were not noted. The following results were obtained: No enhanced revertant rates compared to concurrent negative controls, induced by the test substance, were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. The test did not induce reverse mutations in the tested strains of Salmonella typhimurium in this bacterial mutagenicity test, neither with nor without metabolic activation by S9 -mix. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) did not induce gene mutations in Salmonella typhimurium strains.

Therefore, Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) is considered not to be mutagenic in this bacterial mutagenicity test in vitro.

 

Gene mutation in mammalian cells:

OECD conform studies:

A study was performed to investigate the potential of Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) to induce gene mutations at the HPRT locus in V79 cells of the Chinese Hamster (OECD 476, 1997) (Henkel, BASF PCN, 1994). The assay was performed in two independent experiments (due to technical reasons, experiment II was performed twice), using identical procedures, both with and without liver microsomal activation. The first realization of experiment II failed and no data are reported, the reported data refer to the second realization of experiment II. The test article was tested with the following concentrations:

Experiment I: without S9 mix: 3.0*, 6.0, 10.0, 30.0, 60.0 and 100.0** µg/mL with S9 mix: 16.5, 60.0, 100.0 and 165.0 µg/mL

Experiment II: without S9 mix: 6.0, 10.0, 30.0, 40.0, 50.0** and 60.0**µg/mL with S9 mix: 16.5, 60.0, 100.0 and 165.0 µg/mL

*not evaluated, culture not continued, **not evaluable, toxic effects

According to the pre-test on toxicity the concentration ranges were selected to yield concentration-related toxic effects. The highest concentration produced a low-level of survival and the survival at the lowest concentration was approximately in the range of the negative control. Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independet experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) is considered to be non-mutagenic in this HPRT assay.

Cytogenetic in mammalian cells:

OECD conform studies:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) was tested in the in vitro mammalian cytogenetic test with Chinese Hamster V79 cells for its potential to induce structural chromosomal aberrations (OECD 473, 1997) (Henkel, BASF PCN, 1997a). Chromosomes were prepared at 7, 20 and 28 hours after start of treatment with the test substance, dissolved in medium. The treatment interval was 4 hours, with as well as without metabolic activation. All cultures were run in duplicate per experimental point. For analysis of chromosomal aberrations, the following concentrations were chosen on the basis of the pre-experiment for toxicity and the determination of the mitotic index in the main experiment: without metabolic activation: 7h ( 200 µg/mL );  20h (40, 200, 400 µg/mL) and 28h (400 µg/mL) with metabolic activation: 7h (800 µg/mL) ; 20h (40, 200, 400 µg/mL) and 28h (400 µg/mL) .The substance was suspended in medium. In the experiment on plating efficiency, strong toxic effects were noticed at 1000 µg/mL and higher with and without metabolic activation. In the experiment without metabolic activation, the highest concentration of 800 µg/mL were toxic at all time points; 7 h after the start of treatment the mitotic index was reduced to about 20 % at 200 µg/mL. In the experiment with S9 -mix, the highest concentration of 800 µg/mL was toxic 20 h and 28 h after treatment; 7 h after start of treatment the mitotic index was reduced to approximately 40 %. In the experiment with metabolic activation, an increase in the number of cells with chromosomal aberrations without gaps was observed at the highest concentration of 800 µg/mL 7 h after treatment. The number of cells with exchanges was slightly increased 20 h after start of treatment at all concentrations chosen for the evaluation of chromosomal aberrations. There was no indication of an increase in the frequency of polyploid metaphases after treatment with the test substance compared to negative controls. In conclusion, it should be stated that in the study described and under the experimental conditions reported, the test substance Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) might induce chromosomal aberrations in the V79 chinese hamster cell line. To confirm this result a second experiment was conducted (see below).

In an additional experiment Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) was tested in the in vitro mammalian cytogenetic test with Chinese Hamster V79 cells for its potential to induce structural chromosomal aberrations (OECD 473, 1997) (Henkel, BASF PCN, 1997b). This test is the repetitive experiment of the assay mentioned above with the study number 9300029 5 performed to verify a questionable positive result of the first experiment. Chromosomes were prepared at 7 and 20 hours after start of treatment with the test substance. The treatment interval was 4 hours, only with metabolic activation. All cultures were run in duplicate per experimental point. For the analyses of chromosomal aberrations, the following concentrations were chosen on the basis of the pre-experiment for toxicity and the determination of the mitotic index in the main experiment: with metabolic activation: 7h (200 µg/mL); 20h (200, 400 µg/mL).The substance was suspended in medium. In the experiment on plating efficiency (PE), strong toxic effects were noticed at 400 µg/mL. Taking into account that the dilution factor (50%) was not taken into consideration in the first experiment the data of the PE are in agreement with the first experiment. In the cytogenetic experiment, the test substance was applied up to 400 µg/mL pure substance 7h and 20h after the start of treatment. The rel. mitotic index 7h after start of treatment was reduced in the highest concentration to approx. 10% whereas there was no reduction 20h after start of treatment. No biological effects with respect to induction of chromosomal aberrations were observed. A positive effect in one of the cultures 20h after start of treatment with 200 µg/mL pure substance could not be confirmed in the parallel culture. Furthermore, no increased aberration rate was detected at a higher concentration, a dose dependent increase could not be observed. Therefore, this effect was not regarded as biologically significant. There was no indication of an increase in the frequency of polyploid metaphases after treatment with the test substance compared to the negative controls. In the experiment with S9-mix, the mitotic index was slightly reduced. Nevertheless, significant increases in cells with chromosomal aberrations were revealed compared to the concurrent negative controls, with and without S9-mix. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce chromosomal aberrations in the V79 chinese hamster cell line in this second experiment.

According to the registrant in the first experiment an increase in the chromosomal aberration rate was observed 7 h after the start of treatment with metabolic activation. The mitotic index in the first experiment at 400 µg/mL was reduced to approx. 10%. This induction could not be confirmed in this second experiment. The highest concentration which could be evaluated in the second experiment did not show any induction of chromosomal aberrations. Therefore the clastogenic effect observed in the first experiment (R9501041) might be due to an artificial effect caused by cytotoxicity. Summarizing the data of both experiments, Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) is considered not to be mutagenic in this chromosome aberration test.

 

GENETIC TOXICITY IN VIVO

In vivo cytogenicity:

OECD conform studies:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (OECD 474, 1997) (Henkel, BASF PCN, 2006). The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a sinlge administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females; NMRI mouse) per test group were evaluated for the occurence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ration between polychromatic and total 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, and 2000 mg/kg b.w. (active substance); 48 h preparation interval: 2000 mg/kg b.w. (active substance). As estimated by a pre-experiment 2000 mg test substance per kg b.w. (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) did not have any cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as a positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia (AS 50%) did not induce micronuclei as determined by the micronucleus test in bone marrow cells of the mouse.

Key study assignment:

For each toxicological endpoint one reliable and suitable study are available. No further reliable information was available, therefore these studies are integrated as key studies.

Assessment of genetic toxicity:

According to the testing strategy for mutagenicity, first in vitro tests were conducted to assess the mutagenic potential. All performed invitro tests assessing the mutagenicity in bactria (Henkel, BASF PCN, 1991),the gene mutation in mammalian cell (Henkel, BASF PCN, 1994) and the cytogenetic in mammalian cells (Henkel, BASF PCN, 1997a+b) are negative. The chromosomal aberration test shows some unusual findings, therefore this was partly repeated with negative result. Nevertheless the repetition of these test rose some doubts according the cytogenicity of the substance. In result the registrant decided to remove this scepsis performing an in vivo micronucleous test. This test shows no increase in mirconuclous compared control.

Summarizing all available information on genetic toxicity of the substance show no mutagenic effects, neither in vitro nor in vivo.

Short description of key information:
GENETIC TOXICITY IN VITRO
Gene mutation in bacteria:
S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538, with and without metabolic activation, OECD 471: negative with and without metabolic activation (Henkel, BASF PCN, 1991)
Gene mutation in mammalian cells:
HPRT, V79, with and without metabolic activation, OECD 476: non mutagenic with and without metabolic activation (Henkel, BASF PCN, 1994)
Cytogenetic in mammalian cells:
Chromosomal aberrations, V79, with and without metabolic activation, two experiments , OECD 473: no mutagenic with and without metabolic activation (Henkel, BASF PCN, 1997a+b)
GENETIC TOXICITY IN VIVO
Micronucleus test in vivo, NMRI mice, 2,000, 1,000 and 500 mg/kg body weight, OECD 474: negative, no increase in the number MN compared to control (Henkel, BASF PCN, 2006).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In summary no hints for mutagenicity obtained from experiments in vitro or in vivo are available therefore the test material does not fulfil the requirement for germ cell mutagens cat. 2 according to GHS (Regulation (EU) 1272/2008) or mutagenic category 3 DPD (67/548/EEC).

Labelling mutagenic:

GHS: no labelling

DSD: no labelling