Fatty acids, C14-18 and C16-18-unsatd., maleated

Endpoint summary

Additional information

Read across justification

The test item is an UVCB and composes mainly of modified fatty acids from sun flower oil (mSOFA) and, to a minor part, of modiefied fatty acids from tall oil (mTOFA). The fatty acids obtained from tall oil and sun flower oil are of comparable chain lenghth and are modified by the same reaction step. In contrast to mTOFA, mSOFA is also produced solely by the very same reaction as the UVCB and consists therefore of the same reaction products and similar impurities. Furthermore, mSOFA was intentively examined for its toxicological properties. Thus, it is acceptable to derive data on gentotoxicity from the read across substance mSOFA.

Procedure and observations

The substance was tested for its mutagenic potential based on the ability to induce point mutations in S.typhimurium and E. coli at concentrations of 33 μg - 5 500 μg/plate (SPT and PIT) in a reverse mutation assay. A standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) was performed. Precipitation of the test substance was found from about 5 500 μg/plate onward with and without S9 mix. A bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2 750 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

The substance was also assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase

(HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, all with and without the addition of liver S9 mix at concentrations up to 4000 ug/ml. Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6- thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The 1st Experiment with and without metabolic activation did not fulfill the requirements of the current OECD Guideline 476 due to strong test substance precipitation in culture medium from the lowest applied concentration onward. Therefore, this experiment was discontinued

directly at the end of exposure period. In this study, in the 2nd and 3rd Experiment in the absence and the presence of metabolic

activation, the highest applied concentrations tested for gene mutations led to clear test substance precipitation in culture medium at the end of exposure period and showed severe cytotoxicity. The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two valid experiments performed independently of each other.

read across CAS 1309959 -24 -7

Three in vitro assays were performed to evaluate the genotoxicity of the read across substance. At first, the material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test item was applied at concentrations of 33 μg - 5500 μg/plate (standard plate test) and 10 μg - 2750 μg/plate (pre-incubation test) in presence and absence of a metabolic activation system (S9 mix). Precipitation of the test item was found at 5500 μg/plate with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

The test item dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations inV79cells of the Chinese hamster in vitro in five independent experiments. In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations. Cytotoxicity was observed at the highest concentrations. In Experiment I in the absence and presence of S9 mix and in Experiment IIB and IID in the absence of S9 mix after 18 and 28 hours continuous treatment no clastogenicity was observed at the concentrations evaluated. In Experiment IIA in the presence of S9 mix at preparation interval 28 hours statistically significant increases in chromosomal aberrations were observed after treatment with 60.5, 120.9 and 241.9 µg/mL (4.5, 4.0 and 6.0 % aberrant cells, excluding gaps). Two of these values (4.5 and 6.0 %) exceeded the range of the laboratory historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps). In the confirmatory experiment IIC, one statistically significant increase above the range of the laboratory historical solvent control data was observed treatment with 250.0 µg/mL (5.3 % aberrant cells, excluding gaps). No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

A micronucleus assay in mouse 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 material, dissolved in 30% DMSO / 70% PEG 400, was administered singly at concentrations of 500, 1000 and 2000 mg/kg bw. 24h and 48h after administration the bone marrow cells were collected for micronuclei analysis. Seven males per test group were evaluated for the occurrence of micronuclei; per animal 2000 PCEs were scored. To investigate a cytotoxic effect of the compound the ratio between polychromatic and normochromatic erythrocytes was determined. After treatment the number of PCEs was not substantially decreased as compared to the vehicle control thus indicating that the substance did not exert a cytotoxic effect 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.

Discussion

In conclusion, the test item as well as the analogue substance is not mutagenic when tested in bacteria and mammalian cells. The analogue did not induce micronuclei and is considered to be non-mutagenic in the in vivo micronucleus assay. In Experiment IIA of the chromosome aberration assay in the presence of S9 mix increases in chromosomal aberrations were observed. In experiment IIC, one increase above the range of the laboratory historical solvent control data was confirmed. Since no dose-dependency was observed and the positive values were observed in a range where strong cytotoxicity and precipitation occurred, is considered to be non-clastogenic.

Short description of key information:
Experimental data on genotoxicity are available for the test item as well as for a structural analogue. The test material is not mutagenic in bacteria and in mammalian cells when tested accoding to the OECD guidelines 471 and 476 under GLP. The structural analogue is also negative for mutagenicity when tested in vitro and did not induce micronuclei in vivo. In Experiment IIA and IIC of a chromosome aberration assay increases in chromosomal aberrations were observed. Since no dose-dependency was observed and the positive values were in a range where strong cytotoxicity and precipitation occurred, the test item is considered to be non-clastogenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC.

 

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.