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A key in vitro gene mutation study in bacteria was identified for alkenes, C24 -28. In this study, strains TA1535, TA1537, TA98, and TA100 of Salmonella typhimuriuma nd strain WP2uvrA of Escherichia coli were exposed toC24-C30, branched and linear at concentrations of 0, 15, 50, 150, 500, 1500, or 5000 µg/plate using the direct plate incorporation method (Thompson, 1998). The test conditions complied with the guideline requirements for this study type. In a preliminary range finding toxicity test, there was no reduction of the background lawn at any dose level (tested up to 5000 µg/plate); therefore, the test material was tested at the maximum dose level (i.e., 5000 µg/plate). The study authors reported that there were no significant increases in the frequency of revertant colonies in any bacterial strain at any dose level, ±S9. All positive, vehicle and negative controls responded appropriately and the S9 fraction was shown to be satisfactory. Based on these test results, the authors concluded that the test material is non-mutagenic to the bacterial strains tested in this study. 

In addition, a read-acrossin vitrocytogenicity study in mammalian cells was identified from alkenes, C20 -24. In a mammalian cell cytogenetics assay (chromosome aberration), human lymphocytes (duplicate cultures) were tested for chromosome aberration at dose levels of 0, 312.5, 625, 1250, 2500, or 5000 µg/mL in the presence and absence of metabolic activation (Wright, 1998). Two independent experiments were conducted with different exposure and harvest times: Experiment 1) 4 hour exposure followed by 16-hours for a total of 20 hour harvest time, ±S9; and Experiment 2) 20 -hour exposure, -S9, or 4-hour exposure, +S9, followed by 16-hours for a total of 20 hour harvest time. Additionally, vehicle (acetone) and positive controls were also utilized in the study design.

 

Treatment with the test material did not result in any statistically significant increase in the frequency of cells with chromosome aberrations. The number of cells with chromosome aberrations in the vehicle control was found to be within the normal range, and the positive controls responded appropriately. Under the conditions of this study, C20 -24 alkenes, branched and linear are considered to be non-clastogenic to human lymphocytes.

 

In a similar study, human lymphocytes (duplicate cultures) were tested for chromosome aberration at dose levels of 0, 3, 6, 12, 24, 48, 96 µg/mL in the presence and absence of metabolic activation (Pickard, 2008). Two independent experiments were conducted with different exposure and harvest times: Experiment 1) 4 hour exposure followed by 20 -hour culture in treatment time, ±S9; and Experiment 2) 24 -hour exposure, -S9, or 4-hour exposure,+S9, followed by 20 hour culture in treatment time. Additionally, vehicle (acetone) and positive controls were also utilized in the study design.Treatment with the test material did not result in any statistically significant increase in the frequency of cells with chromosomeaberrations. The number of cells with chromosome aberrations in the vehicle control was found to be within the normal range, and the positive controls responded appropriately. Consequently, under the conditions of this study, C20-C24 alkenes, branched and linear are considered to be non-clastogenic to human lymphocytesin vitro.

 

Read across from linear alpha olefins was conducted for in vitro gene mutation assays in mammalian cells. For this study, Chinese hamster ovary cells (CHO-K1) were treated to study its potential to induce point mutations in the HGPRT gene in the CHO-K1 cell line in the absence and presence of metabolic activation (±S9) (Papciak et al., 1983). Mutagenicity was evaluated at 4, 16, 128, 512, 1024, and 2048 ug/mL Gulftene 12-16 (±S9); however, results were only provided for concentrations =128 ug/mL.  In the absence of S9, there were an insufficient number of cells to sub-culture 1 million cells per dish at the 1024 and 2048 ug/mL test concentrations and cell counts for these concentrations were also reduced with metabolic activation (+S9). In addition, the cloning efficiency was depressed at 1024 and 2048 ug/mL, indicating that the immediate toxic effect also delayed growth of surviving cells. There was no increase in the frequency of mutant colonies at any test concentration with or without metabolic activation (±S9). The vehicle control was well within the <90% toxicity level, while the two positive control groups (ethyl methane sulfonate & benzo(a) pyrene) exhibited a positive response indicating that the assay was functional. Based on these results, the study authors concluded that there was no increase in the frequency of mutant colonies in treated cells.

Read across within category from alkenes, C20-24 for in vivo gene mutation assays. In this study, mice dosed intraperitoneally with 500, 1000, or 2000 mg/kg bw of alkenes, C20-24 showed no evidence of increased incidence of micronucleated polychromatic erythrocytes (Durward, 1998).

 

Based on the lack of observed mutagenic effects in in vitro and in vivo studies with multiple carbon number isomerised olefins and linear alpha olefins, it is concluded that alkenes, C24 -28 are not mutagenic. Based on these findings, alkenes, C24 -28 do not meet the EU criteria for classification and labelling (Dangerous Substances Directive 67/548/EEC and CLP EU Regulation 1272/2008) for mutagenicity.

Justification for Read Across:

Several criteria justify the use of the read across approach to fill data gaps for multiple carbon number isomerised olefin substances using linear alpha olefin substances. Studies indicate that changing the carbon number, the location of the double bond, or adding branching does not measurably alter effects on mammalian health endpoints. There is a consistent toxicity potency pattern for alpha olefins and alpha olefins with range of carbon numbers supported by a low toxicity concern for acute oral, dermal and inhalation exposure. These materials are slightly irritating to skin and mildly irritating to non-irritating to eyes of rabbits. Screening studies indicate that they are not genotoxic. Study results for the aforementioned endpoints indicate a low hazard potential for human health. Since the addition of branching does not measurably alter the results of studies on mammalian health endpoints, there should not be any significant toxicological differences between substances in multiple carbon number isomerised olefins and linear alpha olefins.  Therefore, read across between these categories can be justified.


Short description of key information:
A key in vitro gene mutation study in bacteria study (OECD 471) was identified. Two read-across in vitro cytogenicity studies in mammalian cells (OECD 473) were identified from alkenes, C20-24. A read-across study (OECD 476) from linear alpha olefins for in vitro gene mutation in mammalian cells was identified. One read-across study (OECD 474) for in vivo gene mutation was identified from isomerised olefins; alpha, internal, linear and branched – multiple carbon numbers (alkenes, C20-24).

All genetic toxicity tests, both in vitro and in vivo, were negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All in vitro genetic toxicity studies (i. e., gene mutation studies in bacteria; cytogenicity studies in mammalian cells; and gene mutation studies in mammalian cells) from linear alpha olefins and multiple carbon number isomerised olefins showed negative results. In vivo mouse micronucleus studies with multiple carbon number isomerised olefins also produced no evidence of mutagenic effects. Based on the weight of evidence approach, alkenes, C24 -28 are unlikely to be mutagenic and does not meet the criteria for classification and labelling as described in EU Dangerous Substances Directive 67/548/EEC or CLP EU Regulation 1272/2008.