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Two key in vitro gene mutation studies in bacteria were identified. In the first study conducted by May (1996), four strains ofSalmonella typhimurium(TA 98, 100, 1535, and 1537) were exposed to SHOP internal olefins C134 dissolved in ethanol at concentrations of 50, 158, 500, 1580, and 5000 µg/plate in the presence and absence of mammalian S-9 metabolic activation using a plate incorporation assay and incubated for 2 days. None of the test chemical treated bacterial strains showed increases in reversions. Based on these results, the study authors concluded that SHOP internal olefins C134 was not cytogenic to the four Salmonella typhimurium strains treated with concentrations up to 5000 µg/plate either in the presence or absence of S9. 

In the second key study, five strains of Salmonella typhimurium and 2 strains of Escherichia coli were exposed to olefin 11/12 in acetone, olefin 13/14 in acetone, olefin HE bleed as an emulsion in acetone at concentrations of 31.25, 62.5, 125, 250, 500, 1000, 2000, or 4000 µg/plate and olefin intermediate recycle in acetone at concentrations of 31.25, 62.5, 125, 250, 500, 1000, or 2000 µg/plate in the presence or absence of rat liverS9 fraction using a plate incorporation assay and incubated for 48 hours (Brooks, 1982). Although there were occasional increases in the reverse mutation rates in bacteria, the results were slight, were not reproducible, and were not dose dependent. Therefore, the authors concluded that the olefin products did not affect the reverse mutation rates in bacteria in the presence or absence of S9 activation. 

In a key cytogenicity assays in mammalian cells, alkenes, C11/C13/C14 were examined in cultures of rat liver cells that were exposed to the different olefin products for 24 hours (Brooks, 1982). Since specifics on the study design were not provided, it cannot be determined whether the test conditions complied with OECD 473 guidelines. Preliminary cytotoxicity tests indicated that neither olefin 11/12 nor olefin 13/14 were cytotoxic at any of the concentrations tested (concentrations ranged from 0.1 to 500 µg/mL). Therefore, concentrations of 125, 250, and 500 µg/mL were used to test for mutagenicity. For the olefin HE bleed, concentrations of 12.5, 25, and 50 µg/mL were used in the mutagenicity assay. Although some abnormalities were observed, there were no significant or dose dependent increases observed for any of these olefin compounds. The first sample of olefin intermediate recycle had highly variable results for cytotoxicity. Concentrations of 25, 50, or 100 µg/mL were used in the mutagenicity assay with increases in chromosomal aberrations, as well as cytotoxicity, noted in the two highest concentrations. Retesting the sample a year later led to an increase in cytotoxicity indicating an alteration in the chemical composition. A second fresh sample was tested at the 25, 50 and 100 µg/mL concentrations with no differences between the control group and the treated groups. Based on the results presented above, the study authors concluded that all test compounds were negative for chromosomal damage in rat liver cells.      

 

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 isomerised olefins; alpha, internal, linear and branched – multiple carbon numbers and linear alpha olefins, it is concluded that alkenes, C11/C13/C14 are not mutagenic. Based on these findings, alkenes, C11/C13/C14 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 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:
Two key in vitro gene mutation in bacteria studies (OECD 471) was identified. A key study for in vitro cytogenicity in mammalian cells (OECD 473) also was identified. 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, C11/C13/C14 are unlikely to be mutagenic and do not meet the criteria for classification and labelling as described in EU Dangerous Substances Directive 67/548/EEC or CLP EU Regulation 1272/2008.