Ethylene polymerisation (triethylaluminium catalysed), distillation residue, C20-40 fraction

Endpoint summary

Additional information

In a reverse gene mutation assay in bacteria, strains (TA1535, TA1537, TA98, and TA100) of S. typhimurium and a strain (WP2 uvrA’) of E. coli were exposed to alkenes C20-24, branched and linear, in acetone at concentrations of 0, 15, 50, 150, 500, 1500, and 5000 μg/plate in the presence and absence of mammalian metabolic activation using the plate-incorporation method (Thompson, 1998). No significant amounts of revertants were noted. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. In a second reverse gene mutation assay in bacteria (Thompson, 2008), strains(TA1535, TA1537, TA98, TA100, and TA 102) of S. typhimurium were exposed to alkenes C20-24, branched and linear, in acetone at concentrations of 0, 15, 50, 150, 500, 1500, and 5000 μg/plate in the presence and absence of mammalian metabolic activation using the plate-incorporation method. No significant amounts of revertants were noted. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In a mammalian cell cytogenetics assay (chromosome aberration) performed with alkenes, C20 -24, 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 hourharvest 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 lymphocytes in 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.

In an in vivo gene mutation assays conducted with alkenes, C20 -24, 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 with a range of carbon numbers, it is concluded that alkenes, C20 -24 are not mutagenic. Based on these findings, alkenes, C20 -24 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 in vitro gene mutation studies in bacteria (OECD 471) and two key in vitro cytogenicity studies in mammalian cells (OECD 473) were identified for alkenes, C20-24. A read-across study (OECD 476) from linear alpha olefins for in vitro gene mutation in mammalian cells was identified. A key study (OECD 474) for in vivo gene mutation also was identified.

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, C20 -24 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.