Alkenes, C6-

Endpoint summary

Additional information

For alkenes, C6, the mutagenicity potential of MRD-91 -937 (alkenes, C6) was tested in five Salmonella tester strains. The high dose selected was 320 µg/plate in the presence and absence of S9. 2-Aminoanthracene (2AA), 2-nitroflourene (2NF), 9-aminoacridine (9AA), and n-methyl-n-nitro-n-nitrosoguanidine (MNNG) served as positive controls. The mutagenicity assay indicated that the test chemical did not exhibit a positive dose related increase in the number of revertants in any of the tester strains used. Toxicity in the form of reduction in the number of revertant colonies or reduction in the background lawn was observed in all five strain tested. The replicate analysis performed to confirm the data observed in the first analysis did not show any deviations in the results. Based on these results it was concluded that the test material is not mutagenic to the five Salmonella strains used in the assay upto and including the highest dose of 320 ug/plate.

 

Read across within category from alkenes, C10/C11/C12/C13 and alkenes, C11/C13/C14 for cytogenicity assays in mammalian cells. For alkenes, C10/C11/C12/C13, two studies were identified. In the first study,cultures of rat liver cells were exposed to Olefin 103 PQ/11 for 24 hours at concentrations equivalent to 5, 10, 15, 20, 25, 30, 35, or 40 µg/mL for the cytotoxicity assay (Brooks et al., 1983). After 24 hours fresh medium was supplied and cells were plated and cultured for 5 days, then colonies containing at least 50 cells were counted. A concentration of 20 µg/mL caused a reduction of 15% in the cloning efficiency and 25 µg/mL caused a reduction of 59% in cloning efficiency; therefore, concentrations of 5, 10, 20, or 25 µg/mL were used for the chromosomal aberration assay. The rat liver assay using Olefin 103 PQ/11 did not induce chromosomal damage under the experimental conditions of the study.

In the second study, both C10/C11/C12/C13 and alkenes, C11/C13/C14 were examined. In this study,cultures of rat liver cells 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 four 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.

A key and supporting in vivo micronucleus assays were identified for alkenes, C6. In the supporting study, 5 male and 5 female mice were orally exposed to 1.25, 2.5, or 5.0 g/kg MRD 91 -938 (i.e., alkenes, C6) dissolved in corn oil (Przygoda, 1991). The positive control, cyclophosphamide, was administered by intraperitoneal injection as a single dose. Animals were sacrificed at 24, 48, or 72 hours post dosing. Animals receiving the positive control were sacrificed 24 hours post dosing. All animals were examined at least once daily for signs of overt toxicity. At the end of study termination, bone marrow from the treated mice were collected at the three points and 1000 polychromatic erythrocytes (PCE) were examined from each exposed animal for the presence of micronuclei. In addition, the ratio of PCEs to NCEs was determined for each animal by counting 1000 erythrocytes (PCEs and NCEs).

The test material did induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes (p < 0.05) at the 5.0 g/kg dose at the 24 hours time point in males and females. MRD 91-938 did not induce a statistically significant decrease in the mean percent of PCEs which is a measure of bone marrow toxicity, hence the study authors concluded that the test material was not toxic to mouse bone marrow under the conditions of this experiment. However, since the test material did induce a statistically significant increase in the mean number of PCEs at the 5.0 g/kg dose level, the study authors concluded that the test material is considered to be clastogenic in the mouse bone marrow at the 5.0 g/kg dose level evaluated at 24 hours after dose administration. The ratio of PCEs to NCEs was not reported.

Because of these positive effects, an additional inhalation in vivo micronucleus test was conducted. In this study, five male and five female B6C3F1 mice were exposed nose only for six hours/day for ten consecutive days to 1000 ppm of MRD 92-321 (C6 alkene) in air (Przygoda, 1991). The positive control, cyclophosphamide in water, was administered via oral gavage at a dose of 40 mg/kg, while air served as the negative control. Bone marrow samples were collected at approximately 24 hours after the last exposure and evaluated for micronucleus formation.

 

MRD 92-321 did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes which is a measure of bone marrow toxicity. The test material also did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. The positive and negative control gave appropriate results. Based on these results, it can be concluded that alkenes, C6 are not clastogenic to the bone marrow of the B6C3F1 mouse.

 

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, C6 are not mutagenic. Based on these findings, alkenes, C6 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 linear alpha olefins 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 multiple carbon number isomerised olefins and linear alpha olefins.  Therefore, read across between these categories can be justified.

Short description of key information:
One key in vitro gene mutation in bacteria study (OECD 471) was identified. Read-across studies for in vitro cytogenicity in mammalian cells (OECD 473) from within isomerised olefins; alpha, internal, linear and branched – multiple carbon numbers were identified. 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 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, C6 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.