Fusel oil

Endpoint summary

Additional information

Fusel oil is a UVCB substance comprising a complex mixture of alcohols, aldehydes, esters and other substances. The constituents and their concentration ranges are known. Fusel oil contains 4 main constituents being above ≥ 10%. In total, the 4 main constituents account for ≥ 80% of all constituents. In order to fulfil the standard information requirements set out in Annex IX in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from surrogate substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from surrogate substances (grouping or read-across).

The physicochemical, toxicological and ecotoxicological properties of the main constituents of Fusel oil determine, to a great extent, the physicochemical, toxicological and ecotoxicological properties of Fusel oil itself. Therefore, having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, 1.5, of Regulation (EC) No 1907/2006, a read-across is appropriate as their physicochemical, toxicological and ecotoxicological properties are likely to be similar. A detailed justification for use of read-across is given in chapter 13 of the technical dossier.

In conclusion, hazard assessment was based on the main constituents, when no experimental data was available with Fusel oil itself. The main constituents are ethanol (CAS No. 64-17-5), 3-methylbutan-1-ol (CAS 123-51-3), 2-methylbutan-1-ol (CAS No. 137-32-6), and 2-methylpropan-1-ol (CAS No. 78-83-1).

Genetic toxicity in vitro

Gene mutations in bacteria

No detailed study reports are available for assessment of bacterial mutagenicity of 3-methylbutan-1-ol (CA 123-51-3). Data from ECB IUCLID indicate that 3-methylbutan-1-ol was not mutagenic, when tested in an Ames assay with and without metabolic activation in the Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA1537 (EC, 2000).

For 2-methylpropan-1-ol (CAS No. 78-83-1) an Ames performed equivalent to OECD 471 test was available (Shimizu et al., 1985). Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) and Escherichia coli (WP2uvrA) strains were incubated with 5, 10, 50, 100, 500, 1000, and 5000 µg/plate 2-methylpropan-1-ol using the preincubation method in the presence and absence of S9 mix. Negative and positive controls were valid. No genotoxicity and no cytotoxicity was observed in Salmonella strains or in E. coli. Furthermore, Zeiger et al. (1988) confirmed that 2-methylpropan-1-ol was not mutagenic in Salmonella typhimurium strains Ta 97, TA98, TA100, TA1535, and TA1537 when tested up to 10000 µg/plate in the presence and absence of metabolic activation.

For ethanol (CAS No. 64-17-5) an Ames test equivalent to OECD 471 was available for assessment (Zeiger et al., 1992). Salmonella typhimurium (TA 97, TA98, TA100, TA1535, and TA 104) strains were incubated with 1, 100, 333, 1000, 3.333, and 10000 µg/plate ethanol (diluted in water) using a preincubation protocol in the presence and absence of S9 mix (10 and 30%, rat and hamster). TA 97 was additionally tested with 3, 10, and 33 µg/plate in the presence of 30% S9-mix. Five replicates in 2 independent experiments were performed. Cytotoxicity was assessed by evaluation of a decrease in the background number of his+ colonies and/or a clearing in the background lawn. Negative and positive controls were valid. No genotoxicity was observed in any experiment and any strain. No information regarding cytotoxicity was reported. Further Ames tests have been performed with ethanol confirming that ethanol is not mutagenic in several Salmonella typhimurium strains (Blevins and Taylor, 1982; Blevins and Sheltons, 1983; Phillips and Jenkinson, 2001; McCann, 1975; de Flora, 1984).

Mutagenicity in mammalian cells

Results from a HPRT gene mutation assay are available with the test substance 3-methylbutan-1-ol (CAS No. 123-51-3; Kreja and Seidel, 2002). Chinese hamster lung fibroblasts were treated similar to OECD guideline 476 with 51.5 mM 3-methylbutan-1-ol, which was the highest non-toxic concentration. No data were provided for additional concentrations. The mutant frequency of the 3-methylbutan-1-ol treated cells was within the range of the negative control in the absence and presence of a metabolic activation system (S9-mix prepared with liver from Aroclor 1254-treated rats). In addition, 2-methylbutan-1-ol (CAS No. 137-32-6) was tested at a concentration of 46 mM, which was the highest non-toxic concentration. No data were provided for additional concentrations. The mutant frequency of the 2-methylbutan-1-ol treated cells was within the range of the negative control in the absence and presence of a metabolic activation system (S9-mix prepared with liver from Aroclor 1254-treated rats). Also 2-methylpropan-1-ol (CAS No. 78-83-1) was tested as a further chemical up to 107 mM. No data were provided for additional concentrations. No genotoxicity and cytotoxicity was observed after treatment of cells with 2-methylpropan-1-ol in the absence and presence of a metabolic activation system (S9-mix).

In conclusion2-methylbutan-1-ol, 3-methylbutan-1-ol, and 2-methylpropan-1-olare not mutagenic in mammalian cells in the absence or presence of a metabolic activation system.

Furthermore, 2-methylpropan-1-ol was not mutagenic in a mouse lymphoma assay (US EPA, 1987).

Four mouse lymphoma assays are available for assessment of the mutagenic properties of ethanol in mammalian cells. In the key study performed equivalent to OECD 476 (Wangenheim and Bolcsfoldi, 1988) L5178Y cells were treated with ethanol (CAS No. 64-17-5) at concentrations of 0.092, 0.184, 0.369, 0.553, and 0.738 mol/L (without activation) and 0.414, 0.465, and 0.517 mol/L (with activation). In the absence of S9-mix treatment with the lowest and highest dose induced a significant increase of the mutation frequency. As no dose-response relationship was observed and the increase was lower than 2-fold, the evaluation criteria for a positive response were not met. The total growth was reduced to 17% of the control after treatment with 0.738 mol/L (highest dose tested). Incubation of cells in the presence of S9-mix yielded a statistically significant increase of the mutation frequency. Again no dose-response relationship was observed and the increase was lower than 2-fold, thus the evaluation criteria for a positive response were not met. In addition, treatment with 0.517 mol/L ethanol was cytotoxic, reducing the total growth to 6% of the control value. In conclusion, ethanol was found to be non mutagenic in mammalian cells, with and without metabolic activation.

Cytogenicity in mammalian cells

Results from an in-vitro micronucleus assay in Chinese hamster lung fibroblasts (V79 cells) are available with the test substance 3-methylbutan-1-ol (CAS No. 123-51-3), 2-methylbutan-1-ol (CA 137-32-6) and 2-methylpropan-1-ol (CAS No. 78-83-1; Kreja and Seidel, 2002). Chinese hamster lung fibroblasts were treated 5, 9, and 23 mM 3-methylbutan-1-ol in the presence and absence of S9-mix. No increase in cells with micronucleus was observed after treatment with 3-methylbutan-1-ol. Furthermore, chinese hamster lung fibroblasts were treated with 23 and 45 mM 2-methylbutan-1-ol in the presence and absence of S9-mix. No increase in cells with micronucleus was observed after treatment with 2-methylbutan-1-ol. In addition chinese hamster lung fibroblasts were treated with 11 and 53 mM 2-methylpropan-1-ol in the presence and absence of S9-mix. No increase in cells with micronucleus and no cytotoxicity was observed after treatment with 2-methylpropan-1-ol. In conclusion, 2-methylpropan-1-ol does not induce micronuclei in mammalian cells in the absence or presence of a metabolic activation system.

In conclusion, 2-methylbutan-1-ol, 3-methylbutan-1-ol, and 2-methylpropan-1-ol did not induce micronuclei in mammalian cells in the absence or presence of a metabolic activation system.

In addition, a comet assay performed in human peripheral blood cells after treatment with 3-methylbutan-1-ol and 2-methylpropan-1-ol revealed no indication of DNA damage by the test substance at non-toxic concentrations (Seidel and Plappert, 1999).

In a review of the genotoxicity of ethanol (CAS No. 64-17-5), the results from using ethanol as a vehicle in a number of guideline clastogenicity (chromosome abberation) assays were reported for human lymphocytes and Chinese hamster lung cells (summarized in Phillips and Jenkinson, 2001). As a control vehicle, ethanol was used in the guideline tests at doses of 1%, well in excess of the maximum dose normally recommended for use.In 1998 studiesit was used as solvent for 19 test materials without metabolic activation and for 11 test materials with metabolic activation. Ethanol was comparable to the 1997 vehicle control history profile (145 assays) for all vehicle controls used at Safepharm Laboratories in this year. In conclusion, ethanol is not clastogenic in-vitro in the presence and absence of a metabolic activation system.

Genetic toxicity in vivo

Several micronucleus tests, chromosome aberration tests and dominant lethal assay are available for ethanol. A conclusion is given based on the OECD SIDS document (OECD, 2004). Many studies can be criticised on the grounds of inadequate numbers of animals or on the methods used to score or evaluate the incidence of early or late foetal deaths or distinguish between early and late deaths. Also, the very high ethanol doses used make interpretation of the effects difficult. The most satisfactory test is the inter-laboratory study performed according to OECD guidelines (James, 1982). In this study a negative result was obtained in mice from which it is concluded that ethanol is negative in the dominant lethal assay in male mice.

Conclusion

No genetic toxicity hazard was observed with the main constituents of Fusel oil, being ethanol (CAS No. 64-17-5), 3-methylbutan-1-ol (CAS No. 123-51-3), 2-methylbutan-1-ol (CAS No. 137-32-6), and 2-methylpropan-1-ol (CAS No. 78-83-1). In addition, the 4 main constituents do not have a structural alert for genetic toxicity. In conclusion, based on available data and the chemical structures it is expected that Fusel oil is also negative for genetic toxicity.

References

EC (2000) ESIS: European Chemical Substances Information System. European Commission Joint Research Centre, Institute for Health and Consumer Protection.

OECD (2004) SIDS Initial assessment report for SIAM 19. Ethanol. CAS No: 64-17-5. UNEP Publications

Mortelmans, K. and Zeiger, E. (2000) The Ames Salmonella/microsome mutagenicity assay. Mutation Research, 455, 29-60.

 

 

Justification for selection of genetic toxicity endpoint

Most reliable data of the main constituents of Fusel oil were used for hazard assessment.

Short description of key information:

No genetic toxicity hazard was observed with the main constituents of Fusel oil, being ethanol (CAS No. 64-17-5), 3-methylbutan-1-ol (CAS No. 123-51-3), 2-methylbutan-1-ol (CAS No. 137-32-6), and 2-methylpropan-1-ol (CAS No. 78-83-1). In conclusion, the negative genetic toxicity studies of the known constituents of Fusel oil suggest that Fusel oil is also negative for genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on available data on the main constituents of Fusel oil on genetic toxicity, Fusel oil does not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.