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Additional information

For unrefined/acid treated oils, the following key in vitro genetic toxicity studies were identified: two bacterial mutation assays; one cytotoxicity assay in mammalian cells; and one gene mutation assay in mammalian cells. With regard to the bacterial mutation assays, a modification to the Ames reverse mutation assay was developed as an adaptation to improve sensitivity to complex hydrocarbon mixtures produced by the refining of petroleum. Modifications included extraction of the oil samples with dimethyl sulfoxide to produce aqueous-compatible solutions that more easily interact with the bacteria and use of only one bacterial tester strain (TA98) most susceptible to poly aromatic hydrocarbon induced mutagenesis (Blackburnet al., 1984). The procedure was further modified by preliminary solubilization of the oil in cyclohexane, substitution of Aroclor 1254-induced hamster liver S-9 for rat S-9, and an increase in the concentrations of both the NADP co-factor and S-9 homogenate levels (Blackburn et al., 1986).   In the Blackburn et al. study (1986), S. typhimurium strain TA 98 was exposed to six DMSO-extracted oil samples (heavy paraffinic distillate, light paraffinic distillate, three separate samples of heavy naphthenic distillate, and heavy naphthenic distillate blend) in the presence of Aroclor 1254-induced hamster liver S9 fraction. All of the test substances showed mutagenicity indices ranging from 4.1 to 10. Base oils with an MI between 1 and 2 may or may not be mutagenic whereas oils with an MI > 2.0 are considered to be potentially mutagenic. As all of the unrefined oils had MI values > 2.0, they are all considered to be potentially mutagenic. This method of testing base oils and related materials for mutagenic potential by the modified Ames test was standardized by the ASTM for use in formulating metal working fluids (ASTM, 2004).

In a mammalian cell cytogenetics chromosome aberration assay, Chinese Hamster Ovary cells were exposed to naphthenic distillate at concentrations of 0.2, 0.1, 0.05, or 0.02 µl/ml under metabolically activated conditions for a 2 -hour exposure period and at test concentrations of 0.3, 0.15, 0.08, or 0.03µL/mL under non-activated conditions for 10 hours (ARCO, 1985a). The test article was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background under activation or non-activation assay conditions. Similar supporting assays were identified and provide further support that unrefined/acid treated oils do not induce chromosome aberrations.

Gene mutation was assessed in mammalian cells on an unrefined light paraffinic distillate CAS No. 64741-50-0, (API, 1986c). This forward mutation assay was carried out in accordance with OECD Guideline 476 using the L5178Y cell line. The mutations in the cells heterozygous for thymidine kinase to the homozygous genotype were scored after selection of mutants with a medium containing bromodeoxyuridine or trifluorothymidine. Each sample was also tested with rat or mouse liver microsomal metabolic activation. Ethyl methane sulphonate was tested as a positive control without metabolic activation, and 2-acetylaminofluorene was tested as a positive control with metabolic activation.   The mouse lymphoma cells were exposed to the test material, dissolved in acetone, for 4 hours with and without metabolic activation with S-9 rat liver homogenate. The test material was visibly insoluble at concentrations exceeding 100 nl/ml. Without metabolic activation, the test material was assayed from 400 to 1000 nl/ml, and little or no toxicity was observed well into the insoluble range. No significant increases in the mutation frequency were observed.   With metabolic activation, however, treatments from 50 to 1000 nl/ml induced significant increases in the mutant frequency at the thymidine kinase (TK+/-) locus. Increases ranged from 2.1-fold to 7.3-fold above background.   It should be noted, however, that survival was 73% of control at 50 nl/ml, 33% of control at 200 nl/ml, and less than 20% of control at all higher levels. The mutation frequency did not increase with concentration at levels above 300 nl/ml. Increased mutant frequencies at low cell survivals are now regarded with scepticism.

One key in vivo genetic toxicity study was identified. In the micronucleus study, unrefined/acid treated oil, either neat or following extraction with dimethyl sulphoxide, was administered to the mice by oral gavage in two consecutive daily doses. There were four mice per group, two males and two females. Cells were harvested 24 hours after the final dose was administered. There were no significant increases in micronuclei using either the neat or extracted material. 

Additional data supports that unrefined/acid treated oils are not mutagenic (Clive et al., 1979; Carver et al., 1984; McKee and Przygoda, 1987; Microbiological Associates, Inc., 1985 1985a,b,c,d,e,f,g; Microbiological Associates, Inc., 1986; Microbiological Associates, Inc., 1988a,b,c,d,e; Pienta et al., 1977; Roy et al., 1988). This information is presented in the dossier.

Short description of key information:
Two key in vitro gene mutation assays in bacteria (OECD 471); results for these studies were positive. One cytogenicity assay in mammalian cells (OECD 473) and one in vitro gene mutation assay (OECD 476) in mammalian cells were identified; both studies were negative. One key in vivo gene mutation assay (OECD 474) was identified which was negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Some oil products containing relatively high concentrations of polycyclic aromatic compounds (PAC) are considered genotoxic carcinogens, and, consequently, are classified and labelled as carcinogens (H350) according to the EU CLP Regulation (EC No. 1272/2008). This classification as carcinogenic does not automatically imply that these substances need also to be classified as mutagenic as defined by the EU CLP Regulation. The EU legislation aims primarily to classify substances as mutagenic if there is evidence of producing heritable genetic damage, i. e. evidence of producing mutations that are transmitted to the progeny or evidence of producing somatic mutations in combination with evidence of the substance or relevant metabolite reaching the germ line cells in the reproductive organs. The PAC in oil products are poorly bioavailable due to their physico-chemical properties (low water solubility and high molecular weight), making it unlikely that thegenotoxic constituents can reach and cause damage to germ cells (Roy, 2007; Potter, 1999). Considering their poor bioavailability, oil products which have been classified as carcinogenic do not need to be classified as mutagenic unless there is clear evidence that germ cells are affected by exposure, consistent with the EU CLP Regulation. Based on in vivo micronucleus tests on five sufficiently refined, solvent extracted/dewaxed paraffinic base oils, one catalytically cracked clarified oil, and one unrefined lubricating oil that were all negative for genotoxicity, unrefined acid treated oils are not classified as mutagens according to criteria for classification under the EU CLP Regulation.