Extracts (petroleum), heavy naphthenic distillate solvent

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A key mouse lymphoma assay (OECD 476) was identified, in which the DAE sample tested positive. In contrast, two in vivo micronucleus tests (OECD 475) were identified that showed negative results in rats that were exposed either dermally or orally.

Two positive read-across studies for in vitro gene mutation in bacteria (OECD 471) were identified.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The following read-across in vitro genetic toxicity studies were identified for 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 sulphoxide 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 (Blackburn et al., 1984). The procedure was further modified by preliminary solubilisation 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 in vitro. 

One key mouse lymphoma assay was identified. In this study, L5178Y cells cultured in vitro were exposed to light paraffinic distillate solvent extract in ethanol at concentrations of 25, 50, 75, 100, 150 and 200µL/mL without activation and at 12.5, 25, 50, 75, 100 and 150µL/mL with activation (API, 1986c).

In the absence of metabolic activation, a dose-response relationship was observed from 75 µL/mL to 200 µL/mL DAE with decreasing relative growth percentages (i.e., 119.7% to 2.2%), decreasing viable colonies (i.e., 661 to 89), and increasing mutant frequency (in 10E-6 units). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 67.9x10-6. Concentrations of 150 µL/mL and 200 µL/mL induced mutant frequencies that exceeded the minimum criterion. Hence the tested material was considered mutagenic without activation. In the presence of metabolic activation, a dose-response relationship was observed from 75 µL/mL to 150 µL/mL DAE with decreasing relative growth percentages and increasing mutant frequency (in 10E-6 units). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 73.5x10-6. Treatments from 25 mL/mL to 150 mL/mL induced increases in the mutant frequency, which ranged from 1.8-fold to 3.2-fold above the minimum criterion.

There was a general trend toward higher mutant frequencies at higher concentrations of the test material. The tested material was therefore considered mutagenic with activation. Light paraffinic distillate solvent extract is therefore mutagenic with and without activation under the conditions specified in this assay.

Two in vivo micronucleus tests also were identified. In one study, rats were dermally exposed to 30, 125, or 500 mg/kg/day of 318 Isthmus Furfural Extract for 90 days (Mobil, 1987). The study also found no significant difference in the number of micronucleated PCEs of the 318 Isthmus Furfural Extract-treated animals in comparison to each other or to the negative controls. 318 Isthmus Furfural Extract was not cytotoxic to red blood cell formation nor did it induce significant increase in the formation of micronucleated PCEs or NCEs in bone marrow of treated rats. 318 Isthmus Furfural Extract does not cause chromosome damage to rats dermally exposed.

In the second study, rats were orally exposed by gavage to 125, or 500 mg/kg/day of 318 Isthmus Furfural Extract for 90 days (Mobil, 1987). Additionally, the study found no significant difference in the number of micronucleated PCEs of the 318 Isthmus Furfural Extract-treated animals in comparison to each other or to the negative controls. 318 Isthmus Furfural Extract was not cytotoxic to red blood cell formation nor did it induce significant increase in the formation of micronucleated PCEs or NCEs in bone marrow of treated rats. 318 Isthmus Furfural Extract does not cause chromosome damage to rats orally exposed.

Justification for Read Across

DAE is produced as a by product in the refining of lubricating oil base stocks and waxes. Straight run vacuum distillates are extracted with solvents such as furfural, phenol, or N-methyl-2-pyrrolidone to selectively remove the undesirable polycyclic aromatic compounds, (especially 3-7 fused ring structures). DAE can be considered “worst case” by comparison to unrefined/acid treated oils, in that the extract contains higher concentrations of biologically active components (poly aromatic hydrocarbons) than the unrefined/acid treated oils. Since unrefined/acid treated oils produce positive results in the in vitro modified assay there is no reason to expect that DAE would not.

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 Carcinogenic, Cat. 1B (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 CLP. 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 the genotoxic 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 CLP. For example, based on two in vivo micronucleus assays, one via oral exposure and the other via dermal exposure, both of which were negative, distillate aromatic extracts are unlikely to be mutagenic under in vivo conditions and do not meet the criteria for classification and labelling as mutagenic under the EU CLP Regulation (EC No. 1272/2008).