Extracts (petroleum), solvent-refined heavy paraffinic distillate solvent

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For genetic toxicity, TDAEs (IP 346 ≥ 3%) were evaluated using read-across information from unrefined/acid treated oils and untreated distillate aromatic extracts.  Information included a modified Ames test (similar to OECD 471) and a mouse lymphoma test (OECD 476).  Results were positive for the modified Ames test, and the in vitro mouse lymphoma test showed a general trend toward higher mutant frequencies at higher concentrations of the test material. In vivo mutagenicity tests (dermal and oral; OECD 475) using read-across data for UDAEs showed negative results for mutagenicity.

TDAEs (IP 346 < 3%) were evaluated using read-across information from other lubricant base oils (IP 346 < 3%).  OLBOs (IP 346 < 3%) were found to be non-mutagenic in a modified Ames test (similar to OECD 471) or a chromosome aberration assay (similar to OECD 473).  In an in vivo micronucleus assay (OECD 474), the test materials were found not to be clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Treated distillate aromatic extracts (TDAEs) are a further processing of untreated distillate aromatic extracts (UDAEs) in an attempt to reduce the amount of 3-7 ring PAC that is present. Since the treatment is mostly a selective reduction of PACs, the data from UDAEs can serve as read across where treatment was insufficient and a significant amount of PACs still remain (≥ 3 wt% DMSO extractables as measured by IP-346). Where treatment was sufficient to reduce the 3-7 ring PACs (<3 wt% DMSO extractables as measured by IP-346), the material is most similar to a lubricating base oil and it is this data that should be used for read across. 

 

In Vitro Genetic Toxicity: Gene Mutation Study in Bacteria

TDAEs (IP 346 ≥ 3 wt%)

The following read-across in vitro genetic toxicity studies were identified for bacterial mutation assays. A modification to the 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 (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. 

TDAEs (IP 346 < 3 wt%)

Two key studies by Blackburn et al. (1984, 1986) used a Modified Ames (1975) procedure to include preliminary solubilisation of the oil in cyclohexane followed by single extraction of dimethylsulphoxide, use of S9 fraction from hamster liver, use of a single strain of S. typhimurium (TA98), and an increase in the NADP concentration. Various samples with IP 346 < 3% were tested. Solvent-refined samples with IP 346 < 3% (CAS 64742-52-5, 64742-65-0, 64742-54-7) were found to have mutagenicity indices of 0.0 and therefore considered non-mutagenic in vitro.

In Vitro Genetic Toxicity: Cytogenicity on Mammalian Cells

TDAEs (IP 346 ≥ 3 wt%)

In a mammalian cell chromosome aberration study (Microbiological Associates, 1987a), CHO cell cultures were exposed to L-06 in DMSO at concentrations of 0.2, 0.1, 0.05 and 0.02µL/mL without S-9 activation and 0.03, 0.15, 0.08 and 0.03µL/mL with S-9 activation for 10 hours for non-activation and two hours for activation. L-06 was tested up to precipitating concentrations. Positive controls induced the appropriate response. The test material did not demonstrate mutagenciity in the chromosomal aberration assay using Chinese hamster ovary cells.

TDAEs (IP 346 < 3 wt%)

In a key mammalian cell chromosome aberration study (Microbiological Associates, 1987b), Chinese hamster ovary cells were exposed to 55/60 pale oil (CAS 64742-53-6, IP 346 < 3%) at concentrations of 0.02, 0.04, 0.08, or 0.15 μL/mL without S9 for 10 hours or 0.05, 0.1, 0.2, or 0.4 μL/mL with S9 metabolic activation for 2 hours. Results from the chromosome aberration assay showed no significant structural or numerical aberrations in CHO cells at any dose level, with or without metabolic activation.

In Vitro Genetic Toxicity: Gene Mutation in Mammalian Cells

 

TDAEs (IP 346 ≥ 3 wt%)

In the key read-across mouse lymphoma assay, 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 untreated distillate aromatic extract with decreasing relative growth percentages (i.e., 119.7% to 2.2%) 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 untreated distillate aromatic extract 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.

TDAEs (IP 346 < 3 wt%)

In a key mammalian cell gene mutation assay (API, 1986d), mouse lymphoma L5178Y cells cultured in vitro were exposed to API 83 -15 (Sufficiently refined lubricant base oil, IP 346 < 3%) in DMSO at concentrations of 400, 500, 600, 800, and 1,000 nL/mL in the absence of mammalian metabolic activation and concentrations of 200, 400, 500, 600, 800, and 1,000 nL/mL in the presence of metabolic activation, for 4 hours. Low to moderate toxicities were observed, thus, this sample was considered to be inactive in the mouse lymphoma assay. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 73.3 x l0-6 and none of the assayed treatments induced this level of mutant action.The negative control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background.

Supporting data from mammalian cell gene mutation assays (Microbiological Associates, 1987c; 1987d; 1987e; 1987f; 1989a; 1989b; 1989c; 1989d; 1989e; 1989f) demonstrated that sufficiently refined other lubricant base oils (IP 346 < 3%) are not mutagenic when tested in vitro on mouse lymphoma cells.

In Vivo Genetic Toxicity

TDAEs (IP 346 ≥ 3 wt%)

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, 1987a). 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, 1987a). 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.

TDAEs (IP 346 < 3 wt%)

A key micronucleus study carried out in accordance with OECD Guideline 474 was conducted in male and female CD-1 mice with five highly refined, solvent extracted / dewaxed paraffinic base oils (McKee, et al. 1990). In this study, the lubricant base oils were administered intraperitoneally at 1.0, 2.5, and 5.0 g/kg. Neither toxicity nor mutagenicity was observed. These data indicate that sufficiently refined (IP 346 < 3%) base oils do not induce chromosomal mutations.

 

Justification for selection of genetic toxicity endpoint

One of 21 in vitro and in vivo genetic toxicity studies available.

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. Based on in vivo micronucleus tests on two untreated distillate aromatic extracts and one solvent-extracted, dewaxed paraffin oils that were all negative for genotoxicity, TDAE are not classified as mutagens according to the EU CLP Regulation (EC No. 1272/2008).