Distillates (petroleum), hydrotreated light paraffinic

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sufficiently refined other lubricant base oils (IP 346 < 3%) were found to be non-mutagenic while insufficiently refined other lubricant base oils (IP 346 ≥ 3%) were observed to be mutagenic in vitro tests.  In vivo micronucleus tests on five sufficiently refined, solvent extracted / dewaxed paraffinic base oils and one unrefined lubricating oil were all negative for genotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Multiple key studies were identified for in vitro genetic toxicity, and two key studies were identified for in vivo genetic toxicity.  

 

In Vitro Genetic Toxicity

 

Sufficiently Refined Other Lubricant Base Oils (IP 346 < 3%)

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 a key mammalian cell chromosome aberration study (Microbiological Associates Inc, 1987b), Chinese hamster ovary cells were exposed to 55/60 pale oil (CAS 64742-53-6, IP346 < 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.

Supporting data from mammalian chromosome aberration assays (Microbiological Associates Inc., 1987c; 1987d; 1987e; 1987f; 1987g; 1987h; 1989i) demonstrated that sufficiently refined other lubricant base oils (IP 346 < 3%) are not mutagenic when tested in vitro on CHO cells.

In a key mammalian cell gene mutation assay (API, 1986d), mouse lymphoma L5178Y cells cultured in vitro were exposed to AP 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^-6and 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 mouse lymphoma assays (Microbiological Associates Inc., 1987i; 1987j; 1987k; 1987l; 1987m; 1987o; 1987p; 1989j; 1989k; 1989l) demonstrated that sufficiently refined other lubricant base oils (IP 346 < 3%) are not mutagenic when tested in vitro.

 

Insufficiently Refined Other Lubricant Base Oils (IP 346 ≥ 3%)

Two key studies by Blackburn et al. (1984, 1986) used a Modified Ames (1975) procedure to include preliminary solubilization 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. One sample with an IP 346 ≥ 3% (CAS 64741-50-0) was tested found to be mutagenic with a mutagenicity index of 17 (1984). Two hydrotreated heavy naphthenic distillates with IP 346 ≥ 3% (CAS 64742-52-5) had mutagenicity indices of 4.9 and 5.9, respectively and were considered mutagenic in vitro.

In a key read across mammalian cell chromosome aberration study (Microbiological Associates Inc., 1987a), CHO cell cultures were exposed to L-06 (a light hydrotreated feedstock, IP 346 ≥ 3%) in DMSO at concentrations of 0.2, 0.1, 0.05, and 0.02 µL/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 mutagenicity in the chromosomal aberration assay using Chinese hamster ovary cells.

In a key mammalian cell gene mutation assay conducted in accordance with OECD Guideline 476 with cell line L5178Y (API, 1986e), Hydrotreated Light Naphthenic (CAS 64742-53-6, IP346 ≥ 3%) was assayed without metabolic activation from 250 to 1200 nL/mL, and a wide range of toxicities were induced. In the presence of metabolic activation, it was assayed from 400 to 1600 nL/mL. Treatments above 1000 nL/mL produced repeatable increases in mutant frequency ranging from 2.5-fold to 8.8-fold above the background level, both in the presence and absence of S9 metabolic activation. Thus, this sample was considered as positive in the mouse lymphoma assay both with and without metabolic activation. However, due to the very low relative growth in all of the treated groups, it is probably more reasonable to consider this result as equivocal.  

 

In Vivo Genetic Toxicity

 

Sufficiently Refined Other Lubricant Base Oils (IP 346 < 3%)

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, Klimisch score=1). 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.

 

Insufficiently Refined Other Lubricant Base Oils (IP 346 ≥ 3%)

Four separate bone marrow micronucleus assays were conducted using two types of petroleum-derived materials: catalytically cracked clarified oil (CCCO) and an unrefined lubricating oil (ULO). 

In the test (experiment 3) with neat or DMSO-extracted ULO, material was administered to CD-1 mice (2/sex/dose) in two consecutive daily doses via oral gavage at dose levels of 0, 1.25, 2.5, or 5.0 g/kg. Bone marrow cells were harvested at 24 hours following the final dose.

In the first study, CD-1 mice (5/sex/dose) were administered CCCO in corn oil in two consecutive daily doses via oral gavage or intraperitoneal injection at dose levels of 0, 0.188, 0.375, or 0.75 g/kg. An additional high dose of 1.50 g/kg was administered to the oral gavage group only. Bone marrow cells were harvested at 24 and 48 hours after the final dose. In a second micronucleus test, CD-1 mice (2/sex/dose) were administered a DMSO extract of CCCO in two consecutive daily doses via oral gavage at dose levels of 0, 1.25, 2.5, or 5.0 g/kg. Bone marrow cells were harvested at 24 hours after the final dose.  In the fourth test, CCCO in corn oil was administered to CD-1 mice (2/sex/dose) in two consecutive daily doses by IP injection at dose levels of 0, 0.75, 1.5, or 3.0 g/kg. Bone marrow cells were harvested at 24 hours after the final dose.

There were no signs of clastogenicity in any of the four studies, even though a lethal response was observed in mice administered DMSO-extracted CCCO where one of four mice in the 2.5 g/kg group and three of four mice in the 5 g/kg group died. The positive and negative controls of all studies induced the appropriate response. 

Overall, the test results above demonstrate that other lubricant base oils are non-mutagenic under the conditions of the in vivo genetic toxicity assays, even if they are known to contain polyaromatic compounds that make them mutagenic in the in vitro assays or in animal carcinogenesis bioassays.

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: May cause cancer) according to 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 (EC No. 1272/2008).

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. 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 (EC No. 1272/2008). 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, other lubricant base oils are not classified as mutagens according to EU classification.