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Genetic toxicity in vitro

Description of key information

In the key in vitro modified bacteria Ames study (similar to OECD 471), there was no evidence of mutagenic activity. This result was supported by other studies with straight run gas oils and related materials, the majority of which were negative. Results in mouse lymphoma and SCE assays proved ambiguous and unreliable and  were considered inconclusive.

In the key in vitro sister chromatid exchange assay (equivalent or similar to OECD 479), Hydrodesulfurised middle distillate did not induce an increase in sister chromatid exchange (SCE) in Chinese hamster Ovary (CHO) cells in the absence of S-9 activation. In the presence of S-9, there was an inverse dose-response trend observed that resulted in the statistically significant increase in the frequency of SCEs at the lower doses. Hydrodesulfurised middle distillate was concluded to be equivocal in the SCE assay.

A key in vivo chromosome aberration assay (OECD 475) was identified, in which straight run middle distillate was not found to be mutagenic in male rat bone marrow cells.  An additional chromosome aberration assay also showed negative results for mutagenicity (OECD 475).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Straight run gas oil studies using the optimised Ames assay and the negative in-vivo assays suggest that straight run gas oils are likely to have little or no genotoxic potential.

 

In vitro gene mutation in bacteria

Mutagenic activity of complex aromatic hydrocarbon mixtures such as mineral oils was inadequately detected by the standard Ames assay. Consequently, modification of the assay was needed. An optimised assay was developed, in which a DMSO extract of the oil instead of the whole oil was tested. DMSO is able to extract the principle carcinogenic components (polycyclic aromatic hydrocarbons) from oils, and allows them to be tested without other ingredients interfering with the mutagenic response. As some components of oils were found to inhibit PAC metabolism, the metabolic activation system was modified by increasing the S9 concentration 8-fold and doubling of the NADP co-factor concentration. Hamster S9 instead of rat S9 is used in this assay and only the most sensitive strain of bacteria for PACs (TA98) is used. 

Two modified Ames Assays with straight run gas oil samples (May 2013) showed no evidence of mutagenic activity. Deininger et al. reported on the mutagenicity of two straight run gas oils in S. typhimurium TA 98 using themodified Ames assay (1991; Klimisch score = 1).Concentrations of 0, 5, 10, 15, 20, 30, 40, 50, or 60 μl/plate were applied in the presence of mammalian metabolic activation (Aroclor 1254-induced hamster liver S9) using the pre-incubation method. One straight-run gas oil (CAS 68814-87-9) gave a mutagenicity index (MI) of 0.8, which is below the normal level seen with a carcinogenic oil (results were considered negative), whereas another straight run gas oil (CAS 64741-44-2) gave a borderline MI result of 1.3 (considered positive and marginally genotoxic). Based upon these scores, both oils would be expected to have no more than borderline genotoxic potential

Supporting data from a study (CONCAWE, 1991 and Nessel et al., 1998) on a straight-run gas oil (CAS 64742 -46 -7) shows a mutagenicity index of 1.0 and was not considered to be genotoxic in the modified AMES assay.

Compositional and physico-chemical data show that VGOs/HGOs/Distillate Fuels and Other Gas Oils are very similar to Straight-Run Gas Oils. It is considered appropriate, therefore, to read across from the Straight-Run Gas Oils data to VGOs/HGOs/Distillate Fuels and Other Gas Oils. In a read-across, modified Ames assay, diesel 2 (OGO) and home heating oil (VGO/HGO/Distillate fuel) in DMSO were tested for their mutagenic potential (McKee et al., 1994; Klimisch score = 2). Diesel 2 produced a greater than 2-fold increase in the number of revertant colonies, but this increase did not show a dose-dependent trend. Home heating oil exhibited a dose-dependent increase in the number of revertant colonies, but the increase was not statistically significant compared to the solvent control. It is not clear whether these results were reported for the test material in the presence or absence of S-9 activation. Based on the results obtained, the study authors concluded that neither of the petroleum middle distillate fractions tested for mutagenicity exhibited any evidence of mutagenic activity in the presence or absence of S-9 activity.

 

In vitro gene mutation in mammalian cells

 

Results of mouse lymphoma and SCE assays with straight-run gas oil samples were equivocal, inconsistent and were considered inconclusive.

Compositional and physico-chemical data show that Straight-Run Gas Oils are very similar to Other Gas Oils. It is considered appropriate, therefore, to read across from the Other Gas Oil data to SRGOs. In a read-across study from Other Gas Oils, American Petroleum Institute completed a mammalian cell gene mutation assay with hydrodesulfurised middle distillates inmouse lymphoma cells, with and without Arochlor 1254-induced S9 (API, 1986a, Klimisch score = 1). Test substanceswere solubilized in acetone for diesel fuel 2, ethyl acetate for home heating oil No. 2 and absolute ethanol for the remaining samples. Ethylmethanesulfonate (EMS) was used as a positive control in the absence of S9 and 2-acetylaminofluorene (2-AAF), dimethylnitrosamine(DMN), 3-methylcholanthrene (MCA) or 7,12 dimethylbenz(a)anthracene (DMBA) in the presence of S9. Results showed that the test substance was weakly positive, with and without activation. 

Additional data are available on mouse lymphoma assays of straight run gas oil (API 1985a, API 1985b). This information is presented in the dossier, and showed mixed results. In the first assay, a positive response was elicited both in the presence and the absence of S-9 metabolic activation, while in the second assay, the test material was positive in the presence of S-9 and negative in the absence of S-9. 

 

In vitro cytogenicity in mammalian cells

 

Compositional and physico-chemical data show that Straight-Run Gas Oils are very similar to Other Gas Oils. It is considered appropriate, therefore, to read across from the Other Gas Oil data to SRGOs. In a read-across study, API completed sister chromatid exchange assay in Chinese hamster ovary (CHO) cells (API, 1988; Klimisch score = 1, key study), which yielded ambiguous results with metabolic activation. Hydrodesulfurised middle distillate (CAS 64742-80-9) did not induce an increase in SCEs in the CHO cells in the absence of S-9 activation. In contrast, in cells treated with the test material in the presence of S-9 activation, there was a statistically significant increase in the frequency of SCEs at two consecutive low dose levels compared to the solvent control. However, an inverse dose-response trend was observed with no significance at the highest two doses tested. The positive control CP induced SCEs as expected. Based on these results, it was concluded that hydrodesulfurised middle distillate did not induce an increase in SCEs in CHO cells in the absence of S-9. However, due to a statistically significant increase in SCE frequency at two consecutive low dose levels, hydrodesulfurised middle distillate was concluded to be equivocal in the SCE assay in the CHO cells in the presence of S-9.

 

In vivo gene mutation in mammalian cells

 

In a rat bone marrow micronucleus assay, male and female Sprague-Dawley rats (5/sex/dose/timepoint) were treated with a single intraperitoneal dose of straight-run middle distillate (CAS# 64741-44-2)at dose levels of 0, 300, 1000, or 3000 mg/kg bw in corn oil. Bone marrow cells were harvested at 6, 24, and 48 hours post-treatment (API, 1985c; Klimisch score = 1). There was no evidence of increased incidence of chromatid or chromosome gaps and breaks, fragments, structural rearrangements, or ploidy in bone marrow cells treated with straight-run middle distillate in comparison to controls. Results were negative for genotoxicity and no toxic effects were seen. 

Another rat bone marrow micronucleus assay by API (1986b; Klimisch score = 1) first dosed Sprague-Dawley rats via IP injection with either 3.0 or 5.0 g/kg straight run middle distillate and observed them for 48 hours (pilot study). No effects were seen. For the final assay, five male and female Sprague-Dawley rats were administered 5.0, 1.7, and 0.5 g/kg neat straight run middle distillate via IP injection and sacrificed either at 6, 24, or 48 hours. A concurrent control group was given an IP injection of deionised water and sacrificed either at 6, 24 or 48 hours after being dosed. Another group of animals received 1.0 mg/kg triethylenemelamine (TEM) as a positive control and was sacrificed 24 hours after dosing. The results were negative for mutagenicity; the test material did not induce a significant increase in the percentage of aberrant cells compared to the control group for either male or female rats, and no test material-related effects in the mitotic index were noted. No toxic effects were seen.

 

Compositional and physico-chemical data show that VGOs/HGOs/Distillate Fuels and Other Gas Oils are very similar to Straight-Run Gas Oils. It is considered appropriate, therefore, to read across from the Straight-Run Gas Oils data to VGOs/HGOs/Distillate Fuels and Other Gas Oils. In a read-across study, an in vivo mammalian cell micronucleus test (gene mutation) was performed on diesel 2 (OGO) and home heating oil (VGO/HGO/Distillate fuel) (McKee et al., 1994; Klimish score = 2). Results were negative for mutagenicity as there was no increase in the frequency of micronuclei for either material. 

Summary

Results are available from a number of studies that have examined the mutagenicity and genotoxicity of straight run gas oils in vitro and in vivo. Straight run gas oils were predominantly negative in the optimised Ames assay, indicating either no or at most very weak genotoxic potential. Due to the uncertain reliability of the in vitro mouse lymphoma assays that have been conducted and the inconsistent (mainly borderline) results obtained with and without metabolic activation, no firm conclusions can be drawn from these studies. The in vivo genotoxicity assays conducted in rats were negative. The studies involved high dose i.p. administration and no carcinogenic oil was included as a positive control in the investigations.

 

Therefore, the studies using the optimised Ames assay and the negative in-vivo assays suggest that straight run gas oils are likely to have little or no genotoxic potential.

 

Additional data support that straight run gas oils are not mutagens (API, 1985d; API, 1985e; Blackburn et al., 1984; Blackburn et al., 1986; CONCAWE, 1991; Jungen et al., 1995; Nessel et al., 1998). This information is presented in the dossier.

Justification for classification or non-classification

Bacterial mutation assay (modified) with straight-run gas oils were predominantly negative and in vivo chromosome aberration assays were negative. Based on the evidence, straight run gas oils are unlikely to be mutagenic in humans and do not meet the criteria for classification and labelling as described in EU CLP Regulation (EC No. 1272/2008) criteria.

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