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In Vitro Genetic Toxicity

In vitro gene mutation in bacteria

One key study (Deininger et al., 1991; Klimisch = 2) and one weight of evidence study (McKee et al., 1994; Klimisch = 2) was identified to evaluate the in vitro genotoxic potential of vacuum gas oils in bacterial cells.

Deininger et al. reported on the mutagenicity of six different types of petroleum-derived middle distillate test substances [blend 55% straight run gas oil, 45% fluid catalytically cracked gas oil substance (MD 57389); blend 54% straight run gas oil, 46% cracked gas oil components substance (MD 57391); blend 50% straight run gas oil, 50% cracked gas oil components substance (MD 57386); straight run gas oil with 59% cracked gas oil (MD 57401); straight run gas oil cracked gas oil blend (MD 57388); and diesel fuel (MD 57393)]inS. typhimuriumTA 98 using themodified Ames assay.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.The test materials were evaluated at concentrations of0, 1, 3, 5, 7, 10, 15, 20, 25, 40, 40, 50, or 60 µL/platein the presence of mammalian metabolic activation (Aroclor 1254-induced hamster liver S9) using the pre-incubation method. The mutagenicity index for these substances ranged from 2.0 to 9.0. These substances are considered genotoxic materials. The diesel fuel (MD 57393) was marginally mutagenic with an MI of 1.7. Based upon these scores, VGOs/HGOs/Distillate Fuels are likely to have some genotoxic potential.

In a non-traditional mutagenicity assay (McKee et al., 1994), home heating oil in DMSO was tested for its mutagenic potential. S. typhimurium strainTA 98 wasexposed to 1, 5, 10, 25, or 50 µL/plate (for cyclohexane extracts) or 5, 10, 15, 20, 30, 40, 50, and 60 µL/plate (for DMSO extracts) to each of the five fractions of the test material with and without metabolic for 72 hours at 37ºC. 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 home heating oil did not exhibit any evidence of mutagenic activity in the presence or absence of S-9 activity.

Additional supporting data from various in vitro bacterial studies conducted using vacuum gas oils indicate inconsistent results.  This information is presented in the dossier. Two studies investigating the mutagenic potential of diesel fuel 2 (API, 1978) and no. 2 home heating oil (API, 1979) demonstrated that diesel fuel was negative with all strains. Equivocal findings were reported with no. 2 home heating oil, with a few treated groups showing a 2-fold increase in revertants both with and without S9, but without any dose response. Studies reported by CONCAWE (1991) and Nessel et al. (1998) showed four gas oils having mutagenicity indices ranging from 1.0 to 6.1. A commercial gas oil (MD11) tested in this study demonstrated a mutagenicity index of 1.2 in TA 98 indicating that it should be regarded as having no more than borderline genotoxic potential. The mutagenicity index of vacuum gas oil (vacuum tower overhead; CAS 64741-49-7) was reported to be 6.7 by Fueston et al., 1994.

 

In vitro gene mutation in mammalian cells

One read across weight of evidence study (API, 1986b) from Other Gas Oils was identified to evaluate the in vitro genotoxic potential of Vacuum Gas Oils in mammalian cells. Compositional and physico-chemical data show that Other Gas Oils are very similar to VGOs/HGOs/Distillate Fuels. It is considered appropriate, therefore, to read across from the Other Gas Oil data to VGOs/HGOs/Distillate Fuels.

In a mammalian cell gene mutation assay (API, 1986b; Klimisch = 2) hydrodesulfurised middle distillates were tested in mouse lymphoma cells, in the presence and absence of Arochlor 1254-induced S9. Test substances were 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 in the presence and absence of metabolic activation.

 

Additional supporting data is available from in vitro mammalian gene mutation studies conducted using vacuum gas oils. This information is presented in the dossier. Diesel fuel was observed to be negative in the mouse lymphoma assay for forward mutation at the thymidine kinase (TK) locus (API, 1978). A sample of no. 2 home heating oil was positive in the mouse lymphoma assay both with and without S9, however findings were limited to dose levels of 1600 and 1200mg/ml that reduced survival to 5% and 56% in the absence of S9, and to dose levels of 25 and 12.5mg/ml that reduced survival to 14% and 49% in the presence of S9 (API, 1979)

In vitro cytogenicity in mammalian cells

One read across weight of evidence study (API, 1988; Klimisch = 1) from Other Gas Oils was identified to evaluate the in vitro genotoxic potential of Vacuum Gas Oils in mammalian cells. Compositional and physico-chemical data show that Other Gas Oils are very similar to VGOs/HGOs/Distillate Fuels. It is considered appropriate, therefore, to read across from the Other Gas Oil data to VGOs/HGOs/Distillate Fuels. 

In the read-across in vitro cytogenicity study (API, 1988) hydrodesulfurised middle distillate was tested at dose levels of 0.008, 0.016, 0.03 and 0.06 µL/mL in the absence of S-9 and at dose levels of 0.13, 0.25, 0.5, and 1µL/mL in the presence of S-9 activation. 

 

Hydrodesulfurised middle distillate did not induce an increase in SCEs in the CHO cells in the absence of S-9 activation. In contrast, there was a statistically significant increase in the frequency of SCEs at two consecutive low dose levels when compared to the solvent control in the presence of metabolic activation. However, an inverse dose-response trend was observed with no significance at the highest two doses tested. The positive control induced SCEs as expected. Based on these results, the study authors 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, the study authors concluded that Hydrodesulfurised middle distillate was equivocal for induction of SCEs in the CHO cells in the presence of S-9.

 

In Vivo Genetic Toxicity

One key read-across study (API, 1985a) and one supporting study (McKee et al., 1994) were identified to evaluate the in vivo genotoxic potential of vacuum gas oils.Compositional and physico-chemical data show that Straight-Run Gas Oils are very similar to VGOs/HGOs/Distillate Fuels. It is considered appropriate, therefore, to read across from the Straight-Run Gas Oil data to VGOs/HGOs/Distillate Fuels.

In the key read-across rat bone marrow micronucleus assay (API, 1985a; Klimsich = 1), male and female Sprague-Dawley rats (5/sex/dose/timepoint) were treated with a single intraperitoneal dose of a straight-run middle distallate (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, 1985a; 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 distallate in comparison to controls. Results were negative for genotoxicity and no toxic effects were seen. 

 

In a supporting micronucleus assay (McKee et al., 1994), fifteen male and female CD-1 mice were treated with 1.0, 2.5, or 5.0 g/kg of home heating oil dissolved in corn oil via oral gavage. A concurrent control group received only corn oil, while another group served as positive control and was treated with 0.04 g/kg cyclophosphamide. There was no increase in the frequency of micronuclei for the test material. In addition, there was no evidence of bone marrow depression. Cyclophosphamide, the positive control, exhibited appropriate results and the vehicle control result was within the normal range. Based on these results the study authors concluded home heating oil did not exhibit a positive response. 

 

Additional supporting data is available from in vivo genotoxicity studies conducted in rats. This information is presented in the dossier.  Diesel fuel was observed to be clastogenic in a chromosome aberration test (API, 1978) when tested intraperitoneally in rats at dose of 0.6, 2.0, and 6.0 mL/kg.  Marginal positive findings were reported in an in vivo cytogenetics when rats were given home heating oil no. 2 at doses of 125, 417 or 1250 mg/kg bw/day for 5 days (API, 1979), with significant increases seen at the top and bottom doses but not at the middle dose. Aberration frequencies were: control, 1.5%; low-dose, 12.5%; mid-dose, 7.5%; high-dose 7.5%. Diesel fuel no. 2 was investigated in a mouse dominant lethal assay (API, 1980cc). Male mice were exposed by inhalation to diesel fuel at airborne concentrations of 100 and 400 ppm, 6 hours per day, 5 days each week for 8 weeks (40 exposures). The results showed that the test material did not cause significant increases in either pre- or post-implantation loss of embryos compared to negative controls. The sensitivity of the assay was confirmed by a significant increase in dominant lethal mutations in the females that had been mated with the males treated with the positive control substance. It was concluded that diesel fuel did not cause dominant lethal mutations at 100 or 400 ppm.

 

Summary

Key and supporting data (including read-across) are available from a number of studies that have examined the mutagenicity and genotoxicity of VGOs/HGOs/Distillate fuels in vitro and in vivo.

 

From the results observed and the difficulties known to arise with the testing of complex hydrocarbon mixtures in the Ames assay, it can be concluded that the standard Ames test and the yeast cell mutation assay are unlikely to give reliable findings. The findings reported by the API on the mouse lymphoma assay must also be regarded as being of questionable reliability for the following reasons: a) Findings were observed to be inconsistent in multiple studies conducted using the same test material; b) Many findings reported as positive were, on inspection of the report, weak or questionable; c) Positive findings were obtained in some cases both with and without S9 whereas in other cases the assay was positive only in the presence of S9; d) If metabolic activation by the standard method is inadequate for the Ames assay, there is no reason to suppose that it would be adequate for mammalian cell assays; e) Positive findings in various mammalian cell in vitro assays at dose levels producing a high degree of toxicity (as occurred in some the tests reported here) were considered unreliable (Scott et al. 1991). 

 

Findings indicate that VGOs/HGOs/Distillate fuel products containing cracked materials are likely to have some genotoxic potential. The degree of activity is likely to be dependent on the amount of cracked material present, the type of cracking involved and other factors.

Additional data support that VGOs/HGOs/Distillate fuels are not mutagens (API, 1984c; Blackburn et al., 1984; API, 1985d; Blackburn et al., 1986; API, 1987a; API, 1987b; API, 1987c; Jungen et al., 1995; Walborg et al., 1998). This information is presented in the dossier.  


Short description of key information:
In a key in vitro modified bacteria Ames study (similar to OECD 471), results indicated that there was evidence of genotoxic activity with gas oils producing mutagenicity indices from 1.7 to 9. An additional modified Ames assay showed negative results for mutagenicity. Additional in vitro mutagenicity tests in mammalian cells showed ambiguous and unreliable results (OECD 476 and OECD 479). A key read-across 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).

These findings indicate that vacuum gas oil products containing cracked materials are likely to have some genotoxic potential. The degree of activity is likely to be dependent on the amount of cracked material present, the type of cracking involved and other factors.

Endpoint Conclusion:

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. 1A or 1B (H350) or Cat. 2 (H351) according to the EU CLP Regulation (EC)1272/2008. This classification as carcinogenic does not automatically imply that these substances need also to be classified as mutagenic as defined by the 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 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 the CLP Regulation. For example, based on in vivo micronucleus tests on home heating oil as well as for read-across substances that were all negative for genotoxicity, vacuum gas oils/hydrocracked gas oils/distillate fuels are not classified as mutagens according to the EU CLP Regulation (EC)1272/2008.

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