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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key modified Ames test (MAY 2013), a sample of a hydrotreated middle distillate (CAS 64742-46-7) showed no evidence of mutagenic activity.This lack of activity in the modified Ames assay is supported by seven other studies, with other gas oils or read across to diesel fuel,  which showed no or marginal activity.

In a series of in-vitro mouse lymphoma assays with samples of hydrodesulphurised middle distillate (CAS 64742-80-9) mixed but inconsistent results were obtained. Some studies were considered unreliable and disregarded. In a sister chromatid exchange assay hydrodesulphurised middle distillate did not induce an increase in SCEs in CHO cells in the absence of S-9 and was equivocal for induction of SCEs in the CHO cells in the presence of S-9 (OECD 479).

In a series of acute, in vivo cytogenetic assays (OECD 475), hydrodesulphurised middle distillate (CAS 64742-80-9) DID not induce chromosome aberrations in bone marrow cells of rats (OECD 475). The lack of activity was also supported by a similar study with diesel fuel.

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

Additional information

Additional information from genetic toxicity in vitro:

Using a weight of evidence approach for in vitro genotoxicity results, studies by the modified assay suggest that Other Gas Oils are likely to have little or no genotoxic potential.

 

In vitro gene mutation in bacteria

In a key genetic toxicity study (May 2013) a sample of a hydrotreated middle distillate (CAS 64742-46-7) showed no evidence of mutagenic activity in a modified Ames test. 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.

In a supporting study (MAY 2013), a sample of a hydrodesuphurised middle distillate (CAS 64742 -80 -9) also showed no evidence of mutagenic activity in a modified Ames test.

In a further supporting study a modified Ames test was conducted using three different types of straight run hydrodesulfurised gas oils, (Klimisch score = 2, Dieninger at al. 1991). A marginal dose-related increase in revertants compared to the mean solvent control value occurred for all the test materials [straight run hydrodesulfurised gas oils]. The mutagenicity indexes ranged from 0.7 to 1.4.These results suggest that some substances in this category may exhibit weak mutagenic activity.

In a supporting modified AMES test (Walborg et al., 1998), the mutagenicity of API 81-10 (a hydrodesulfurized petroleum middle distillate; CAS No. 647420-80-9) was assayed using a Salmonella typhimuriumstrain TA98 in the presence of metabolic activation (+S9). The mutagenic index was 3.6 and the test material was considered to be mutagenic in the presence of metabolic activation.

In an additional supporting modified AMES assay (CONCAWE, 1991), a straight-run hydrotreated gas oil was evaluated for potential to cause genetic mutation in Salmonella typhimuirum TA98 strain cells in the presence of metabolic activation (±S9). A Mutagenicity index of 1.0 was determined.

In a read-across study, diesel 2 in DMSO was tested for its mutagenic potential in a modified Ames assay (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. Based on these results, the study authors concluded that the petroleum middle distillate fraction tested for mutagenicity did not exhibit any evidence of mutagenic activity in the presence or absence of S-9 activity.

In two other supporting studies, 14 Gas oils were examined for mutagenic activity in one histidine dependent auxotroph of Salmonella typhimurium, strain TA98 using modified AMES assay conducted according to the ASTM Standard Test Method E 1687. Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay. Both test substance, CAS 64742-46-7 and other test substance CAS 64742-80-9 were negative with metabolic activation and had a Mutagenicity Index (MI) of 0.01 and 0.05, respectively.

These results show no or marginal activity are consistent with the presence of only low levels of 3-7 ring PACs in the samples

 

In vitro gene mutation in mammalian cells

Hydrodesulfurised middle distillate was examined in a series of mouse lymphoma assays.The first mouse lymphoma study (API, 1984c) was reported to produce weak positive findings in the presence of S9 but not in its absence. In this study, increases in mutant frequency of 2.1 fold and 3.4 fold were seen at concentrations of 31.3 and 62.5 nl/ml respectively, at which relative survival was 25% and 11.1%. However, because the positive results were found at such low survival rates, the study is considered unreliable.

 

The second study (API 1986b) was judged by the authors as producing weak positive findings both in the presence or absence of S9. In this study, in the absence of S9, treatment levels from 50-300 nl/ml produced low to high toxicity and all gave rise to an increase in mutant frequency but not in a dose related manner.In the presence of S9, a barely 2-fold increase was seen at the top 2 doses (200 and 250 nl/ml) with survival of 47% and 32% respectively. When the +S9 part of the study was repeated all dose levels gave positive findings but not in a dose-related manner. In view of the positive findings both in the presence and absence of S9, the poor repeatability and the absence of a dose response relationship, this study is considered unreliable.

 

The third study (API 1987a) produced positive findings in the presence but not the absence of S9. All of the treatment groups (75-400 nl/ml) in the presence of S9 exceeded the calculated positive mutation frequency of 81.5, the degree ranging from 2-8 fold the control frequency. Survival as a measure of toxicity ranged from 15% at the top dose to 70% at the lowest dose. In this case a dose-response relationship was observed.

 

Hydrodesulfurised middle distillate sample API 81-10 was also fractionated into a saturated (API 81-10 Sat) and an aromatic (API 81-10 Aro) fraction and these were tested in the mouse lymphoma assay (API, 1987b,c). Both fractions gave negative findings both in the presence or absence of S9. In an additional supporting mouse lymphoma forward mutation assay on hydrodesulfurised middle distillate (API, 1985a), the increases in mutant frequency were small and only occurred at a dose that caused high toxicity, so the result was judged to indicate borderline activity.

  

In vitro cytogenicity in mammalian cells

In a sister chromatid exchange assay in chinese hamster ovary (CHO) cells (API, 1988; Klimisch score = 1), hydrodesulfurised middle distillate

gave ambiguous results with metabolic activation. Hydrodesulfurised middle distillate 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, 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 gene mutation in mammalian cells

Compositional and physico-chemical data show that Other Gas Oils are very similar to Straight-Run Gas Oils. It is considered appropriate, therefore, to read across from the SRGO data to Other Gas Oils.

In two bone marrow cytogenetic assays, samples of hydrodesulphurised middle distillate (CAS 64742 -80 -9) did not induce chromosomal aberrations in rat bone marrow cells. Rats were administered a single IP dose of test material at levels of 0.3, 1.0 or 3.0 g/kg. Animals were sacrificed either 6, 24 or 48 hrs after treatment and bone marrow cells harvested for examination.

In a read-across bone marrow chromosome aberration assay, 5 Sprague-Dawley rats/sex/dose were treated intraperitoneally with a straight run gas oil (API 83 -11) at doses of 0, 0.3, 1.0, or 3.0 g/kg bw.Bone marrow cells were harvested at 6, 24, and 48 hours post-treatment (Klimisch score = 2, API 1985b). A 4% body weight loss was observed in male rats 24 hours post exposure to 3.0 g/kg and a 2% weight loss in females 48 hours post exposure to 3.0 g/kg of the test material. No mortality or signs of clinical toxicity were observed at any dose level in either sex through the study period.There were no signs of toxicity during the study. The positive control (triethylenemelamine) induced the appropriate response.There was no significant increase in the frequency of chromosome aberrations in bone marrow after any treatment time. Under the conditions of the study, the test substance did not induce chromosome aberrations in bone marrow cells of male or female rats.

  

In an additional read-across study, an in vivo mammalian cell micronucleus test (gene mutation) was performed on diesel 2 (McKee et al., 1994; Klimish score = 2). Results were negative for mutagenicity as there was no increase in the frequency of micronuclei after exposure to the test material. 

Due to the uncertain reliability of the in vitro mamalian cell genotoxicity assays conducted by API 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 by the API were negative. Most of the studies involved high dose i.p. administration. No carcinogenic oil was included as a positive control in these investigations and, in one instance where it was, it failed to produce clear positive findings. 

  

Using a weight of evidence approach for in vitro genotoxicity results, studies by the modified assay suggest that Other Gas Oils are likely to have little or no genotoxic potential. Additional data supports that Other Gas Oils are not mutagens and should not be classified (Walborg et al., 1998; CONCAWE 1991; Jungen et al., 1995; Nessel et al., 1998).This information is presented in the dossier.

Justification for selection of genetic toxicity endpoint

One of a number of in-vitro assays available. Lack of activity supported by results of other in-vitro and in-vivo studies.

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 Carc. 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 the EU 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 PAHs 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, Other Gas Oils 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 criteria laid out in the EU CLP Regulation. For example, based on in vivo micronucleus tests on three other gas oils as well as for read-across substances that were all negative for genotoxicity, Other Gas Oils are not classified as mutagens according to EU classification.