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In Vitro Genetic Toxicity

In vitro gene mutation in bacteria

A total of 13 in-vitro gene mutation assays have been identified. In a series of studies (May 2013) nine substances from the VHGO category were tested in a modified 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, referred to as the Modified or Optimised Ames Assay, is recognised as an ASTM method (E1687-95) and substances with an Mutagenic index (MI) greater than 1 are considered to be mutagenic. In these nine studies eight of the materials tested showed no evidence of mutagenic activity (MI values between 0.2 and 0.7). One sample gave an ambiguous, weak response (MI 1.07). Additional bacterial studies proved either negative for gave weak/ambiguous results. In-vitro SCE and mouse lymphoma studies gave equivocal results.


A work program is underway to provide additional evidence and justification for the use of the optimised Ames assay instead of the standard test for petroleum substances.


In vitro gene mutation in mammalian cells

A sample of diesel fuel was found to be negative in the mouse lymphoma assay for forward mutation at the thymidine kinase (TK) locus (Jagannath, 1978). The test was carried out both in the presence and absence of mouse liver microsome fraction and dose ranged between 0.064 and 0.5 ul/ml.


In vitro cytogenicity in mammalian cells

In a 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

Additional testing is planned for four substances in the VHGO category; combined in vivo Comet assay and micronucles tests are proposed. These will help to fill data gaps and confirm conclusions on genetic toxicity.


In a 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. Diesel fuel was observed to be clastogenic in a chromosome aberration test (Jagannath, 1978) when tested intraperitoneally in rats at dose of 0.6, 2.0, and 6.0 mL/kg. 

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.



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 may 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.


Short description of key information:

The genotoxicity of members of the vacuum gas oil, hydrocracked gas oil and distillate fuel category has been investigated in a number of in-vitro and in-vivo studies. In-vitro studies include 13 bacterial mutation assays, a mouse lymphoma assay and an SCE assay in Chinese hamster ovary cells. Genotoxicity in-vivo has been investigated in 3 bone marrow cytogenicity studies and a male dominant lethal assay.


Although some ambiguous results were seen the majority of in-vitro studies showed no, or little mutagenic activity. In the in vivo micronucleus test home heating oil showed no evidence of genotoxic activity. This lack of activity was supported by further in-vivo studies including a male dominant lethal assay.


Endpoint Conclusion: No adverse effect observed (negative)

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.