Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

RAEs’ mutagenic potential was evaluated by modified Ames assays, in vitro mammalian cell gene mutation assays and an in vivo mutagenic assays. Results of those studies are summarized below. One of the tests used for RAEs is the modified Ames assay, which was developed to be an effective screening tool for petroleum products which had low mutagenic activity. Modifications to the standard Ames assay involved increasing the amount of hamster derived metabolic fraction and testing in a single bacterial strain sensitive to Polycyclic Aromatic Hydrocarbons, in an effort to maximize the effect to allow for discrimination amongst weak responders in a standard assay. Investigating the correlation of results from modified Ames assays and dermal carcinogenicity studies for RAEs revealed that a mutagenicity index (MI) of 0.4 allowed for discrimination of the carcinogenic potential; those with an MI less than 0.4 were non carcinogenic while those with MI greater than or equal to 0.4 could be considered carcinogenic, presumably by a genotoxic mechanism of action.

Modified Ames assays were conducted on a number of residual aromatic extract samples. In studies conducted by Petrolabs S. typhimurium T98 was exposed to a various RAE test oils in DMSO at 0, 12, 24, 36, 48, and 60 µL in the presence of mammalian metabolically active S9 liver fractions using the plate-incorporation method. For all tests, the positive controls induced the appropriate responses . In the samples tested 3 samples were negative with mutagenicity indices (MI) less than 0.4. Two samples were positive with this assay with MIs greater than 0.4 (0.43, and 0.89). An additional two modified bacterial reverse mutation assays were identified (Exxon Mobil, 1997), in which various residual aromatic extracts (MRD 96 -657 and 96 -601) were diluted (1:5) in DMSO, shaken, centrifuged, and separated into two fractions. Each extracted oil sample (0, 5, 10, 15, 20, 30, 40, 50, or 60 uL/plate), tester bacteria (TA98), and hamster S9 metabolizing mixture were combined in suspension, incubated for 20 minutes at 37 degrees Celsius, poured into a Petri plate, incubated for 48 hours at 37 degrees Celsius, and revertant colonies were counted. The mutagenicity index was calculated from the initial slope of the linear portion of the dose-response curve. Under conditions of the study, both residual aromatic extract samples are considered not mutagenic with MIs of 0.0 and 0.3. 

In two modified AMES tests according to ASTM E 1687, 3 bitumen and 2 RAE samples were examined for mutagenic activity in one histidine dependent auxotroph of Salmonella typhimurium, strain TA98, using a modification of the pour-plate assay designed to detect mutagenicity mediated by polynuclear aromatic compounds derived from petroleum. A lower threshold value of 0.4 has been selected as the cut-off for Residual Aromatic Extracts (s), based on the results of skin-painting studies (Blackburn et al, 1996): RAEs with a MI >0.4 demonstrated carcinogenic potential upon dermal application to mouse skin with chronic exposure, whereas RAEs with a MI <0.4 did not demonstrate a carcinogenic potential. The results demonstrated that the test substances, CAS 91995-70-9 and 64742-10-5 had a Mutagenicity Index (MI) of 0.47 and 0.29, respectively.

In a mouse lymphoma assay, L5178Y TK +/- cells were treated with residual aromatic extract dissolved in acetone at test concentration of 1.17, 2.34, 4.69, 9.38, 18.75 or 37.5 µg/ml at 3 hours for the first experiment and 24 hours for the second experiment. (CIT, 2001). There were no significant differences noted in the cytotoxicity or cloning efficiencies of the treated cells versus the controls. The residual aromatic extract did not induce a significant increase in mutant frequency as compared to vehicle controls. Therefore, under the conditions of the study, the test material is considered negative.

In an in vivo micronucleus study, male and female Sprague Dawley rats (10/sex/dose) were treated dermally with residual aromatic extract once daily, five days per week for 13 weeks at dose levels of 0, 500, or 2000 mg/kg/day (Mobil, 1988). At the end of the 13 -week treatment period, bone marrow was harvested from the femur of 5 animals per sex per dose and analyzed for induction of micronucleus formation. There was no difference in the percentage of micronucleated cells in the treated animals versus the controls when analyzed by SAS ANOVA and SAS GLM (General Linear Model). Therefore, residual aromatic extract does not induce a significant increase in micronucleus formation over untreated controls.

Additional data supports that RAE is not mutagenic (API, 2003; Blackburn et al., 1984; Blackburn et al., 1986; CIT, 2003; Kuwait Petroleum, 2001; Total, 2000). This information is presented in the IUCLID dossier.

Short description of key information:

Ambiguous results from modified Ames assays (modified OECD 471), and negative results from both an in vitro mammalian cell gene mutation assay (OECD 476) as well as an in vivo micronucleus study were identified for RAE.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Evaluation of the mutagenic potential of RAE with modified Ames assays produced mixed results with some samples testing positive in a bacterial assay and other not. When evaluated in a mammalian cell in-vitro gene mutation study the results were negative. In addition the results for the in vivo investigation revealed that RAEs are not mutagenic. 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. Due to the weight of evidence, RAEs are unlikely to be mutagenic under in vivo conditions and do not meet the criteria for classification and labelling as mutagenic under the CLP Regulation, (EC)1272/2008. The polycyclic aromatic compounds (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.