Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

A large number of published in-vitro and in-vivo genetic toxicity studies were identified. In-vitro studies included Ames, or modified Ames tests, chromosomal aberration studies, micronuclei  and mouse lymphoma assays and Comet assays to investigate DNA damage. In-vivo studies included transgenic studies, cytogenicity investigations, micronucleus assays,  Comet assays (for DNA damage) and investigation of  DNA adduct formation and gene expression profiling.

In-vitro studies were undertaken on condensates of bitumen, or solutions of bitumen in solvent whereas in-vivo studies were carried out following inhalation exposure to bitumen fumes, dermal exposure to fume condensates/solvent solutions, oral exposure to bitumen solutions or intratracheal administration of condensate.

Two inhalation transgenic animal model studies (non-guideline) were identified as key in vivo studies. Both studies (one in mice and another in rats) produced negative results. Results of a key ‘read across’ micronucleus assay are available for a mixture of air-rectified bitumen and vacuum residue which was negative. Supporting oral cytogenetic assays were also negative. In vitro gene mutation studies in bacteria (OECD 471) were identified on bitumen and showed mixed results.  

In conclusion, although the in-vitro and in-vivo studies identified showed some mixed results the  large body of data did not show clear, consistent evidence of genotoxic activity.

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

Additional information

Additional information from genetic toxicity in vivo:

It is important to recognize that toxicity studies involving exposure to fume or fume condensates from bitumens represent only the volatile fraction of the whole material.

One key gene mutation study in bacteria (Klimisch score=2) was identified. In a reverse gene mutation assay in bacteria, strains TA98, TA100, YG 1041 and YG 1042 of S. typhimurium were exposed to bitumen fume condensates in DMSO at amounts not exceeding 10 µL and 0.1 mL of the overnight culture in the presence and absence of mammalian metabolic activation using pre-incubation method (De Meo et al., 1996). There was no evidence of mutagenic activity in any samples without metabolic activation. All samples and strains with metabolic activation showed varying degrees of mutagenic activity from 268 (x 0.08) to 11748 rev/ µL. The positive controls induced the appropriate responses in the corresponding strains.

In three supporting studies, 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, based upon the principles of the ASTM Standard Test Method E 1687. A clear indication of mutagenic activity is indicated by an MI value >/=1. The results showed, that the test substance, CAS 8052-42-4 had a Mutagenicity Index (MI) of 2.63, test substance, CAS 64741-56-6 had a Mutagenicity Index (MI) of 0.84, and the test substance, CAS 92062-05-0 had a Mutagenicity Index (MI) of 2.84. 

In a key gene mutation study (Kriech et al., 2007, Klimisch score = 2), four samples of fume condensate originating from paving bitumen were tested in a modified Ames assay. The four samples were collected from bitumen storage tanks (indicated by TR).  Three of the four samples were negative whilst one showed a marginally positive response.

In a mammalian micronucleus assay, Chinese hamster cell V79 cultures were exposed to Type I and Type III roofing (oxidized) asphalt fume condensates (generated at temperatures similar to actual roofing operation (316 ± 10°C)in DMSO at concentrations of 0, 62.5, 125, 187.5, and 250 μg/mL for 24 hours (Qian et al., 1996). The results were expressed as the mean number of cells with micronucleus per 1000 cells. Fume condensates of both types I and III oxidized asphalt were found capable of causing micronucleus formation in mammalian cells in vitro. The genotoxic potential appears to be similar for both types of oxidized asphalt condensate. Both condensates caused a similar dose-related increase in the frequency of micronucleated cells. The increase was statistically significant for all four concentrations tested. These findings indicate that both Type I and Type III roofing oxidized asphalt fumes are capable of causing cytogenetic damage, principally to the spindle apparatus in cultured mammalian cells.

With regard to in vivo studies, the genotoxic effects of fumes from bitumen were studied in transgenic (Big Blue) mice (Micillino, 2002) and rats (Bottin et al., 2006). Animals were exposed, nose only, to 100 mg/m3 (TPM) bitumen fumes for 6 hours a day for five consecutive days. Mutation properties of bitumen were evaluated by analyzing the mutation frequency and spectrum of neutral receptor gene cII inserted into the rodent genome. For the mouse study, DNA adducts and cII mutant frequencies in the isolated lung DNA were not different in the exposed mice compared to controls. The mutation spectra were also very similar in the exposed and control animals and indicated that exposure to about 100 mg/m3 (TPM) of fumes from bitumen similar to those produced during road paving did not induce any genotoxic effect. Contrasting those results found in mice, rat DNA adducts could be detected using 32P-postlabelling in the transgenic rats and there was a clear increase in excretion of 1-hydroxypyrene in the exposed animals. However, like in the mice, the cII mutant frequencies were not changed but a small, albeit statistically not significant, change in incidence of transversions was observed. Consequently, it was concluded that, despite the presence of DNA adducts, there was no mutagenic potential from fumes generated from bitumens heated to temperatures used during road paving activities.

In animal studies, it is clearly shown that bitumen-induced DNA adducts are not necessarily linked to mutagenic effects. Overall, there is no convincing evidence from studies in animals that exposure to fumes from bitumen causes mutagenic or clastogenic effects.

Additional data supports that bitumen is not mutagenic including API, 1984a, b; Gate et al., 2006; Ma et al., 2002; Machado et al., 1993; Robinson et al., 1984, Halter 2007, Booth 1998).This information is presented in the dossier.

Justification for selection of genetic toxicity endpoint

An in-vivo transgenic study was selected but other studies eg in-vivo micronucleus studies would also be valid.

Justification for classification or non-classification

Some oil products containing relatively high concentrations of polycyclic aromatic compounds (PAC) are considered genotoxic carcinogens. 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 do not need to be classified as mutagenic unless there is clear evidence that germ cells are affected by exposure, consistent with DSD.

A large number of in-vitro and in-vivo genotoxicity studies are available for bitumens or air-rectified bitumen. Although the database provides some mixed results, overall the data do not provide clear consistent evidence of genotoxic activity. Chronic inhalation studies with oxidized (air-rectified) asphalt, together with comparative fume composition information, indicate that read across to the bitumen category, is appropriate. Based on these in vivo animal studies, it clearly is shown that bitumen-induced DNA adducts are not necessarily linked to mutagenic effects. Consequently, bitumens are unlikely to be mutagenic and do not meet the criteria for classification and labelling under CLP Regulation, (EC)1272/2008.