Asphalt, oxidized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

In-vitro and in vivo genotoxicity studies are available for samples of oxidized asphalt. Although results show weak activity in some in vitro studies, this is not supported by results of an in vivo study. In the key in vivo study, animals exposed by inhalation for 28 days showed no evidence of micronucleus formation in the bone marrow.

Several supporting in vitro and in-vivo studies are available for samples of paving bitumen. Most in-vitro studies were undertaken on fume condensates, whilst in-vivo studies were carried out following inhalation exposure to fumes. Read across from studies on straight run bitumen, is justified based on weight of evidence from all studies and that the oxidation process increases molecular weight. Furthermore, fumes of air-rectified (partially oxidized) asphalt have been shown to be comparable with fumes of straight-run paving bitumen; in a comparative analytical study, fumes were closely similar in terms of composition and physical properties

Most of the read-across studies with paving bitumen show no evidence of genotoxic activity. Where positive results were seen, this was generally weak and occurred in isolated in vitro assays.

In conclusion, results of a range of in vivo genotoxicity studies show no convincing evidence that exposure to oxidized asphalt presents a genotoxic hazard.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Most genetic toxicity studies have been carried out on samples of paving grade bitumen. Read across from studies on straight run bitumen, is justified based on weight of evidence from all studies and that the oxidation process increases molecular weight. Furthermore, fumes of air-rectified (partially oxidized) asphalt have been shown to be comparable with fumes of straight-run paving bitumen; in a comparative analytical study and fumes from a straight-run and an air-rectified (partially oxidized) asphalt were closely similar in terms of composition and physical properties. It is important to also recognize that toxicity studies involving exposure to fume or fume condensates represent only the volatile fraction of the whole material.

In Vitro Genotoxicity

In a key bacterial gene mutation study (Kreich et al 2007), five samples of fume originating from Type III BURA (oxidized asphalt) were tested in a modified Ames assay. The first four samples were collected from the headspace of a bitumen storage tanks (indicated by TR) and the last was generated in a laboratory fume generation device from one of the same bitumen’s to be used for comparison. The laboratory generated sample LR-A was compared against its tank collected counterpart TR-A and the results indicate that generating the fume in a laboratory device results in a material that has greater mutagenic potential. In contrast, the tank collected fumes exhibited a range of activity (negative, equivocal and weak) in the mutagenicity assay.

In a supporting bacterial gene mutation study (Machado et al 1993) with roofing fume condensate, generated at either 232 or 316^oC, weak to moderate mutagenic activity was seen in the modified Ames test.

Several ‘read-across’, supporting bacterial gene mutation studies have been carried out on samples of paving bitumen. Most studies showed no evidence of mutagenic activity, although in one assay (De Meo et al 1996), weak positive responses, with metabolic activation, were observed with fume condensates.

In a key 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 can cause cytogenetic damage, principally to the spindle apparatus in cultured mammalian cells.

Supporting, ‘read-across’ in vitro, mammalian genotoxicity studies (micronucleus, chromosome aberration, mouse lymphoma and DNA damage) with samples of bitumen are available. In these studies mixed results were obtained; chromosome aberration and DNA damage studies were negative, whilst mouse lymphoma and micronucleus studies showed weak positive responses.

In Vivo Genotoxicity

In a key bone marrow micronucleus assay, (5 rats/sex/dose) rats were treated via inhalation with oxidized asphalt fume condensate (CAS No. 64742-93-4) at doses of 0, 30, 100, or 300 mg/m3 THC (total hydrocarbons). Bone marrow smears of cells prepared from the femur of each rat were examined microscopically (Fraunhofer, 2009,Parker et al., 2011). Results show that following 28 days of exposure there was no repression of red blood cell formation in male and female animals, as compared to controls. This does not mean that fume condensate was not systemically available, because in a range finding study, using a concentration of 1000 mg/m3 THC, blood formation was significantly depressed in female animals, thus showing the material could reach the bone marrow (target organ for the mammalian erythrocyte micronucleus test). There was no evidence of a significantly enhanced mean frequency of micronucleated erythrocytes (MN per 2000 PCE) due to exposure, as compared to the respective clean air groups.

In a supporting study (Qian 1998), rats were exposed intratracheally to roofing asphalt condensates. Unidentified DNA adducts were found in lung tissue from exposed animals, confirming exposure and interaction with DNA.

Several supporting, in vivo genotoxicity studies with paving bitumen were identified. The genotoxic effects of fumes from bitumen were investigated in 'read across' studies 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. Genotoxic effects were evaluated by examining the mutation frequency and spectrum of neutral receptor gene cII inserted into the rodent genome. In the mouse study, DNA adducts and cII mutant frequencies in isolated lung DNA were no different in 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 paving bitumen did not induce any genotoxic effect. In contrast, DNA adducts were detected in rats, using32 P-postlabelling and there was a clear increase in excretion of 1-hydroxypyrene in 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. It was concluded that, despite the presence of DNA adducts, there was no mutagenic activity from exposure to fumes from bitumen heated to temperatures used during road paving activities.

In supporting micronucleus (Ma 2002, Halter 2007, Zhao 2004) and chromosomal aberration (API 1984) assays results showed no clear evidence of genotoxic activity, following exposure to paving bitumen.

Supporting in vivo studies (Gate 2006, Schoket 1998, Genevois 1996, Booth 1998, Genevois 2001, Halter 2007) show that exposure to paving bitumen can result in formation of DNA adducts and altered gene expression. This provides confirmation of exposure but is not linked directly with mutagenic or clastogenic effects in the exposed animals.

Justification for selection of genetic toxicity endpoint

Key study with oxidized asphalt. Supported by a large number of in vitro and in vivo studies with oxidized asphalt and paving bitumen.

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.

An in vivo micronucleus test on oxidized asphalt was negative for genotoxicity. Based on these in vivo data and other animal studies, it clearly is shown that oxidized asphalt-induced DNA adducts are not necessarily linked to mutagenic effects. Consequently, oxidized asphalts are not considered mutagenic and do not meet the criteria for classification and labelling under CLP EU Regulation 1272/2008.