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GENOTOXICITY IN VITRO

Mutagenicity in bacteria:

Brams et al. (1987), Chung and Adris (2003), Cline and McMahon (1977), Glatt et al. (1992), Haworth et al. (1983), Ishidate et al. (1984), NTP (1979), and Pagano et al. (1983) are the available Klimisch 2 studies. These studies cover all relevant tester strains, with and without S9 mix, including negative and positive controls. All studies, including those assigned a Klimisch 3 or Klimisch 4 score, demonstrate that biphenyl is negative in the Ames test under all conditions. A weight of evidence approach was presented using all Klimisch 2 studies.

Chromosome aberration:

Chromosome aberration tests with biphenyl were performed with Chinese hamster lung cells (CHL), Syrian Hamster cell line and Hamster Lung fibroblasts. In the Klimisch 2 studies (Ishidate and Odashima, 1977; Ishidate et al., 1984), used in a weight of evidence approach, biphenyl tested negative for chromosome aberration in the absence of metabolic activation. No positive control substances were tested under similar conditions. No data on cytotoxicity were reported. One Klimisch 3 study (Abe and Sasaki, 1977) and one Klimisch 4 study (Kawachi et al., 1980) reported negative results without metabolic activation. In another Klimisch 4 study (Sofuni et al., 1985) biphenyl tested negative with and without metabolic activation (at a dose range of 0-0.125 mg/mL), but biphenyl tested positive when tested in the presence of metabolic activation (at a dose range of 0-0.02 mg/mL).

Mouse lymphoma assay:

Biphenyl tested negative in the absence of metabolic activation and positive with metabolic activation when tested in the mouse lymphoma assay (Wangenheim and Bolcsfoldi, 1988). Cytotoxicity was observed at the two highest concentrations tested. Positive and negative controls were tested as well. The genotoxicity results were supported by the Klimisch 4 study of Wangenheim and Bolcsfoldi (1986). The former study was selected as a key study with the remark that not too much weight should be given to the weak positive results observed in the presence of metabolic activation.

Unscheduled DNA synthesis:

Unscheduled DNA synthesis was tested in primary cultures of rat hepatocytes and human lung fibroblast cells. Biphenyl tested negative in the Klimisch 2 study of Probst et al. (1981). No data on cytotoxicity were reported. Four Klimisch 4 studies were available in which biphenyl tested negative. All unscheduled DNA synthesis studies with biphenyl were used as supporting evidence for the in vitro genotoxicity endpoint.

DNA repair:

Biphenyl tested negative for DNA damage in a Nick Translation Assay without metabolic activation (Klimisch 3 study of Snyder and Matheson, 1985). No cytotoxicity data were reported. Negative results for DNA damage in bacteria (E. coli strains 343/636, 343/591, WP2, WP2 urv A, CM 571 and WP100) were also reported in two Klimisch 4 studies (Hellmer and Bolcsfoldi, 1992; Nishioka and Ogasawara, 1978). No data on cytotoxicity were reported in this study. All studies can be used as supporting evidence for the in vitro genotoxicity endpoint.

Gene mutation assay:

One Klimisch 2 supporting gene mutation assay was identified (Glatt et al., 1992). Biphenyl tested negative in absence of a metabolic activation system and positive with metabolic activation in Chinese Hamster Lung fibroblasts (V79). No cytotoxicity was reported, however biphenyl was tested up to concentrations resulting in precipitation.

Other:

Biphenyl tested negative in a bioassay for genetic activity profiling with and without metabolic activation (Klimisch 4 study of Garrett et al., 1986). No data on cytotoxicity were reported. This study was used as supporting information.

Conclusion on genetic toxicity in vitro:

Bacterial genotoxicity studies, unscheduled DNA synthesis as well as DNA repair tests have been uniformly negative. These results were supported in a bioassay for genetic activity profiling. Testing in mammalian cells has produced mixed results with limited positive results for gene mutation and clastogenicity reported only in the presence of metabolic activation. Positive results were observed in the Klimisch 4 chromosome aberration test of Sofuni et al. (1985) in the presence of metabolic activation (at a dose range of 0-0.02 mg/mL). However, no positive or negative controls were tested simultaneously and no data on cytotoxicity were reported. As the reliability of this study is not assignable, these positive results should be neglected in determining whether biphenyl is a mutagenic substance. In the mouse lymphoma assay (Wangenheim and Bolcsfoldi, 1988) a weakly positive response was only observed at a single concentration that significantly inhibited cell growth (12% of control values) only in the presence of S-9 following a 4-hour exposure. However, it was not mentioned if the positive control was valid and therefore, not too much weight should be given at these results as it is not clear if the test system was valid. In the Chinese hamster V79 gene mutation assay, although no appreciable toxicity was observed during the treatment period, as determined from the number of cells harvested at subcultivation during the expression period (> 80% of control value), cloning efficiencies were not reported, hampering interpretation of the obtained results.

GENOTOXICITY IN VIVO

Mouse micronucleus test:

Biphenyl tested negative in the (CD-1) mouse micronucleus test (Klimisch1 key study of Gollapudi et al., 2007). Cytotoxicity was reported at the highest dose level tested (800 mg/kg bw/day).

Chromosome aberration:

Biphenyl tested negative in male Sprague-Dawley rats in a Klimisch 2 supporting study (Johnston et al., 1976). No data on cytotoxicity were reported. Similar results were observed in a Klimisch 4 chromosome aberration test with rats (Kawachi et al., 1980).

Comet assay:

A Klimisch 2 comet assay was performed with male CD-1 male mice (Sasaki et al., 1997) at 2000 mg/kg. Biphenyl tested positive in this comet assay. No toxic effects were observed in this study.

Conclusion on genetic toxicity in vivo:

Biphenyl tested negative in two chromosome aberration tests in rats. These results were confirmed in a mouse micronucleus test

(CD-1 strain). A Comet Assay was performed in CD-1 males where biphenyl tested positive. No toxicity was reported for the animals used in the Comet Assay, although it was reported in the micronucleus test at 800 mg/kg (Gollapudi et al., 2007, Klimisch 1 study); both studies were performed in CD-1 mice. Although Comet Assay is scientifically accepted, many questions have been raised regarding the validity of the test results in Sasaki et al., (1997) report: no data on controls was given, only males were tested, only one very high concentration was tested (2000 mg/kg). Since data from lower doses are missing in the Comet Assay and there are significant methodological insufficiencies found with the study, DNA fragmentation secondary to cell death should be considered as a source of DNA comets. Also, weight of evidence from in vitro assays suggests that biphenyl is not DNA reactive, including results from USD and DNA repair assays. Furthermore, the substance was entered into the OECD QSAR application toolbox (v.1.1.02). After profiling the substance did not answer any of the categorization criteria for DNA binding and protein binding. This implies that the substance does not bind to DNA nor to associated proteins. In order to exert a genotoxic effect, a substance should be able to bind to DNA and/or to proteins, which biphenyl seems not to be capable of. Finally, in the available carcinogenicity studies, non-genotoxic mechanisms seem to underlie the observed carcinogenic effects. In conclusion, it can be assumed that biphenyl is not genotoxic in vivo.


Short description of key information:
Genotoxicity in vitro:
Mutagenicity in bacteria:
Brams et al. (1987), Chung and Adris (2003), Cline and McMahon (1977), Glatt et al. (1992), Haworth et al. (1983), Ishidate et al. (1984), NTP (1979), and Pagano et al. (1983) indicated that biphenyl does not present mutagenic activity in bacteria.
Chromosome aberration:
Ishidate and Odashima (1977) and Ishidate et al. (1984) indicate that biphenyl does not significantly increase chromosome aberration in the absence of metabolic activation.
Mouse lymphoma assay:
Wangenheim and Bolcsfoldi (1988) observed no significant mutagenic effects without metabolic activation. Not too much weight should be given to the weak positive results in the presence of metabolic activation.
Unscheduled DNA synthesis:
The results of all available studies were negative and used as supporting information for the in vitro genotoxicity endpoint.
DNA repair:
The results of all available studies were negative and used as supporting information for the in vitro genotoxicity endpoint.
Gene mutation assay:
The study of Glatt et al. (1992) was used as supporting information for the in vitro genotoxicity endpoint.

Genotoxicity in vivo:
Biphenyl tested negative in a mouse micronucleus test, which was assigned a Klimisch 1 score and considered as the key study (Gollapudi et al., 2007). These results were supported by two chromosome aberration assays (Johnston et al., 1976; Kawachi et al., 1980). A Comet assay (Sasaki et al., 1997) yielded positive results but not too much weight should be given to these results as explained in the discussion.

In conclusion, biphenyl should not be considered as a substance exerting genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Taken together all data on in vitro genotoxicity, biphenyl is regarded as negative both with and without metabolic activation systems for mutagenicity and clastogenicity. In reliable in vivo experiments, biphenyl tested as negative. This is strengthened by the non-genotoxic modes of action in carcinogenicity studies, where species-specific and sex-specific carcinogenic effects were observed, not related to binding to DNA or specific proteins. In conclusion, biphenyl does not need to be classified for mutagenic effects.