Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro data:

2-Phenylphenol (OPP) was non-mutagenic in the majority of bacterial tests (San, R. H. C. and Springfield, K. A., 1989; Pagano, G. et al., 1988; Shirasu, Y. et al., 1978; Moriya, M. et al., 1983; Probst, G. S. et al., 1981; McMahon, R. E. et al., 1979; Cline, J. C. and McMahon, R. E., 1977; Ishidate, M. Jr et al., 1984; National Toxicology Program, 1986a; Bomhard, E. M. et al., 2002 and Brusick, D., 2005). In mammalian cells, OPP was non-mutagenic in the CHO-HGPRT assay (Brendler, S., 1992; Bomhard, E. M. et al., 2002 and Brusick, D., 2005) and mutagenic at cytotoxic concentrations after metabolic activation in the sensitive TK+/– assay in L5178Y mouse lymphoma cells (Harbell, J. W., 1989; Bomhard, E. M. et al., 2002 and Brusick, D., 2005).

In the human RSa cell strain, OPP induced mutations that gave rise to ouabain resistance (Suzuki, H. et al., 1985; Bomhard, E. M. et al., 2002 and Brusick, D., 2005). However, this test system not commonly accepted or validated and therefore, the result has to be considered carefully.

In some mammalian cell lines, OPP caused micronucleus formation and sister chromatid exchanges, but no chromosomal aberrations (Tayama, S. et al., 1989; Ishidate, M. Jr et al., 1984; Tayama-Nawai, S. et al., 1984; Ishidate Jr., M. et al., 1988; Tayama, S. and Nakagawa, Y., 1991; National Toxicology Program, 1985; Bomhard, E. M. et al., 2002 and Brusick, D., 2005).

No unscheduled DNA synthesis was evident in primary rat hepatocytes treated with OPP (Probst, G. S. et al., 1981; Bomhard, E. M. et al., 2002 and Brusick, D., 2005).

Induction of DNA single-strand breaks could be detected after treatment of V79 cells with the OPP metabolites PHQ and PBQ but not after treatment with OPP itself (Henschke, P. et al., 2000; Bomhard, E. M. et al., 2002 and Brusick, D., 2005).

In vivo data:

OPP did not produce dominant lethal mutations in gametes of male mice (Kaneda, M. et al., 1978) and was not clastogenic in the chromosome aberration test conducted in bone marrow cells (Shirasu, Y. et al., 1978). Furthermore, OPP was negative in the Sex-Linked Recessive Lethal Test in Drosophila melanogaster (National Toxicology Program, 1986b).

Ambiguous results were obtained from in vivo Comet assays in mice. One study found evidence for transient DNA damage in stomach, liver, bladder, lung and kidney cells but not in bone marrow and brain cells after a single oral dose of 2000 mg/kg that caused no mortality (Sasaki et al., 1997). These results could not be reproduced in a more recent and GLP compliant study (Brendler-Schwaab, S., 2000; Bomhard, E. M. et al., 2002 and Brusick, D., 2005) where two out of twelfe mice died after a single oral dose of 2000 mg/kg but DNA tailing from liver and kidney cells was not higher than in controls. The article by Sasaki et al. has areas where technical aspects of the assay may be questioned. This refers to, for example, purity of the compound, dose selection, adequate controls, toxicity in the whole animal as well as cytotoxicity measurements in the organs. This may have led to erroneous conclusions on the genotoxicity of certain compounds, including OPP. In the repeat study these pitfalls were avoided. OPP induced strong clinical symptoms in the highest dose of 2000 mg/kg, which were not reported in the Sasaki article. Instead of cell nuclei intact organ cells were isolated from liver and kidneys therefore making it possible to determine cytotoxicity in these cells. In addition, a concurrent positive control was used. In this respect, there is more confidence in the negative result of OPP in the Comet assay by Brendler-Schwaab, S. (2000).

A micronucleus test in vivo is available for OPP (Balakrishnan, S. and Eastmond, D.A., 2006). Fisher 344 rats were daily treated with the test item at dietary doses of 2000, 4000, 8000, or 12,500 ppm, respectively, for 15 days. Animals were evaluated for micronuclei formation in urinary bladder epithelial cells. Cytotoxicity in the target tissue indicated as increased cell proliferation was examined by means of BrdU incorporation. For comparison, further animals were treated with 8000 ppm OPP and bone marrow cells were evaluated for OPP-induced micronuclei. Increased micronuclei formation in urinary bladder epithelial cells was observed only in male F344 rats dosed with 8000 and 12,000 ppm OPP, which were shown to produce cytotoxic effects in the target tissue. At the same time, bone marrow cells of animals treated with 8000 ppm OPP did not show increased micronuclei formation. No positive control group was included into the test to clarify whether the test item reaches the bone marrow. However, Bomhard, E.M. et al. (2002) report, that there are toxicokinetic data, allowing the conclusion that OPP, as well as ist sodium salt SOPP and their metabolites, reach the bone marrow in sufficient quantities. Thus, the test item was concluded to be not clastogenic under the conditions of this test.

DNA adducts were examined in urinary bladders of male rats after subchronic treatment with OPP (Christenson, W.R., Wahle, B.S. and Cohen, S.M., 1996;Bomhard, E. M. et al., 2002 and Brusick, D., 2005). Male CDF[F-344]/BR rats were given OPP at dietary levels of 800, 4000, 8000, and 12,500 ppm for 13 weeks. During weeks 12-13 and 13-14 of the study, urine was collected for metabolite and general urinalysis determinations, respectively. In addition, urinary bladders were collected during week 14 to perform a 32P-postlabelling analysis for the DNA adduct analysis on the urothelium, while histopathological evaluations included determination of a BrdU-labelling index as well as light and scanning electron microscopy (SEM only on 0 and 8000 ppm group). ESI-LC/MS and GC/MS analysis revealed that glucuronide and sulphate conjugates of OPP and the hydroxylated metabolite, 2,5-phenylhydroquinone (PHQ), were the major urinary metabolites. For all dose groups, the major conjugate present was the sulphate conjugate of OPP. Minute levels of free OPP and PHQ were observed in all dose groups, with free PHQ comprising 0.6-1.5% of the total metabolites measured. Only at 8000 and 12,500 ppm, signs of systemic toxicity were noted, indicated by reduced body weights without changes in food consumption. Animals of these dose groups showed simple hyperplasia of the urothelium at histopathological examination significant bladder changes during SEM analysis. Cell proliferation of the bladder epithelium, examined by means of BrdU-labelling index, was also increased in these animals. 32P-postlabelling of rat urothelial DNA did not show any evidence for formation of OPP-DNA adducts. Thus, OPP caused an increase of mitotic activity and hyperplasia of the urothelium at dose levels ≥ 8000 ppm that also caused evident toxicity. As no DNA adducts were formed by OPP or its metabolites, genotoxicity caused by direct interaction of OPP or its metabolites with DNA is unlikely.

Bomhard, E. M. et al.(2002) and Brusick, D. (2005) summarisevarious further studies investigating genotoxicity of OPP, its sodium salt SOPP or their metabolites in vivo. They come to the conclusion thatOPP does not possess a clastogenic potential relevant under in vivo conditions. The mostly slight increases in chromosomal aberrations after treatment of different cell lines essentially under S9 mix activation in vitro occurred at concentrations that were severely toxic. On the basis of the upper cytotoxicity limit defined in current guidelines, the results of most of these studies would be estimated as unreliable. Bomhard, E. M. et al. (2002) suggest that the induction of chromosome aberrations could be inhibited by the addition of glutathione or cysteine. The various in vivo studies looking for chromosomal aberrations under different, partly longterm high-dose treatment conditions gave no indication of a clastogenic effect in the bone marrow of rats and mice. Although the bone marrow is not the target of carcinogenicity, the available toxicokinetic data allow the conclusion that OPP, its sodium salt SOPP and their metabolites have reached it in sufficient quantities. The increase in micronuclei in the urinary bladder after high-dose feeding of OPP and SOPP (and NaCl) over 14 days does not contradict the aforementioned thesis. Under these conditions they could have developed as a secondary response to toxic changes (Bomhard, E. M. et al., 2002).

Metabolites:

OPP or its hydroxylated metabolite PHQ did not cause DNA damage when injected directly into the urinary bladder of rats. In contrast, the oxidation product of PHQ, PBQ, produced DNA damage. A comprehensive evaluation of genotoxicic properties of metabolites of OPP is presented by Bomhard, E. M. et al. (2002) and Brusick, D. (2005).

Overall evaluation:

Bomhard, E. M. et al. (2002) and Brusick, D. (2005) summarise and extensively discuss numerous genotoxicity studies in vitro and in vivo conducted with OPP, its sodium salt SOPP or any of their enzymatic or non-enzymatic breakdown products. More than 130 studies are available to determine if OPP, SOPP or its metabolites directly react with DNA to induce mutation, changes in chromosome structure or number, DNA repair, or nonspecific DNA damage including strand breakage or covalent binding. Genotoxicity data for OPP and SOPP are not only numerous but heterogeneous, requiring weight-of-evidence methods to arrive at a conclusion regarding their genotoxic properties and potential. Evidence derived from the available studies leads to the conclusion that study results showing OPP/SOPP directly interacting with DNA are equivocal. Clastogenicity was the most consistent type of genetic toxicity produced by OPP/SOPP (and their breakdown products) and was consistently associated with other intracellular pre-neoplastic toxicity produced at super-threshold concentrations. The weight of evidence from the combined database supports the hypothesis that OPP/SOPP-induced DNA damage is a threshold-dependent response associated with target tissue toxicity, most likely induced by their breakdown products phenylhydroquinone and phenylbenzoquinone. It is possible that this threshold-dependent clastogenicity could contribute to the carcinogenic mode of action for OPP or SOPP (Brusick, D., 2005).

This view is in general agreement with the evaluations of FAO-WHO (1999), US-EPA (2005) and EU EFSA (2008) which come to the overall conclusion that OPP is not genotoxic.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vivo and in vitro genetic toxicity data refer to different endpoints and all key studies were negative. The selected key studies are the most adequate and reliable ones based on the overall assessment of quality. Most key studies are GLP and guideline compliant or were at least conducted similar to current guidelines.

Short description of key information:
Gene mutation in bacteria in vitro (Ames test): negative with and without metabolic activation in all strains tested (similar to OECD 471).
Gene mutation in mammalian cells in vitro (HGPRT test): negative with and without metabolic activation in CHO-WB1 cells (OECD 476).
Cytogenicity in mammalian cells in vitro (Chromosomal aberration): negative with metabolic activation in CHO-K1 cells (similar to OECD 473).

DNA damage in vivo (Alkaline Comet Assay): negative in mouse kidney and liver (no guideline available).
Micronucleus Test in vivo: negative in bone marrow and in urinary bladder epithelium (similar to OECD 474).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

2-phenylphenol does not meet the criteria to be classifed for genetic toxicity according to the criteria of EU Directive 67/548/EEC or Regulation (EC) No 1272/2008.