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Introduction

1. 2-Phenylphenol and its sodium salt are broad spectrum fungicides that are approved in the UK for use as wood preservatives. They are also used as surface biocides in a number of areas. The COM have advised on the mutagenicity of these compounds, specifically in the context of the mechanism of the bladder tumours seen in male rats fed high doses of these compounds, on a number of occasions, the most recent being in 1997. Significant new data are now available. Furthermore an EU review of the use of 2-phenylphenol and its sodium salt in wood preservation is shortly to be initiated under the Biocidal Products Directive (98/8/EC). It would thus be timely to update the COM view on the mutagenicity of these compounds. This information would be helpful in developing the UK position with regard to the EU in the context of the Biocidal Products Directive.

2. The advice of the COM was thus sought on the mutagenicity of 2-phenylphenol and its sodium salt, and specifically whether the induction of bladder tumours at high dose levels in male rats in the chronic bioassay is likely to have arisen from a genotoxic mechanism. If this were the case the risk assessment would need to adopt a non-threshold approach.

Background

3. The COM gave detailed consideration to a comprehensive review of the mutagenicity of 2-phenylphenol in 1992 and agreed the following conclusions:-

i. Data from several assays to investigate the ability of 2-phenylphenol or its sodium salt to produce gene mutation in Salmonella typhimurium were consistently negative.

ii. Positive results were obtained in in-vitro metaphase analysis studies in CHO cells. Mouse lymphoma assays in the presence of exogenous metabolic activation also gave positive results in the form of small colonies. These results suggested that a metabolite of 2-phenylphenol had clastogenic potential. These findings conflicted with an earlier in-vitro metaphase analysis in a single study.

iii. Negative results were obtained in bone marrow assays for clastogenicity in vivo and also in germ cells (dominant lethal assay), indicating that any possible clastogenic potential was not expressed in the whole mammal.

iv. Conflicting results appeared to be obtained in in-vivo assays for effects on DNA in bladder epithelium, the target tissue of concern. The Committee therefore wished to see data from a more sensitive in-vivo method to investigate adduct formation. There was insufficient evidence to recommend a departure from the use of the safety factor approach for regulation of this compound at the present time.

[Note not included in the 1992 conclusions. The positive results seen in the in vitro metaphase study in CHO cells were in the presence of an exogenous metabolic activation system.]

4. In the light of this advice the Advisory Committee on Pesticides (ACP) requested further data relating to DNA adduct formation in the urinary bladder epithelium of the male rat with particular emphasis on the dose response relationship at levels known to produce hyperplastic and neoplastic changes in male rats.

5. In 1997 industry provided these data to the registration authority (HSE) which was referred to COM for an opinion. The following conclusions were drawn by the COM:-

The Committee expressed concern regarding the limitations of methodology used in the study entitled "³²P-post-labelling study of technical grade 2-phenylphenol to examine the potential for the formation of DNA adducts in the urinary bladder of the male rat." The Committee requested additional analyses of the bladder epithelial samples from this study using an appropriate sensitive adduct enrichment method for the detection ³²P-postlabelled adducts (namely both nuclease P₁ and butanol extraction) and appropriate control experiments to evaluate the fate of 2-phenylphenol DNA adducts during the extraction and enrichment procedures.

6. The ACP considered the COM opinion, together with additional data from specialist feeding studies/carcinogenicity bioassays in 1998 and concluded that the post-review data requirements had been met and confirmed that a threshold based approach remained relevant for risk assessment purposes.

Consideration of new data published since the comprehensive review in 1992

7. A number of papers relating to the metabolism of 2-phenylphenol and its sodium salt and the production of reactive species were considered together with DNA adduct studies in rat bladder epithelium, mutagenicity data on the quinone metabolites and other data relevant to the mutagenicity of 2-phenylphenol. This new information is summarised below.

Metabolism

8. The metabolic profile of 2-phenylphenol is now well established^{(1, 2, 3)}. Recent data on the metabolism of 2-phenylphenol in the rat and the mouse confirms that at relatively low dose levels (around 20mg/kg bw/day) the compound is metabolised and excreted in the urine as sulphate, or to a lesser extent, the glucuronide conjugate⁽¹⁾. At higher doses increased levels of conjugates of PHQ were present, but very little non-conjugated PHQ or PBQ. Studies on the comparative metabolism in the rat and the mouse indicate only minor differences ⁽²⁾. It is difficult to see how these could explain the marked species difference in the induction of bladder tumours in the male rat and not the mouse. It is possible that localised deconjugation of PHQ glucuronide (or sulphate) may play a role at high dose levels in the male rat⁽²⁾.

Formation of DNA adducts in male rat bladder epithelium

9. Further data are now available on the ability of 2-phenylphenol or its sodium salt to induce DNA adducts in the rat bladder in vivo. The earlier studies had given conflicting results, with negative results using radioactivity detection following a very high acute exposure with ¹⁴C-2-phenylphenol, but positive results using ³²P-postlabelling in a subchronic dietary study using a single high dose level (2%) and examining total bladder DNA (rather than epithelial DNA)^{(4, 5)}.

10. The study considered by the COM in 1997, and criticised because of its insensitivity, has now been published, and a rationale for the chosen methodology was given⁽⁶⁾. The Committee did not agree with this and maintained their view that an appropriate sensitive enrichment method for the detection of ³²P-post labelled adducts should have been used. However a study to investigate both DNA and protein binding in the liver, kidney and bladder of male F344 rats has now been published using the highly sensitive AMS technique⁽⁷⁾. An extensive dose response was investigated, (6 single doses by gavage over the range 15 - 1000mg/kg 2-phenylphenol). A clear dose-related and essentially linear response was seen with protein binding in the liver and kidney, but protein binding was seen in the bladder only at high dose levels (about 500mg/kg and above). There was no evidence for DNA binding at any dose level in bladder tissue.

11. The Committee agreed that the weight of evidence from in-vivo studies is now sufficient to conclude that 2-phenylphenol does not produce significant DNA binding in the male rat bladder. However most of the available data were from short term studies and the one, limited, subchronic study had provided some evidence of adduct formation in bladder DNA. The Committee thus felt that the possibility of prolonged high level exposure producing DNA adducts could not be entirely discounted.

Genotoxicity of PHQ and PBQ

12. A number of studies have investigated the ability of the PHQ or PBQ metabolites to induce chromosome damage, micronuclei or DNA damage in HL60 or V79 cells^(8,9,10). There was also some evidence for the induction of aneuploidy⁽⁸⁾. Positive results were obtained, sometimes only in the presence of arachidonic acid supplementation. Members noted that these cells were readily stimulated to generate an oxidative environment. The relevance of positive data with high concentrations of metabolites rather than the parent compound, was felt to be questionable. Members felt that it was not possible to exclude oxidative damage arising from PBQ or PHQ contributing to the induction of bladder tumours at high dose levels, but they agreed that such effects would not be expected to occur at low dose levels.

Additional In-vivo Mutagenicity Data

13. Members considered data from a study to investigate micronucleus induction cell proliferation and hyperdiploidy in bladder epithelium cells of rats treated with 2-phenylphenol at 2% in the diet⁽¹¹⁾. An increase in micronuclei, but no effect on chromosome number, was reported. However, in view of the limitations of this study, particularly the failure to be able to distinguish micronuclei from cell debris, it was felt that no conclusions could be drawn from the reported micronucleus induction.

14. The Committee also considered the results obtained in a study in rats to investigate DNA damage in stomach, liver, kidney, bladder, brain and bone marrow using a modified version of the COMET assay based on isolated nuclei⁽¹²⁾. The results were suggestive of high dose activity in the stomach, liver, kidney and lung but not the bladder. There were concerns however as to whether the method used at that time would have adequately distinguished between genotoxicity and cytotoxicity, and it was felt that no definite conclusions could be drawn.

Conclusions

15. The Committee agreed the following overall conclusions regarding the mutagenicity of 2-phenylphenol and its sodium salt.

i. Data from several assays to investigate the ability of 2-phenylphenol or its sodium salt to produce gene mutation in Salmonella typhimurium were consistently negative.

ii. Positive results were obtained in in-vitro metaphase analysis studies in CHO cells and also in mouse lymphoma assays in both cases in the presence of an exogenous metabolic activation system. The induction of small colonies in the latter assay is consistent with the compound having clastogenic potential,

iii. Phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), which are metabolites of 2-phenylphenol, PHQ usually present as conjugates, have been shown to produce oxidative DNA damage and single strand DNA breaks in-vitro using HL60 and V-79 cells.

iv. Negative results were obtained in bone marrow assays for clastogenicity in-vivo and also in germ cells (dominant lethal assay), suggesting that any possible clastogenic potential was not expressed in the whole mammal.

v. The weight of evidence from in-vivo studies to investigate 2-phenylphenol binding to DNA in the male rat bladder was negative: a recent study using a highly sensitive AMS techniques was particularly important in this regard. However the possibility of prolonged high level exposure producing DNA adducts cannot be entirely discounted.

vi. Although a contributory role of oxidative DNA damage cannot be excluded when considering the mechanisms of bladder tumour induction in the male rat, this would not be expected to occur at low dose levels.

vii. The committee concluded that it would be reasonable to adopt a threshold based risk assessment for 2-phenylphenol and its sodium salt.

June 2003

References

1. Stouten H Toxicological profile for o-phenylphenol and its sodium salt. J. Appl. Toxicol 18 261-70 (1998).

2. Bartels MJ, McNett DA, Timchalk C et al. Comparative metabolism of ortho-phenylphenate in mouse, rat and man. Xenobiotica 28 579-94 (1998).

3. Appel, KE. The carcinogenicity of the biocide ortho-phenylphenol. Arch Toxicol 74 61 - 71 (2000).

4. Reitz RH, Fox TR, Quast JF et al. Molecular mechanisms involved in the toxicity of orthophenylphenol and its sodium salt. Chem. Biol Interact 43 99-119 (1983).

5. Ushiyama K, Nagai F, Nakagawa A and Kano I. DNA adduct formation by o-phenylphenol metabolite in vivo and in vitro. Carcinogenesis 13 1469-73 (1992).

6. Smith RA, Christensen WR, Bartels MJ et al. Urinary physiologic and chemical metabolic effects on the urothelial cytotoxicity and potential DNA adducts of o-phenylphenol in male rats. Tox. Appl. Pharmacol 150 402 - 13 (1998).

7. Kwok ESC, Buchholz BA, Vogel JS et al. Dose-dependent binding of ortho- phenylphenol to protein but not DNA in the urinary bladder of male F344 rats. Toxicol Appl Pharmacol 159 18-24 (1999).

8. Lambert A and Eastmond DA. Genotoxic effects of the o-phenylphenol metabolites phenylhydroquinone and phenylbenzoquinone in V79 cells. Mut Res 322 243-56 (1994).

9. Murata M, Moriya K, Inove S and Kawanishi S. Oxidative damage to cellular and isolated DNA metabolites of a fungicide ortho-phenylphenol. Carcinogenesis 20 851-7 (1999).

10. Henschke P, Almstadt E, Luttgert S and Appel KE. Metabolites of the biocide o-phenylphenol generate oxidative DNA lesions in V79 cells. Arch. Toxicol 73 607-10 (2000).

11. Balakrishnan S, Uppala PJ, Rupa DJ et al. Detection of micronuclei, cell proliferation and hyperdiploidy in bladder epithelial cells of rats treated with o- phenylphenol. Mutagenesis. 17 89-93 (2002).

12. Sasaki YF, Saga A, Akasaka M et al. In-vivo genotoxicity of ortho-phenylphenol, biphenyl and thiobendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay. Mut. Res. 395 189 - 98 (1997).

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