1.1.1. Nomenclature

1.1.2. Structural and molecular formulae and relative molecular mass

ortho-Phenylphenol

Sodium ortho-phenylphenate

1.1.3. Chemical and physical properties of the pure substance

1.3.1. Natural occurrence

ortho-Phenylphenol and sodium ortho-phenylphenate are not known to occur naturally.

1.3.2. Occupational exposure

According to the 1981–83 National Occupational Exposure Survey (National Institute for Occupational Safety and Health, 1998), approximately 620 000 and 56 000 workers in the United States were potentially exposed to ortho-phenylphenol and sodium ortho-phenylphenate, respectively. Occupational exposure to ortho-phenylphenol and/or its salt may occur during their production and use as chemical intermediates, fungicides, germicides, preservatives and disinfectants.

1.3.3. Environmental occurrence

According to the Environmental Protection Agency Toxic Chemical Release Inventory for 1987, 1400 kg ortho-phenylphenol were released into the air, 120 kg were discharged into water and 110 kg were released onto the land from manufacturing and processing facilities in the United States. By 1996, 1900 kg were released into the air and 110 kg were released onto the land (National Library of Medicine, 1998).

ortho-Phenylphenol has been found in some groundwater and drinking-water samples and in some fruits and juices (IARC, 1983).

3.1. Oral administration

Mouse: Groups of 50 male and 50 female B6C3F₁ mice, six weeks of age, were fed diets containing sodium ortho-phenylphenate (97% pure) at concentrations of 0, 0.5, 1 or 2%; the actual concentrations achieved were 0.41, 0.82 and 1.6%. The mice were fed for 96 weeks and then continued on control diet for an additional eight weeks (total experimental period, 104 weeks). The survival rate of males but not females at the high dose was decreased, and the body weights of the males were significantly reduced throughout the experiment, while those of females were reduced from week 13. The body weights of females at 1% were reduced from week 26 and those of females at 0.5% from week 38. The mean body weight of males at 0.5% was significantly reduced in weeks 1–90. Five haemangiomas and five leiomyosarcomas of the uterus were found in female controls but only one haemangioma was found in treated females. Males given 1% had increased incidences of haemangiosarcomas of the liver, with none in controls and three, five and three in the three treated groups, respectively; and hepatocellular carcinomas were observed in 4, 9, 13 and 14 males and 4, 5, 7 and 0 females given 1 or 2%; however, the authors concluded on the basis of data for their historical controls that there was no treatment-associated carcinogenic effect (Hagiwara et al., 1984).

Rat: Groups of 20–24 male F344/DuCrj rats, 38–39 days of age, were given ortho-phenylphenol (purity > 98%) in the diet at concentrations of 0 (control), 0.625, 1.25 or 2.5% for 91 weeks. Rats at the high dose consumed significantly less food and had a 17–24% lower weight gain compared to controls. The numbers of rats with bladder tumours were reported as: 0/24, 0/20, 23/24 (p < 0.001) and 4/23 in the controls and rats at the three doses, respectively. The 23 bladder tumours in rats at 1.25% were described as three papillomas, 15 non-invasive carcinomas and five invasive carcinomas (Hiraga & Fujii, 1984).

Groups of 50 male and 50 female Fischer 344/DuCrj rats, five weeks of age, were fed a pelleted diet containing 0, 0.7 or 2% (males) or 0, 0.5 or 1% (females) sodium ortho-phenylphenate (purity, 95.5% with 3.75% water, 0.72% free alkali as sodium hydroxide and 0.028% organic substances) for 104 weeks followed by two weeks on basal diet. Groups of 25 rats of each sex were fed the test diets containing 0, 0.25, 0.7 or 2% (males) and 0, 0.25, 0.5 or 1% (females) for 104 weeks and then basal diet until they died or were sacrificed in a moribund state. The mean body weights of males at 2% and females at 1% were lower than that of the controls throughout the first study; in the second study, males at 2% also showed lower mean body weights, but the growth of females at 1% after treatment was stopped was comparable to that of controls. The survival rate of the males but not females at the high dose decreased during weeks 50–100. The total numbers of rats with urinary bladder tumours were 0, 2 and 47 among males and 0, 1 and 4 among females treated for 106 weeks. In the second study, the numbers of rats with bladder tumours were 0, 0, 3 and 23 among males and 0, 0, 0 and 2 among females. Most of the tumours were carcinomas (Fujii & Hiraga, 1985).

3.2. Administration with known carcinogens or modifying factors

Mouse: Groups of 50 male and 50 female Swiss CD-1 mice, seven to eight weeks of age, were treated by dermal application of 55.5 mg ortho-phenylphenol (purity, > 99%, with water as the major impurity) in 0.1 mL acetone on three days per week for 102 weeks; with a single dermal application to the dorsal interscapular region of 0.05 mg dimethylbenz[a]anthracene (DMBA) in 0.1 mL acetone and then, one week later, with dermal applications at the site of DMBA application of either acetone (vehicle), ortho-phenylphenol (55.5 mg in 0.1 mL acetone) or 0.005 mg 12-O-tetradecanoylphorbol-13-acetate (TPA) in 0.1 mL acetone on three days per week for the remainder of the experiment; or dermal applications of acetone alone three times per week. All groups were treated for 103 weeks except males (85 weeks) and females (74 weeks) given DMBA plus TPA, which were killed before the end of the study because of the large number of deaths. The mean body weights of male mice receiving ortho-phenylphenol and those given DMBA plus ortho-phenylphenol were generally 5–10% lower than those receiving acetone after week 44. The mean body weights of females given DMBA plus TPA were higher than those of the other female groups during the first year of the study. The mean body weights of the remaining groups were similar to those of the corresponding controls. The survival rates of male and female mice given DMBA plus TPA were significantly lower than that of the controls, but the rates of all other groups were not significantly different from those of controls. The incidences of squamous-cell papillomas and carcinomas of the skin in males and females given DMBA plus TPA (18/50 and 31/50) were significantly greater than those of mice given DMBA (5/50 and 7/50) or DMBA plus ortho-phenylphenol (5/50 and 5/50). The incidence of basal-cell tumours in males given DMBA plus ortho-phenylphenol (4/50) was significantly greater than that in the controls (0/50) but was not significantly increased over that of mice given DMBA alone (1/50). No significant increase in the incidence of basal-cell tumours or carcinomas was observed in females given DMBA plus ortho-phenylphenol over that in mice given DMBA alone. No basal-cell tumours were seen in mice given either ortho-phenylphenol or the vehicle alone. The incidences of squamous-cell papillomas and carcinomas in mice given ortho-phenylphenol were not increased over those in vehicle controls, and those in mice given DMBA plus ortho-phenylphenol were no higher than those in mice given DMBA alone. No squamous-cell papillomas or carcinomas occurred in mice given ortho-phenylphenol or acetone alone. The authors concluded that ortho-phenylphenol is not a complete carcinogen or promoter when administered by the dermal route to mice (National Toxicology Program, 1986).

Eight groups of 20 female CD-1 mice, eight weeks of age, received dermal applications of 10 mg sodium ortho-phenylphenate (technical grade Dowicide A; purity, 97%) in 0.1 mL dimethyl sulfoxide (DMSO), followed by TPA (10 µg in 0.1 mL acetone); 10 mg sodium ortho-phenylphenate followed by acetone; 10 µg DMBA in 0.1 mL DMSO followed by 5 mg sodium ortho-phenylphenate in 0.1 mL acetone; DMSO followed by sodium ortho-phenylphenate; DMBA followed by TPA; DMBA followed by acetone; DMSO followed by TPA; or DMSO followed by acetone. The initiation treatment was given twice weekly for five weeks, and the promotion treatment twice weekly for 47 weeks. The survival rate was significantly decreased only in the group treated with DMBA plus TPA, because of the growth of skin tumours. All mice survived beyond 26 weeks of the experiment. The numbers of mice in the eight groups with skin tumours were 1, 0, 15, 0, 20, 5, 2 and 0, respectively, and the average numbers of skin tumours per mouse were 0.05, 0, 1.25, 0, 2.9, 0, 0.30, 0.15 and 0, respectively. The incidences and numbers of tumours were significantly increased (p < 0.01) in mice receiving DMBA plus sodium ortho-phenylphenate and in those receiving DMBA plus TPA, indicating that sodium ortho-phenylphenate can promote but not initiate skin tumours in mice (Takahashi et al., 1989).

Rat: Two groups of 30 male Fischer 344 rats, five weeks of age, were given drinking-water treated with 0.01% N-nitrosobutyl(4-hydroxybutyl)amine (NBHBA), while a third received untreated drinking-water for four weeks. Then, one of the treated groups and the untreated group received 32 weeks of treatment with 2% sodium ortho-phenylphenate (purity, 97%) in the diet, and the other nitrosamine-treated group received basal diet. The only evidence of toxicity was a slight retardation in the growth of rats given sodium ortho-phenylphenate. The incidences of bladder carcinoma were 2/28, 1/30 and 0/29, respectively, in the groups given NBHBA plus sodium-ortho-phenylphenate, NBHBA alone and the phenylphenate alone, and the incidences of papillomas were 9, 8 and 5, respectively. In a second experiment, with the same overall protocol, 30 rats received NBHBA followed by 2% sodium ortho-phenylphenate in the diet; 30 rats received 2% ortho-phenylphenol (purity, 98%) in the diet; 30 rats received NBHBA followed by basal diet; 15 rats received untreated drinking-water followed by sodium ortho-phenylphenate; and 15 rats received untreated drinking-water followed by ortho-phenylphenol. The only evidence of toxicity was mild growth retardation with sodium ortho-phenylphenate and ortho-phenylphenol and brown discolouration of the external genital area due to continuous micturition. The numbers of rats with bladder carcinoma were 27, 6, 2, 1 and 0 in the five groups, respectively. The authors concluded that sodium ortho-phenylphenate, but not ortho-phenylphenol, promotes bladder tumours (Fukushima et al., 1983).

Groups of 30 male Fischer 344 rats, six weeks of age, were fed diets containing 2% sodium ortho-phenylphenate [purity not stated] after treatment with either 0.01% NBHBA in the drinking-water or untreated drinking-water for four weeks; a group of 10 rats received NBHBA only followed by untreated diet. The experiment lasted 68 weeks. In another experiment, groups of 30 rats were fed 2% ortho-phenylphenol [purity not stated] in the diet after either NBHBA pretreatment or untreated water. A group of 30 rats served as controls and were treated with NBHBA only. Slight retardation of growth was seen, which was more pronounced with ortho-phenylphenol than with sodium ortho-phenylphenate. The incidences of bladder carcinoma were 15/29 with NBHBA followed by sodium ortho-phenylphenate, 3/10 with NBHBA alone and 6/28 with sodium ortho-phenylphenate alone. Following treatment with ortho-phenylphenol after NBHBA, 21/28 rats developed bladder cancer, whereas none developed bladder cancer without prior NBHBA treatment; of the rats treated with NBHBA alone, 19/29 developed bladder carcinoma. The authors concluded that sodium ortho-phenylphenate, but not ortho-phenylphenol, promotes bladder carcinogenesis. In a further experiment, sodium ortho-phenylphenate was administered in the diet at various concentrations and tumour incidences were calculated in 10 rats at each dose at 36 weeks and in 7–9 rats at 104 weeks. The incidences of bladder carcinoma were 0/10 at 36 weeks and 2/5 at 104 weeks with 2% sodium ortho-phenylphenate; at 104 weeks, two rats also had papillomas. No tumours were found in groups of 10 rats given sodium ortho-phenylphenate at concentrations of 1, 0.5 or 0.25% in the diet or in the untreated controls (Fukushima et al., 1985). [The Working Group noted the small numbers of animals in these experiments, especially in the two-year study.]

As part of a bioassay of 17 environmental chemicals, sodium ortho-phenylphenate [purity not stated] was fed for 20 weeks in the diet of male Fischer 344 rats which had received 0.05% NBHBA in the drinking-water for two weeks. One week after the start of administration of sodium ortho-phenylphenate, the lower section of the left ureter was ligated. The rats were six weeks of age at the beginning of the experiment, and the experiment lasted 24 weeks. A second group received NBHBA and one week later underwent unilateral ureteral ligation, and a third group was treated with sodium ortho-phenylphenate with unilateral ureteral ligation without prior NBHBA treatment. In the first group, 7/19 rats developed bladder papillomas in contrast to 1/15 in the group not pretreated with NBHBA. No bladder tumours occurred in rats given only NBHBA (Miyata et al., 1985). [The Working Group noted the complexity of the experimental protocol and the short period of administration of the chemical.]

Six groups of 15 male and 15 female Fischer 344 rats, five weeks of age, were either untreated; received 0.2% thiabendazole (purity, 98.5%) in the diet; received 1% sodium ortho-phenylphenate (purity, 95.5% with 3.75% free water, 0.72% free sodium hydroxide and 0.028% organic substances and no detectable residues of heavy metals) in the diet; received 2% sodium ortho-phenylphenate alone; received 1% sodium ortho-phenylphenate and 0.2% thiabendazole; or received 2% sodium ortho-phenylphenate plus 0.2% thiabendazole. Treatment was continued for 65 weeks except for males and females receiving 1% sodium ortho-phenylphenate. The mean body weights of animals of each sex were significantly reduced throughout the study. The survival rates of males given 2% of the phenylphenate with or without thiabendazole and females given 1% phenylphenate and thiabendazole were slightly but not statistically significantly reduced. The incidences of bladder papillomas plus carcinomas in the six groups were 0, 1, 0, 15, 12 and 14, respectively, for males and 0, 0, 0, 2, 1 and 12, respectively, for females. The authors concluded that sodium ortho-phenylphenate is carcinogenic to male and female rats at 2% in the diet but not at 1% and that thiabendazole enhanced its tumorigenicity in females (Fujii et al., 1986). [The Working Group noted the small numbers of animals in each group and the relatively short length of the experiment.]

Groups of 30–31 male Fischer 344/CuCrj rats, four weeks of age, received ortho-phenylphenol at a concentration of 1.25% in the diet; received 1.25% ortho-phenylphenol in the diet plus 0.4% sodium bicarbonate in the drinking-water; received 2% sodium ortho-phenylphenate in the diet; received 2% sodium ortho-phenylphenate in the diet plus ammonium chloride in the drinking-water; or were untreated. Treatment was continued for 26 weeks. The water consumption of rats given 1.25% ortho-phenylphenol plus sodium bicarbonate or 2% of the sodium salt plus ammonium chloride was increased in comparison with the groups receiving the phenylphenol or its salt alone, and that of rats given the sodium salt was increased as compared with the control group. Urinary pH measured at week 25 of the experiment was 6.4, 7.0, 7.0, 5.9 and 6.4 for the five groups, respectively. There was no effect on survival. A significant decrease in body weight was seen only in the group given 2% sodium salt plus ammonium chloride. The numbers of rats with bladder papillomas were 12, 20, 21, 3 and 0, respectively, in the five groups. Only one bladder carcinoma was seen in this experiment, in a rat given 2% sodium ortho-phenylphenate (Fujii et al., 1987). [The Working Group noted the short duration of the experiment and the relatively small number of animals evaluated.]

Groups of 30–31 male Fischer 344 rats, six weeks of age, received 2% sodium ortho-phenylphenate (purity, 72.02%, with 26.78% water and 1.25% sodium hydroxide) in the diet; received 1.25% ortho-phenylphenol (purity, 99.45% with 0.55% inert ingredients) in the diet plus 0.64% sodium bicarbonate; received 1.25% ortho-phenylphenol plus 0.32% sodium bicarbonate; received 1.25% ortho-phenylphenol plus 0.16% sodium bicarbonate; received 1.25% ortho-phenylphenol; received 0.64% sodium bicarbonate; or served as untreated controls. The experimental period was 104 weeks. There was no significant effect on survival, but body weights were decreased by > 10% in comparison with controls at the end of the experiment, except in the group given sodium bicarbonate, which had a decrease of approximately 7%. The incidences of bladder carcinoma were 12/29, 9/29, 4/29, 4/26, 0/27, 1/28 and 0/27 in the seven groups, respectively. The incidences in the first four groups were significantly increased in comparison with controls or with rats given ortho-phenylphenol alone. In associated studies, increasing doses of sodium bicarbonate increased the urinary pH in the groups given ortho-phenylphenol plus sodium bicarbonate, so that the two highest doses of sodium bicarbonate produced a urinary pH similar to that produced by sodium ortho-phenylphenate (Fukushima et al., 1989).

Groups of 27 male Fischer 344 rats, five weeks of age, were subjected to freeze-ulceration of the bladder, and two weeks later were given a diet containing 0.5% sodium ortho-phenylphenate [purity not stated] for 76 weeks; were subjected to freeze-ulceration and 12 weeks later given 0.5% sodium ortho-phenylphenate in the diet for 66 weeks; were subjected to freeze-ulceration and then given control diet for 78 weeks; were sham-operated and two weeks later given 0.5% sodium ortho-phenylphenate; or were sham-operated and given control diet for 78 weeks. There were no significant differences between the groups in terms of body-weight gain or survival. Three rats in the first group developed a bladder papilloma, and one in the second group developed a bladder carcinoma (p < 0.24). In a second experiment, 25 male Fischer 344 rats were subjected to freeze-ulceration of the bladder and six weeks later given 2% sodium ortho-phenylphenate in the diet for 30 weeks; 25 rats underwent freeze-ulceration and six weeks later were given 1% sodium ortho-phenylphenate; 20 rats underwent freeze-ulceration and were given control diet for 36 weeks; and 20 rats were sham-operated and six weeks later were given 2% sodium ortho-phenylphenate in the diet for 30 weeks. The growth of rats that received 2% of the compound with or without freeze-ulceration was significantly decreased when compared with freeze-ulceration alone. Seven rats subjected to freeze-ulceration and given the phenylphenate developed bladder papillomas, and 12 other rats had carcinomas (total bladder tumour incidence, 76%). One rat given the compound alone had a bladder carcinoma (5%). No bladder tumours occurred in the other groups (Hasegawa et al., 1989).

A group of 20 male Fischer 344 rats, six weeks of age, were given sodium ortho-phenylphenate [purity not stated] at a concentration of 2% in the diet for 16 weeks after pretreatment with 20 mg/kg bw N-methyl-N-nitrosourea (MNU) administered intraperitoneally (dissolved shortly before each treatment) twice a week for four weeks. A second group of 23 rats received MNU without further treatment, and a third group of 14 rats received sodium ortho-phenylphenate without MNU. The tumour incidences in the thyroid, forestomach, kidney and urinary bladder were enhanced in the group given MNU plus sodium ortho-phenylphenate when compared with those given MNU only. No tumours were observed in the thyroid, lung, liver, pancreas, oesophagus, fore-stomach, small intestine, kidney or urinary bladder of rats that received sodium ortho-phenylphenate only, but four of these rats had papillary or nodular hyperplasia of the urinary bladder (Uwagawa et al., 1991). [The Working Group noted the complex treatment protocol of this experiment, the small number of animals and the short treatment period.]

3.3. Carcinogenicity of metabolites

Mouse: Ten groups of 25 female CD-1 mice, eight weeks of age, were treated as follows: the first seven groups received initiation treatment for five weeks and promotion for 34 weeks, with a one-week period of no treatment between the two phases; groups 8–10 received continuous treatment for the entire 40 weeks. Group 1 received 10 µg DMBA in 0.1 mL DMSO twice a week, followed by 2.5 µg TPA in 0.1 mL acetone twice a week; group 2 received 2 mg 2-phenyl-1,4-benzoquinone [purity unspecified] in DMSO, followed by TPA in acetone; group 3 received 20 mg 2,5-dihydroxybiphenyl in DMSO, followed by TPA in acetone; group 4 received DMSO followed by TPA in acetone; group 5 received DMBA followed by 1 mg 2-phenyl-1,4-benzoquinone; group 6 received DMBA followed by 10 mg 2,5-dihydroxybiphenyl; group 7 received DMBA followed by acetone; group 8 received 1 mg 2-phenyl-1,4-benzoquinone; group 9 received 10 mg 2,5-dihydroxybiphenyl; and group 10 received DMSO (0.1 mL). There were no differences in survival except for a reduction in group 1 due to extensive growth of skin tumours; in addition, five mice in group 1 were accidently lost during the early period of the experiment. There was no statistically significant increase in the incidence of skin tumours in any group except group 1 when compared with mice receiving DMBA only. No skin tumours were observed in group 8 or 9 (Sato et al., 1990).

Rat: Seven groups of 20 female Fischer 344 rats, six weeks of age, were treated in two phases of 5 and 31 weeks. In the first phase, the chemicals were instilled intravesically twice a week for five weeks; during the second phase of 31 weeks, the animals were fed the appropriate dietary treatments. 2-Phenyl-1,4-benzoquinone (prepared fresh; purity, > 99%) was instilled into the bladders of groups 1 and 4, phenylhydroquinone [purity not specified] was instilled into those of groups 2 and 5, and saline was instilled into those of groups 3 and 6. The chemicals were dissolved as 0.1% solutions in saline, and a total volume of 0.2 mL was instilled. Sodium saccharin was fed at a concentration of 5% in the diet to groups 1–3, and untreated diet was fed to groups 4–6 in the second phase. Group 7 served as a positive control and was treated with 0.05% NBHBA in drinking-water followed by 5% sodium saccharin. The length of the entire experiment was 36 weeks. There were no significant differences in body-weight gain and no significant effect on survival. The only bladder tumours detected were two papillomas in group 7. Papillary or nodular hyperplasia occurred in 3/18 animals in group 1 and 9/20 rats in group 7, but not in the other groups (Hasegawa et al., 1990a). [The Working Group noted the small number of animals, the short period of administration and the lack of tumours as an end-point.]

4.1.2. Experimental systems

[¹⁴C]ortho-Phenylphenol was administered by gavage to 10 male B6C3F₁ mice at a dose of 15 or 800 mg/kg bw in 0.5% aqueous Methocel^® and to two male and two female Fischer 344 rats at a dose of 28 or 27 mg/kg bw. Urine was collected at 12-h intervals for 24 h (rats) and 48 h (mice) after exposure. After administration of 15 or 800 mg/kg bw, 84 and 98% of the administered ortho-phenylphenol was recovered in the urine of mice and 86 and 89% in that of male and female rats. Sulfation of ortho-phenylphenol was the major metabolic pathway at low doses, accounting for 57 and 82% of the urinary metabolites in male mice dosed with 15 mg/kg bw and rats dosed with 28 mg/kg bw, respectively. Conjugates of 2-phenylhydroquinone accounted for 12 and 5%, respectively. Little or no free ortho-phenylphenol was present in the urine, and no free 2-phenylhydroquinone or 2-phenyl-1,4-benzoquinone was detected in either species. Dose-dependent shifts in metabolism were observed in mice for conjugation of ortho-phenylphenol, suggesting saturation of the sulfation pathway. Dose-dependent increases in total 2-phenylhydroquinone were observed in mice. The authors noted that their findings did not provide a metabolic explanation for the difference in carcinogenicity in rats and in mice (Bartels et al., 1998).

The metabolism of the sodium salt of ortho-phenylphenol was investigated in male and female Fischer 344 rats dosed at 2% in the feed from the age of five weeks for 136 days. Urinary metabolites accounted for 55% of the dose in males and 40% of the dose in females. The main metabolites were ortho-phenylphenol-glucuronide and 2,5-dihydroxybiphenyl-glucuronide. Male rats excreted 1.8 times as much ortho-phenylphenol-glucuronide and nearly eight times as much 2,5-dihydroxybiphenyl-glucuronide as the females (Nakao et al., 1983).

The free metabolites phenylhydroquinone and phenylbenzoquinone were also identified as minor urinary metabolites of sodium ortho-phenylphenate administered at 0.5, 1 or 2% in the diet to male and female Fischer 344/DuCrj rats. The concentration of phenylhydroquinone represented 1/60 of the 2% dose (93 µmol/g diet), while phenylbenzoquinone was excreted only in traces (10–100-fold lower amounts than phenylhydroquinone). The concentration of phenylhydroquinone in the urine of male rats was approximately 25 times greater than that in the urine of female rats (1500 versus 62 nmol/mL) (Morimoto et al., 1989). Metabolism was investigated in the fifth month of this five-month study, mainly to investigate induction of DNA strand breaks (see section 4.5).

Male Fischer 344 rats were given ortho-phenylphenol at doses of 0, 1000, 4000, 8000 or 12 500 ppm (0, 140, 580, 1100 and 1800 mg/m³) in the diet for 13 weeks and placed in urine collection cages overnight. The urinary volume of rats at the two highest doses was increased, with corresponding decreases in osmolality and the concentrations of creatinine and other solutes. The total urinary excretion of ortho-phenylphenol metabolites increased with dose, and the metabolites consisted almost entirely of conjugates of ortho-phenylphenol and 2-phenylhydroquinone; free ortho-phenylphenol and its metabolites accounted for less than 2% of the total excreted metabolites (Smith et al., 1998).

ortho-Phenylphenol was converted to phenylhydroquinone by microsomal cytochrome P450 in vitro. Phenylhydroquinone was oxidized to phenylquinone by cumene hydroperoxide-supported microsomal cytochrome P450, and phenylquinone was reduced back to phenylhydroquinone by cytochrome P450 reductase, providing direct evidence of redox cycling of ortho-phenylphenol (Roy, 1990).

Male rats were given 1000 mg/kg bw ortho-phenylphenol orally, and their bile was collected for 6 h. In addition to the glucuronide conjugates of ortho-phenylphenol and phenylhydroquinone, phenylbenzoquinone and the glutathione conjugate of phenylhydroquinone were identified in the bile, the latter amounting to 4% of the administered dose (Nakagawa & Tayama, 1989).

ortho-Phenylphenol was shown to be converted to phenylhydroquinone by mixed-function oxidases in vitro, and conversion of phenylhydroquinone to phenylbenzoquinone was shown to be mediated by prostaglandin (H) synthetase in the presence of arachidonic acid and hydrogen peroxide as cofactors (Kolachana et al., 1991). The authors suggested that this pathway may play an important role in ortho-phenylphenol-induced bladder and kidney carcinogenesis in rats, since the activity of prostaglandin (H) synthetase is high in the kidney and bladder, the target organs of ortho-phenylphenol.

Over the pH range 6.3–7.6 observed in the urine, phenylhydroquinone was shown to be auto-oxidized to phenylbenzoquinone in vitro, with an average yield of 0.92 ± 0.02. The rate of phenylhydroquinone auto-oxidation increased rapidly at pH above 7 (Kwok & Eastmond, 1997).

4.2.1. Humans

No data were available to the Working Group.

4.2.2. Experimental systems

ortho-Phenylphenol and sodium ortho-phenylphenate induced similar levels of macromolecular binding in the bladder (and also in the liver and kidney) when administered by gavage at a dose of 500 mg/kg bw to groups of four male Fischer 344 rats. The experiment was terminated 17 h after gavage, and macromolecular binding was detected in all tissues, with a marked, non-linear dose–response relationship. Administration of 200 mg/kg bw of ortho-phenylphenol or sodium ortho-phenylphenate did not increase macromolecular binding in the bladder significantly above control values, while ortho-phenylphenol and sodium ortho-phenylphenate at 500 mg/kg bw induced 130-fold and 210-fold increases, respectively (Reitz et al., 1984).

The effects of ortho-phenylphenol and sodium ortho-phenylphenate were investigated in Fischer 344 rats after administration in the diet over 8–24 weeks at a concentration of 2%. Urinary pH and sodium concentrations were increased only by sodium ortho-phenylphenate, which also consistently induced simple (diffuse thickening of the epithelium with four to eight cell layers) and nodular or papillary hyperplasia of the bladder epithelium at all times investigated (8, 16 and 24 weeks) (Fukushima et al., 1986).

Sodium ortho-phenylphenate and ortho-phenylphenol were administered in the diet at a concentration of 2% for four or eight weeks to groups of 10 male Fischer 344 rats. DNA synthesis in the bladder (assessed after four weeks), urinary pH, sodium content, volume and crystalluria were all increased by sodium ortho-phenylphenate but not by ortho-phenylphenol. Furthermore, sodium ortho-phenylphenate but not ortho-phenylphenol induced morphological changes in the urothelium characteristic of those induced by other genotoxic and non-genotoxic bladder carcinogens, including formation of pleomorphic or short, uniform microvilli and ropy or leafy microridges. [The Working Group noted that similar alterations were induced by the bladder tumour promoter sodium-l-ascorbate but not by the parent compound l-ascorbic acid which lacks tumour promoting activity in the bladder.] Sodium ortho-phenylphenate but not ortho-phenylphenol induced hyperplasia in the renal pelvis of rats treated for four weeks. The authors commented that the observed differences might be due to changes in urinary Na⁺ and pH, since sodium ortho-phenylphenate induced natriuresis and urinary alkalinization but ortho-phenylphenol did not (Shibata et al., 1989a,b).

Administration of ortho-phenylphenol at concentrations of 0, 1000, 4000, 8000 or 12 500 mg/kg of diet (ppm) to male Fischer 344 rats for 13 weeks slightly increased the urinary volume and correspondingly decreased its osmolality and creatinine concentration at the two highest doses. Increased urinary solids (precipitate, crystals or calculi) or abnormal crystals were not detected at any dose. At 8000 and 12 500 ppm, increased urothelial hyperplasia of the bladder was seen, with an increased bromodeoxyuridine labelling index and features of increased proliferation, as detected by scanning electron microscopy. In addition, superficial cell necrosis and exfoliation were observed at these doses, indicating that ortho-phenylphenol induced cytotoxicity with subsequent regenerative hyperplasia (Smith et al., 1998).

Species differences in urinary bladder hyperplasia induced by sodium ortho-phenylphenate were investigated in groups of 30 male Fischer 344 rats, B6C3F₁ mice, Syrian golden hamsters and Hartley guinea-pigs. The compound was administered in the diet at a concentration of 2% for 4, 8, 12, 24, 36 or 48 weeks. Simple and nodular or papillary hyperplasia was observed by light microscopy, and pleomorphic microvilli were seen by scanning electron microscopy only in rats, the lesions becoming more marked over time. In mice, guinea-pigs and hamsters, no proliferative lesions were observed. The urinary pH of treated rats was elevated at 12 weeks in comparison with controls, but there was virtually no difference at week 48. The treatment did not affect the urinary pH of animals of the other species, which is normally higher than that of the rat (Hasegawa et al., 1990b).

Male and female Fischer 344 rats were given diets containing 1.25% ortho-phenylphenol or 2% sodium ortho-phenylphenate alone or in combination with 3% sodium bicarbonate or 1% ammonium chloride for eight weeks. Administration of ortho-phenylphenol alone did not cause proliferative effects, but combination with 3% sodium bicarbonate induced marked urothelial hyperplasia in the urinary bladders of both male and female rats, the response being more severe in males. Sodium bicarbonate alone induced only a borderline effect. Sodium ortho-phenylphenate alone significantly increased the incidence of hyperplasia only in males, which was less pronounced than that seen after concomitant treatment with ortho-phenylphenol and sodium bicarbonate. The hyperplastic effect of sodium ortho-phenylphenate in male rat bladders was completely prevented by co-administration of ammonium chloride, indicating the involvement of alkalinization of the urine in the induction of the observed cell proliferation and hyperplasia. Increased urinary pH and sodium concentrations were positively associated with the induction of hyperplasia in males. There was no significant difference between the sexes in terms of pH, but the sodium concentration was elevated only in males treated with sodium ortho-phenylphenate alone. The urinary concentrations of non-conjugated ortho-phenylphenol metabolites (phenylhydroquinone and phenylbenzoquinone) did not correlate with the development of hyperplasia, suggesting that these metabolites are not important for urinary bladder carcinogenesis induced by sodium ortho-phenylphenate (Hasegawa et al., 1991).

Groups of BALB/c mice were given intraperitoneal injections of 600 mg/kg bw sodium ortho-phenylphenate or 100 mg/kg bw phenylbenzoquinone, a metabolite of ortho-phenylphenol. Maximal decreases in the concentrations of protein and non-protein reduced thiols were observed in the bladder to 66–76% of the control values, in the kidney to 26–72% of control values and in the liver, only by phenylbenzoquinone, to 25–44% that of controls. The concentrations of non-protein disulfide and protein disulfide were increased in a similar manner. Increased contents of both protein and non-protein disulfides after administration of sodium ortho-phenylphenate acounted for only 33% of the entire loss of non-protein reduced thiol, so that direct reaction of a metabolite (probably phenyl-2,5-para-benzoquinone) with glutathione probably contributed to the decrease (Narayan & Roy, 1992).

4.3.1. Humans

No data were available to the Working Group.

4.3.2. Experimental systems

Groups of 25–35 Sprague-Dawley rats received 0, 100, 300 or 700 mg/kg bw ortho-phenylphenol (commercial-grade Dowicide^® containing 99.69% ortho-phenylphenol) per day orally in cottonseed oil on days 6–15 of gestation. The fetuses were examined on day 21 of gestation. One female at the high dose died, and the body-weight gain of dams was reduced on days 6–9 and maternal liver weight on day 21. There were no effects on the numbers of implantation sites, live fetuses or resorptions or on litter size or fetal development. Significant increases were seen in the incidence of delayed ossification of the sternebrae, foramina and the bones of the skull in fetuses at the high dose (John et al., 1981).

4.4.1. Humans

No data were available to the Working Group

4.4.2. Experimental systems (see Tables 1–3 for references)

Table 1

Genetic and related effects of ortho-phenylphenol and sodium ortho-phenylphenate.

Table 2

Genetic and related effects of phenylhydroquinone.

Table 3

Genetic and related effects of phenylbenzoquinone.

Incubation of supercoiled pUC18 DNA with phenylhydroquinone, the proximate metabolite of ortho-phenylphenol, produced a strand scission to the linear form that was dose-dependent; in contrast, DNA cleavage by ortho-phenylphenol and its ultimate metabolite phenylbenzoquinone was barely detectable. The hypothesis that oxygen radicals generated in the process of oxidation of phenylhydroquinone are responsible for the DNA cleavage is supported by the finding of inhibition of DNA strand scission by superoxide dismutase, catalase and several oxygen radical scavengers.

ortho-Phenylphenol induced DNA repair in various strains of Escherichia coli, but did not cause DNA cleavage in plasmid DNA. The compound did not induce differential toxicity in Bacillus subtilis. ortho-Phenylphenol was consistently non-mutagenic in tests for reversion in five strains (TA100, TA1535, TA1537, TA1538 and TA98) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli in the presence and absence of metabolic activation; the only exception was a weakly positive response in strain TA1535 in the absence of exogenous metabolic activation, but the addition of metabolic activation from rat and hamster liver eliminated this effect.

It was reported in an abstract that sodium ortho-phenylphenate induced aneuploidy in Aspergillus (Kappas & Georgopoulos, 1975).

ortho-Phenylphenol did not induce sex-linked recessive lethal mutations in Drosophila. It did not induce unscheduled DNA synthesis in cultured rat hepatocytes in the absence of an exogenous metabolic system.

At cytotoxic concentrations, ortho-phenylphenol was weakly mutagenic in mouse lymphoma L5178Y/tk^+/− cells, both in the absence and presence of exogenous metabolic activation from rat liver.

Studies on the ability of ortho-phenylphenol to induce sister chromatid exchange and chromosomal aberrations in Chinese hamster ovary cells provided contradictory results. In one study performed in the absence of metabolic activation, dose-dependent increases in the incidence of both chromosomal aberrations and sister chromatid exchange were detected after a 27-h post-treatment incubation; the presence of only chromosomal aberrations after 42-h suggested that DNA damage resulting in sister chromatid exchange can be repaired during the longer incubation time. In a second study, a borderline increase in the frequency of chromosomal aberrations occurred in both the presence and absence of exogenous metabolic activation from rat liver, but no sister chromatid exchange was seen. In a third study in the presence of metabolic activation, an increased frequency of sister chromatid exchange occurred, which was not inhibited by several scavengers of oxygen reactive species. Finally, in the presence of 15% metabolic activation, ortho-phenylphenol increased the incidences of both chromosomal aberrations and sister chromatid exchange; both these cytogenetic effects were inhibited by cysteine and glutathione, and the frequency of sister chromatid exchange was found to correlate with the formation of the reactive metabolite phenylhydroquinone.

Phenylhydroquinone and phenylbenzoquinone caused sister chromatid exchange in Chinese hamster ovary cells, but the activity of the latter metabolite was lower in the presence of an exogenous metabolic activating system. Both metabolites also caused chromosomal aberrations in the same cell type, phenylhydroquinone requiring metabolic activation.

ortho-Phenylphenol caused a dose-dependent increase in the number of ouabain-resistant mutants in an ultra-violet-sensitive human RSa cell strain in the absence of metabolic activation. Gene mutation was not induced in a host-mediated assay in which mice were injected intraperitoneally with S. typhimurium and then given oral doses of ortho-phenylphenol.

In the urinary bladder epithelium of male rats, no DNA damage was detectable by the alkaline elution assay after intravesicular injection of ortho-phenylphenol, but it was present in rats of each sex injected with solutions of phenylhydroquinone or phenylbenzoquinone. DNA damage was observed in the urinary bladder epithelium of male rats fed 2% sodium ortho-phenylphenate in the diet for three to five months. In male CD-1 mice given a single oral dose of ortho-phenylphenol, DNA damage, as detected by the Comet assay, was present in stomach, liver, lung kidney and bladder but absent from brain and bone marrow.

ortho-Phenylphenol did not induce chromosomal aberrations in rat bone marrow after exposure in vivo and did not give rise to dominant lethal mutations in mice or rats.

Several studies were carried out in vitro and in vivo to investigate the covalent binding of ortho-phenylphenol to DNA. Reaction of DNA with ortho-phenylphenol or its hydroxylated metabolite phenylhydroquinone produced four major adducts when carried out in the presence of rat liver microsomes and NADPH. The formation of adducts was drastically decreased by cytochrome P450 inhibitors and did not occur in the absence of microsomes, except at high doses. The same major adducts were detected by the ³²P-postlabelling technique in deoxyguanosine-3′-phosphate or DNA reacted with the reactive metabolite of ortho-phenylphenol, phenylbenzoquinone. [¹⁴C]ortho-Phenylphenol was found to bind covalently to calf thymus DNA in the presence but not in the absence of microsomes, indicating that its conversion to an activated metabolite is essential; this was confirmed by the formation of adducts, detected by ³²P-postlabelling analysis, in calf thymus DNA incubated with phenylhydroquinone and phenylbenzoquinone. ³²P-Postlabelling analysis revealed one major adduct in whole urinary bladder DNA of rats fed a diet containing ortho-phenylphenol for 13 weeks, but the presence of DNA adducts was not confirmed in a subsequent study, in which only the bladder epithelium was evaluated. Topical application to female CD-1 mice of sodium ortho-phenylphenol or phenylhydroquinone produced adducts in skin DNA, as detected by ³²P-postlabelling; the levels of these adducts were reduced in mice pretreated with inhibitors of cytochrome P450 or of prostaglandin synthase. The dose of sodium ortho-phenylphenate applied to the mouse skin was far in excess of the concentrations attained in urine by feeding it to mice or rats at high doses. Incubation of DNA with ortho-phenylphenol or phenylhydroquinone in the presence of cytochrome P450 activation or prostaglandin synthase activation systems in vitro produced adducts similar to those detected in vivo.

Formation of 8-hydroxyguanosine, which reflects oxidative DNA damage, occurred in calf thymus DNA incubated with phenylhydroquinone, the major metabolite formed from ortho-phenylphenol by P450 monooxygenase, but was absent after incubation with ortho-phenylphenol and minimal after incubation with the ultimate metabolite phenylbenzoquinone; these findings indicate that DNA damage is likely to be due to the production of oxygen radicals during the conversion of phenylhydroquinone to phenylbenzoquinone.

2,5-Dihydroxybiphenyl, an intermediate of ortho-phenylphenol metabolism, was found to alkylate calf thymus DNA in the absence of metabolic activation (Grether et al., 1989).

In the presence of Cu(II)⁺⁺, ortho-phenylphenol did not induce damage in DNA fragments from the protooncogene c-Ha-ras-1, whereas DNA lesions were observed under the same experimental conditions with the two ortho-phenylphenol metabolites 2,5-dihydroxybiphenyl and 2-phenyl-1,4-benzoquinone (Inoue et al., 1990).

Dose-dependent formation of DNA adducts, as detected by ³²P-postlabelling, was observed in human HL-60 cells exposed to ortho-phenylhydroquinone and ortho-phenylbenzoquinone at 25–250 µmol/L; reaction of calf thymus DNA with ortho-phenylbenzoquinone resulted in the formation of one DNA adduct, which did not correspond to the major adduct produced in HL-60 cells.

Phenylhydroquinone at 31–187 µmol/L induced the formation of CREST-positive micronuclei (which represent whole chromosomes that fail to segregate during mitosis) in an arachidonic acid-supplemented prostaglandin H synthase-containing V79 Chinese hamster cell line. Treatment with phenylbenzoquinone had only a minor effect on micronucleus formation in unsupplemented cells. Neither phenylhydroquinone nor phenylbenzoquinone increased the frequency of mutation at the hprt locus in the same cells. The results suggest that phenylhydroquinone is oxidized to phenylbenzoquinone by prostaglandin H synthase.

Phenylbenzoquinone was found to act as initiatior in the two-stage transformation of BALB/c 3T3 cells, in which the cells were subsequently treated with TPA.

Induction of 8-hydroxy-2-deoxyguanosine, an index of oxidative DNA modification, was not observed in Chinese hamster ovary CHO-K1 cells exposed to phenylhydroquinone. Weakly positive results were observed after inhibition of catalase activity.