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    Pesticide residues in food -- 1999



    Sponsored jointly by FAO and WHO
    with the support of the International Programme
    on Chemical Safety (IPCS)



    Toxicological evaluations




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group

    Rome, 20-29 September 1999

    2-PHENYLPHENOL AND ITS SODIUM SALT

    First draft prepared by
    Jens-Jorgen Larsen
    Institute of Food Safety and Toxicology
    Ministry of Food, Agriculture and Fisheries, Soborg, Denmark

            Explanation
            Evaluation for acceptable daily intake
            Biochemical aspects 
                    Absorption, distribution, and excretion
                    Biotransformation
                    Effects on enzymes and other biochemical parameters
                Toxicological studies
                    Acute toxicity
                    Short-term studies of toxicity
                    Long-term studies of toxicity and carcinogenicity
                    Genotoxicity
                    Reproductive toxicity 
                        Multugeneration reproductive toxicity
                        Developmental toxicity
                    Special studies: Mechanisms of carcinogenicity in rat 
                      urinary bladder
                Observations in humans
            Comments 
            Toxicological evaluation
            References 


    Explanation

         2-Phenylphenol and its sodium salt were evaluated by the 1969,
    1983, 1985, 1989, and 1990 Joint Meetings (Annex 1, references  12, 
     40, 44, 56, and  59). A temporary ADI of 0-0.02 mg/kg bw was
    allocated in 1983, which was extended in 1985 and 1989. An ADI of
    0-0.02 mg/kg bw was established in 1990. Since that Meeting, studies
    have become available on biochemical aspects, biotransformation,
    effects on enzymes and other biochemical parameters, acute toxicity,
    short-term toxicity, long-term toxicity, genotoxicity, reproductive
    toxicity, dermal and ocular irritation and dermal sensitization, and
    on the mechanism of the carcinogenic effect in the rat urinary
    bladder. The compound was reviewed by the present Meeting within the
    periodic review programme of the Codex Committee on Pesticide
    Residues. 

         The toxicological data on the sodium salt of 2-phenylphenol were
    not used to establish the ADI, since the salt rapidly dissociates to
    2-phenylphenol. These data were, however, considered of value for the
    review and are therefore included. 

    Evaluation for Acceptable Daily Intake

    1.  Biochemical aspects

    (a)  Absorption, distribution, and excretion

         Groups of four male Fischer 344 rats were given single oral doses
    of [14C]2-phenylphenol (purity, 99.8%; specific activity, 19
    mCi/mmol) or [14C]sodium 2-phenylphenol (purity, 98.7%) by gavage at
    a dose of 500 mg/kg bw and were immediately placed in glass metabolism
    cages. About 90-95% of the administered radiolabel on both compounds
    was recovered in urine and 5-6% in faeces, mainly during the first 24
    h. The rates of urinary excretion were virtually identical in the two
    groups. In a second experiment, animals were fed diets containing
    13 000 ppm of 2-phenylphenol or 20 000 ppm of the sodium salt
    (equimolar amounts) for 2 weeks before administration of single oral
    doses of the labelled compounds. The animals still eliminated most of
    the radiolabel (88-94%) in urine and small amounts in faeces (3-5%).
    Preconditioning did not greatly affect the disposition of radiolabel,
    although the sodium salt appeared to have been eliminated somewhat
    more rapidly than 2-phenylphenol (Reitz et al., 1983).

         Groups of four male Fischer 344 rats were given single oral doses
    of [14C]2-phenylphenol (purity not given; specific activity, 1.6
    mCi/mmol) at 160 mg/kg bw or [14C]sodium 2-phenylphenol at 250 mg/kg
    bw (equimolar levels; purity not given; specific activity, 1.6
    mCi/mmol). The animals were fasted overnight before and for 6 h after
    dosing. Urine and faeces were collected daily for 7 days. The
    excretion patterns in the two groups did not differ significantly, and
    82-98% of the dose was recovered in urine and only 2-5% in faeces
    within 24 h of dosing. Two male rats received bile duct cannulae, and
    bile was collected for 3 days after a single oral dose of 250 mg/kg bw
    of radiolabelled sodium salt. Excretion of radiolabel in the bile
    began within the first hour of dosing, reached a peak within 3-6 h,
    and was almost complete by 8 h' about one-fourth of the dose was
    recovered in bile over the 3-day collection period. The authors
    interpreted these results as indicating rapid absorption from the
    intestine and enterohepatic circulation of 2-phenylphenol metabolites.
    The pattern of distribution in organs and tissues, examined on days 1,
    3, and 7 after administration of the sodium salt and on days 1 and 7
    after administration of 2-phenylphenol, showed little difference.
    Little radiolabel was retained in organs and tissues. including the
    urinary bladder (Yamaha et al., 1983; Sato et al., 1988).

         In a comparative study, [12C/13C/14C]2-phenylphenol
    (purity, 99.5%; specific activity, 48 mCi/mmol) was given to 10 male
    B6C3F1 mice as a single oral dose of 15 or 800 mg/kg bw, to 10 male
    and 10 female Fischer 344 rats as a single oral dose of 28 or 27 mg/kg
    bw, and to six male volunteers as a dermal dose of approximately 6
    g/kg bw on the forearm for 8 h. The compound was well absorbed in the
    mice, 84% and 98% of the two doses being recovered in urine collected
    over 48 h. Extensive absorption and rapid elimination were also seen
    in the rats, 89 and 86% of the dose being found in the urine of males

    and females, respectively, within 24 h. 2-Phenylphenol was also
    rapidly eliminated by the volunteers, 99% of the absorbed dose being
    collected in urine within the first 48 h of exposure (Bartels et al.,
    1998). 

         The skin of the forearm of six male volunteers aged 19-27 and
    weighing 58-98 kg was exposed to 100 l of a 0.4% isopropanol solution
    of [13C/14C]2-phenylphenol providing a dose of approximately 6
    g/kg bw and 42 Ci for 8 h. Samples of blood, urine, and faeces were
    collected at various times for five days, and blood samples were also
    taken during exposure. High concentrations of radiolabel in blood were
    observed within the first 2 h of the start of exposure in all
    subjects, indicating rapid absorption. The rate diminished fairly
    rapidly at the end of the exposure period, and little or no radiolabel
    was present in venous blood samples collected 2 days after termination
    of exposure. About 43% of the applied dose of 2-phenylphenol was
    absorbed, about 58% of which was recovered in swabs, skin rinse,
    gauze, and the protective enclosure. The majority (99%) of the
    absorbed compound was excreted in urine, and faeces represented a
    minor route of elimination (1% within 5 days). A mean of 0.04% of the
    administered radiolabel was found in the tape strips covering the
    application site, indicating no accumulation in the superficial layers
    of the skin (Selim, 1996).

         The plasma concentrations peaked within 4 h of dosing and then
    declined rapidly, virtually all of the absorbed dose being excreted in
    urine within 24 h. A one-compartment model was used to describe the
    pharmacokinetics of absorption and clearance of [14C]2phenylphenol
    in these volunteers. Approximately 43% of the applied dose was
    absorbed through the skin, with an average absorption half-time of
    10 h. Once absorbed, its renal clearance was rapid, with an average
    elimination half-time of 0.8 h. Overall, the pharmacokinetics of
    [14C]2-phenylphenol was similar in the individual volunteers, and
    the model parameters were in excellent agreement with the experimental
    data. The rapid excretion in urine indicates that 2-phenylphenol is
    unlikely to accumulate in humans exposed repeatedly (Timchalk, 1996).

    (b)  Biotransformation

         Groups of 10 male B6C3F1 mice were given a single oral dose of
    [14C]2-phenylphenol (specific activity, 48 mCi/mmol) at 25 or 1000
    mg/kg bw or five daily doses of unlabelled compound (purity, 99.5%) at
    1000 mg/kg bw per day followed by a single oral dose of labelled
    compound at 1000 mg/kg bw and were killed 48 h after dosing. For
    comparison, groups of two male and two female Fischer 344 rats were
    given a single oral dose of labelled compound at 25 or 125 mg/kg bw
    and were killed 24 h after dosing. The excretion of
    [14C]2-phenylphenol in mice was rapid and was complete by 12-24 h
    after dosing, with 74-98% of the recovered radiolabel in urine and
    6-13% in faeces; < 1% was recovered in the tissues and carcass. Eight
    radiolabelled metabolites were detected in the urine of both mice and
    rats, with no major differences in distribution by species, by sex in
    the rats, or single or repeated dosing in mice. A small amount (0.4%)

    of free 2-phenylphenol was detected only in urine of female rats given
    the single dose of 125 mg/kg bw. Four major urinary metabolites were
    identified: phenylhydroquinone glucuronide, phenylhydroquinone
    sulfate, 2-phenylphenol sulfate, and 2-phenylphenol glucuronide,
    accounting for about 98% of the recovered dose in mice and 102% in
    rats. An additional metabolite which accounted for about 2.7% of the
    recovered dose in rat urine was tentatively identified as the sulfate
    conjugate of 2,4'-dihydroxybiphenyl. No qualitative difference in
    metabolites was observed in male mice, but a dose-dependent,
    quantitative difference was noted in the extent of sulfation and
    glucuronidation of 2-phenylphenol. After a single dose of 25 mg/kg bw
    to mice, the sulfate was the major urinary metabolite, accounting for
    56% of the recovered radiolabel, while the glucuronide accounted for
    29%. After single or repeated doses of 1000 mgkg bw, the glucuronide
    was the major metabolite, accounting for 48-60% of the urinary
    radiolabel, while the sulfate accounted for 20-27%. In rats given a
    single oral dose of 25 mg/kg bw, 2-phenylphenol sulfate was the major
    metabolite, accounting for 91% of the recovered radiolabel, while the
    glucuronide accounted for only 7%. Formation of phenylhydroquinone
    glucuronide and sulfate represented minor metabolic pathways,
    accounting for 11-23% and 2-7% of the radiolabel in mice and rats,
    respectively. The extent of conjugation was not dose-dependent in mice
    given a single oral dose of 25 or 1000 mg/kg bw of 2-phenylphenol. The
    authors concluded that 2phenylphenol is completely metabolized in mice
    and rapidly eliminated in the urine, predominantly as the sulfate and
    glucuronide conjugates. The extent of metabolism was qualitatively
    comparable in mice and rats, although quantitative differences were
    seen in the extent of conjugation (McNett et al., 1997).

         In the comparative study of Bartels et al. (1998) described
    above, sulfation of 2-phenylphenol was found to be the major metabolic
    pathway at low doses in all three species, accounting for 57% of the
    urinary radiolabel in male mice given 15 mg/kg bw, 82% in male rats
    given 28 mg/kg bw, and 69% in the male volunteers given 0.006 mg/kg
    bw. The glucuronide was also found, representing 29, 7, and 4% of the
    total urinary metabolites at these low doses in the three species,
    respectively. Conjugates of phenylhydroquinone accounted for 12, 5,
    and 15% of the dose in mice, rats, and humans, respectively. Little or
    no free 2-phenylphenol was found in any species, and no free
    phenylhydroquinone or phenylbenzoquinone was found in any species,
    with a limit of detection of 0.1-0.6%. A novel metabolite, the sulfate
    conjugate of 2,4'-dihydroxybiphenyl, was identified in rats and
    humans, comprising 3 and 13% of the low doses, respectively.
    Dose-dependent shifts in the conjugation of parent 2-phenylphenol were
    seen in mice, indicating saturation of the sulfation pathway after the
    high dose of 800 mg/kg bw. Dose-dependent increases in the total
    amount of phenylhydroquinone were also observed in the mice. 

         The major metabolites identified in the urine of five male and
    five female Fischer 344 rats fed 20 000 ppm of sodium 2-phenylphenol
    (purity not given) in the diet for 136 days were glucuronide
    conjugates of 2-phenylphenol and 2,5-dihydroxybiphenyl. Trace amounts
    of phenyl-1,4-benzoquinone were also tentatively identified.

    Unconjugated phenolic metabolites accounted for only 1% of the
    phenolic metabolites excreted; no other metabolites were found. By 24
    h after feeding, 55% of the dose had been recovered in males and 40%
    in females. A sex difference was found in the proportion of urinary
    metabolites, male rats excreting 1.8 times as much conjugated
    2-phenylphenol and more than 7 times as much conjugated
    2,5-dihydroxybiphenyl as female rats in 24-h urine samples. No
    explanation was given for the inability to find the sulfate ester of
    2-phenylphenol in urine in this study. As only 40-55% of the
    administered dose was recovered, it may have been present but not
    identified (Nakao et al., 1983).

         Single oral doses of 5, 50, or 500 mg/kg bw of
    [14C]2-phenylphenol (purity, 99.8%; specific activity, 19 mCi/mmol
    per L) or [14C]sodium 2-phenylphenol (purity, 98.7%; specific
    activity, 19 mCi/mmol/L) were administered to groups of four male
    rats, and the urinary metabolites were identified and quantitified. At
    the two lower doses, the major metabolites of both compounds were the
    glucuronide and sulfate ester conjugates of 2-phenylphenol, and
    unconjugated 2-phenylphenol and 2,5-dihydroxybiphenyl accounted for
    < 2% of the total radiolabel recovered in urine at a limit of
    detection of 1-2%. Nearly identical high-performance liquid
    chromatograms were obtained for the two compounds. At 500 mg/kg bw, a
    further metabolite of both compounds was identified, which accounted
    for 20-30% of the urinary radiolabel and appeared to be a conjugated
    dihydroxybiphenyl molecule, most likely with glucuronide and/or
    sulfate groups. The authors hypothesized that this metabolite is
    formed only at high doses as a result of saturation of normal
    glucuronide and sulfate ester conjugation pathways. Incubation of
    [14C]2-phenylphenol with purified microsomes  in vitro in the
    absence of conjugating substrates yielded large amounts of a material
    which co-chromatographed with 2,5-dihydroxybiphenyl. The semiquinone
    and quinone were not identified in these studies, but their formation
    was proposed on the basis of the results of similar studies on benzene
    (Reitz et al., 1983).

         In a study of toxicity in male Fischer 344 rats given diets
    containing 0, 800, 4000, 8000, or 12 500 ppm of 2-phenylphenol
    (purity, 99.5%) for 13 weeks, the DNA of the urothelium was isolated
    at the end of the study and examined for covalent adducts of
    2-phenylphenol by the 32P-postlabelling assay. The concentrations of
    2-phenylphenol metabolites were also measured in overnight urine
    samples collected from the animals at the end of the study. The
    glucuronide and sulfate conjugates of 2-phenylphenol and the
    hydroxylated metabolite, 2,5-phenylhydroquinone, were found to be the
    major metabolites. The major conjugation in all samples was with
    sulfate. The formation of this metabolite appeared to be saturated at
    8000 ppm, while the concentrations of the remaining three conjugated
    metabolites increased in a dose-dependent fashion up to the high dose.
    Traces of free 2-phenylphenol and phenylhydroquinone were observed at
    all doses, free phenylhydroquinone comprising 0.6-1.5% of the total
    metabolites measured. The concentrations of creatinine were comparable
    in all groups (Bartels & McNett, 1996).

         Mature cats and dogs were given [14C]sodium 2-phenylphenol
    (purity and specific activity not given) at single oral doses < 3
    g/kg bw. The amount of radiolabel in plasma was higher in dogs than in
    cats, and the dogs metabolized and excreted three times more
    radiolabel in urine than cats. The urinary metabolites were unchanged
    2-phenylphenol, glucuronide and sulfate conjugates, and phenol derived
    from cleavage of the phenylphenol bond and ring hydroxylation. The
    phenol metabolites were derived from both 2-phenylphenol ring moieties
    (Oehme & Smith, 1972).

         Urine samples were collected weekly after single oral doses every
    second day of [14C]2-phenylphenol (purity, 95%) to three male and
    three female mature beagle dogs (0.3 mg/kg bw per day), three male and
    three female immature beagle dogs (2.0 mg/kg bw per day), three male
    and three female mature domestic cats (1.2 mg/kg bw per day), and
    three male and three female immature domestic cats (2.0 mg/kg bw per
    day) for 8 weeks. The main urinary excretion product was unchanged
    2-phenylphenol, representing 70-90% of the radiolabel in dogs and
    95-98% in cats. Dogs excreted significantly more glucuronide- and
    sulfate ester-conjugated 2-phenylphenol than cats, and immature dogs
    excreted four times as much glucuronide conjugate as mature dogs. The
    age differences did not affect the rate of excretion of the sulfate
    ester conjugate in either species (Savides & Oehme, 1980).

         In the study of Selim (1996) in volunteers treated dermally,
    described above, 99% of the absorbed dose of 2-phenylphenol was
    eliminated in urine, primarily as polar conjugates or hydroxylated
    metabolites. The major urinary metabolite was the sulfate conjugate,
    which accounted for 68% of the absorbed dose; conjugation with
    glucuronic acid accounted for only 3%. Hydroxylation of the phenol or
    phenyl ring, followed by conjugation, was also significant,
    phenylhydroquinone glucuronide representing 14% of the absorbed dose
    and 2,4-dihydroxybiphenyl sulfate, 12%. Traces of unmetabolized
    parent compound (0.5% of absorbed dose) were found only in samples
    taken shortly after administration. No free phenylhydroquinone or
    phenylhydroquinone-sulfate was found in urine (Bartels et al., 1997;
    Timchalk et al., 1998).

         The proposed metabolic pathways of 2-phenylphenol in rodents and
    humans are shown in Figure 1.

    (c)  Effects on enzymes and other biochemical parameters

          2-Phenylphenol 

         2-Phenylphenol was converted to 2,5-dihydroxy biphenyl
    (phenylhydroquinone) by microsomal cytochrome P450 enzymes. Depending
    on the cofactor used, the microsomal enzymes catalysed either
    oxidation and/or reduction of the metabolite. Phenylhydroquinone was
    oxidized to phenyl-1,4-benzoquinone by cumene
    hydroperoxide-supported enzymes, and this compound was reduced to
    phenylhydroquinone by cytochrome P450 reductase. This study provides
    direct evidence of cytochrome P450-catalysed redox cycling of

    FIGURE 1

    2phenylphenol, which may play a role in the induction of bladder
    cancer by this substance (Roy, 1990).

         Activation of the 2-phenylphenol metabolite phenylhydroquinone by
    prostaglandin (H) synthase in the presence of arachidonic acid and
    hydrogen peroxide was studied to test the hypothesis that
    prostaglandin synthase in rat urinary bladder transitional epithelium
    and kidney medullar papilla is responsible for activation of the
    metabolite to reactive intermediates in the bladder and kidney.
    Phenylhydroquinone was found to be metabolized by the peroxidase
    activity of prostaglandin synthase and by other peroxidases such as
    horseradish peroxidase and myeloperoxidase, suggesting that the
    peroxidative metabolism of phenylhydroquinone could play a role in
    urinary bladder and kidney carcinogenesis in rats (Kolachana et al.,
    1991).

         In a study of the effect of pH on nonenzymatic oxidation of
    phenylhydroquinone, the effects of phenylbenzoquinone and oxygen
    concentration on autoxidation of phenylhydroquinone, and the
    nonenzymatic conversion of phenylbenzoquinone to phenylhydroquinone, a
    curvilinear relationship was found between the rate of oxidation of
    phenylhydroquinone and pH over the range 6.3-7.6. Phenylbenzoquinone
    was formed during autoxidation of phenylhydroquinone, with a yield of
    0.92  0.02. The results indicate that the production of reactive
    metabolites from phenylhydroquinone involves both a pH-independent
    (i.e. oxygen-dependent) and a pH-dependent pathway and that the
    presence of phenylbenzoquinone enhances the rate of pH-dependent
    phenylhydroquinone autoxidation. The authors suggested that ionization
    of phenylhydroquinone semiquinone is a key step in production of
    reactive species in the pH-dependent pathway. They found a good
    correlation between the proposed reaction pathway and the induction by
    2-phenylphenol of bladder lesions in rats. Thus, pH-dependent
    autoxidation of free phenylhydroquinone in urine may be responsible
    for the tumorigenic effects of 2-phenylphenol and sodium
    2-phenylphenol in the rat bladder (Kwok & Eastmond, 1997).

         Groups of eight female B6C3F1 mice were given 0, 1, 10, or 200
    mg/kg bw per day of 2-phenylphenol (purity, > 98%) by gavage on 5
    days per week for 2 weeks. As a positive control, mice were given 45
    mg/kg bw of cyclophosphamide intraperitoneally for 4 days. The weights
    of the body, liver, spleen, kidney, and thymus were recorded, and
    samples were prepared for histopathological examination. Haematology
    and clinical chemistry were conducted, and bone-marrow cellularity and
    colony formation, lymphoproliferative responses, delayed
    hypersensitivity responses, immunoglobulin, antibodies, response to
     Listeria monocytogenes challenge, and tumour susceptibility were
    studied. None of the treated animals died or showed signs of toxicity.
    Histopathological examination revealed no significant lesion in any
    tissues. The weight of the thymus and the relative weight of the
    spleen were slightly increased at 200 mg/kg bw per day. The slight
    haematological alterations seen did not show a dose-response
    relationship and were within the normal range of biological variation.
    A slight increase in serum cholesterol concentration and a

    corresponding decrease in triglyceride concentration were seen in mice
    at 200 mg/kg bw per day. The activity of alanine aminotransferase and
    total protein in serum were not affected, although the
    albumin:globulin ratio was slightly decreased at the high dose.
    Bone-marrow cellularity, lymphoproliferative responses, immune
    function, and host susceptibility were not altered. In contrast,
    treatment with cyclophosphamide resulted in marked alterations. The
    authors concluded that 2-phenylphenol, even at relatively high doses,
    did not alter immune function or host susceptibility (Luster et al.,
    1981).

          Sodium 2-phenylphenol 

         Binding of sodium 2-phenylphenol metabolites to macromolecules
     in vitro was studied by incubating [14C]sodium 2-phenylphenol
    (specific activity, 19 mCi/mmol) with purified liver microsomes from
    male rats in the presence of a NADPH regenerating system and bovine
    serum albumin, which served as a 'protein acceptor'. Macromolecular
    binding of radiolabel to protein, which was dependent on the presence
    of both active microsomes and NADP, was observed. In order to study
    the binding of metabolites of 2-phenylphenol and its sodium salt to
    macromolecules in the liver, kidney, and urinary bladder  in vivo, 
    groups of four male rats were given single oral doses of
    14C-labelled compounds at doses of 50, 100, 200, or 500 mg/kg bw,
    and tissues were excised 16-18 h later for measurement of
    macromolecular binding, which was determined as nanomoles of bound
    material per milligram of protein. The extent of binding was not
    linearly related to the administered dose. Disproportionate increases
    were seen in each tissue at doses of sodium 2-phenylphenol > 200
    mg/kg bw and in liver and bladder at doses of 2-phenylphenol at
    200-500 mg/kg bw (Reitz et al., 1984).

         Sodium 2-phenylphenol (purity not given) was administered in the
    diet at a concentration of 20 000 ppm to 4-week-old male and female
    Fischer 344 rats for 136 days. Urine was collected periodically. At
    the end of treatment, the rats were killed, blood samples were
    collected, and livers and kidneys were removed. The amounts of cyclic
    nucleotides (cAMP and c-GMP) were determined in urine, plasma, liver,
    and kidneys, and adenylate cyclase activity was measured in liver and
    kidneys. In male rats, the c-AMP levels in urine and plasma were
    decreased whereas the c-GMP levels were increased. In females, c-AMP
    levels were decreased only during the first 3 days of feeding, and the
    levels of c-AMP and cGMP in liver and kidneys were unchanged. The
    decreased urinary c-AMP in male rats was probably the result of
    decreased adenylate cyclase activity in liver and kidneys. A similar
    change in adenylate cyclase activity was observed in liver but not in
    kidneys of female rats treated with sodium 2-phenylphenol. The
    sex-related alterations in cyclic nucleotide levels were postulated to
    be involved in the sex-dependent induction of urinary bladder tumours
    by sodium 2-phenylphenol (Nakagawa et al., 1984).

         In male and female Fischer 344 Du Crj rats given 20 000 ppm of
    sodium 2-phenylphenol in the diet for 20 weeks, urinary gamma-glutamyl
    transpeptidase activity decreased immediately after the start of
    treatment and remained low throughout the study. The activities of
    this enzyme and of alkaline phosphatase in kidney homogenate were
    found to have decreased to about 80% of the control values at 20
    weeks, but the activity of glucose-6phosphate dehydrogenase was
    significantly increased; that of Na/K-ATPase was unchanged. In liver
    homogenate, however, gamma-glutamyl transpeptidase activity was
    increased by about eight times and that of glucose-6-phosphate
    dehydrogenase was significantly increased, but the activities of
    alkaline phosphatase and Na/K-ATPase were not significantly different
    from the control values. The glutathione concentration in the livers
    of treated rats was significantly reduced (Nagai & Nakao, 1984).

    2.  Toxicological studies

    (a)  Acute toxicity

         The results of studies of the acute toxicity of 2-phenylphenol
    and its sodium salt are summarized in Table 1. The clinical signs of
    toxicity were generally nonspecific.

        Table 1. Acute toxicity of 2-phenylphenol and its sodium salt

                                                                                               
    Species                  Sex    Route        LD50 (mg/kg bw)   Reference
                                                 or LC50 (mg/L)
                                                                                               

     2-Phenylphenol 
    Mouse                    M      Oral               1200        Taniguchi et al. (1981)
                             F                         1100
    Mouse                    M      Oral               3500        Tayama et al. (1983, 1984)
                             F                         3200
    Rat                      M      Oral               2600        Tayama et al. (1980)
                             F                         2900
    Rat                      M      Oral               2800        Gilbert & Crissman (1994)
                             F                         2800
    Rat                      M&F    Inhalation
                                    (4 h)              > 36        Landry et al. (1992)
    Rabbit                   M&F    Dermal           > 5000        Carreon & New (1981)

     Sodium 2-phenylphenol 
    Mouse                    M      Oral                900        Ogata et al. (1979)
                             F                          800
    Rat                      M      Oral               1700        Taniguchi et al. (1981)
                             F                         1600
    Rat                      M      Oral               1100        Tayama et al. (1979)
                             F                         1100
    Rat                      M      Oral                850        Gilbert & Stebbins (1994)
                             F                          590
                                                                                               
    
    (b)  Short-term studies of toxicity 

          2-Phenylphenol 

          Rats 

         2-Phenylphenol (purity, 99.8%) was administered to 30 male
    Fischer 344 rats (Charles River) in the diet at a concentration of
    20 000 ppm, equal to 1000 mg/kg bw per day, for up to 90 days. Interim
    sacrifices were performed at 3, 7, 30, and 65 days. Only seven rats at
    each dose were permitted to live to 90 days, at which time they were
    killed. Food consumption and body weight were markedly reduced within
    the first week and remained low throughout the study. The renal
    lesions observed in these rats included focal cortical cysts,
    significantly decreased urine specific gravity (at 65 and 90 days),
    small amounts of blood in the urine, focal tubular collapse and
    atrophy in the cortex, and cystic degeneration (at 65 and 90 days). No
    treatment-related urinary bladder lesions were observed. A NOAEL could
    not be identified since the body weight was reduced at the only dose
    tested (Reitz  et al., 1983).

         Groups of 10 male and 10 female Fischer 344 rats were fed diets
    containing 0, 1300, 3100, 6300, 13 000, or 25 000 ppm of
    2-phenylphenol (purity not given), equal to 0, 180, 390, 760, 1700,
    and 2800 mg/kg bw per day for males and 0, 200, 410, 800, 1700, and
    3000 mg/kg bw per day for females, for 12 weeks. Body weight and
    body-weight gain were severely depressed in males and females at
    25 000 ppm and to a lesser extent (14%) in males fed 13 000 ppm. No
    significant treatment-related effects were seen in analyses of urine
    performed at weeks 9 and 13. Haematological and blood chemical values
    were generally normal, other than a slight decrease in haemoglobin
    concentration in male and female rats at the highest dose. The
    absolute and relative weight of many organs in male rats at this dose
    were significantly decreased. The NOAEL was 6300 ppm, equal to 760
    mg/kg bw per day, on the basis of reduced body weight and body-weight
    gain at 13 000 ppm (Iguchi  et al., 1984).

         Groups of five male and five female Fischer 344 rats received
    dermal applications of 0, 100, 500, or 1000 mg/kg bw per day of
    2-phenylphenol (purity, 99.8%) once daily on 5 days per week for a
    total of 15 applications over 21 days. The amount applied per animal
    was adjusted weekly on the basis of the body weight of individual
    animals and was applied to a 5 cm  5 cm area of clipped skin on the
    back, covered with a nonabsorbent cotton patch held in place by an
    elastic wrap secured with adhesive tape. The wraps and patches were
    removed not less than 6 h after treatment, and the treated area was
    wiped with a wettened gauze pad to remove residual test material.
    Control animals were handled in the same way. All animals were
    acclimated to the wraps for 2 days before the beginning of treatment.
    They were observed at least daily and were given a complete clinical
    examination weekly. The skin at the site of treatment was examined
    after removal of the wrap on the last day of dosing, each week, and on
    the day before necropsy. Body weights were measured weekly and feed

    consumption and feed efficiency were calculated weekly. Urine was
    analysed on day 19. The rats were fasted overnight before necropsy,
    when haematological and serum clinical chemical parameters were
    evaluated, all animals were examined for gross pathological changes,
    selected organs were weighed, and tissues were preserved. Selected
    tissues and all gross lesions from animals in the control and
    high-dose groups were examined histologically.

         No deaths occurred at any dose. Treatment-related effects
    indicative of dermal irritation were observed at the site of
    application in animals of each sex at 500 and 1000 mg/kg bw per day.
    Female rats appeared to be slightly more sensitive than males, but the
    severity of lesions increased with duration of exposure and dose in
    both sexes. The irritating effects ranged from scaling to fissures.
    Body weights and feed consumption were not affected, and no
    significant treatment-related effects were found in the
    haematological, clinical chemical, and urinary parameters evaluated.
    No treatment-associated alterations were found in the liver or kidneys
    (Zempel & Szabo, 1993).

          Guinea-pigs 

         2-Phenylphenol was evaluated in 10 male Hartley albino
    guinea-pigs for dermal sensitization potential by a modified Buehler
    method. The animals received three dermal applications of 0.4 g of
    2-phenylphenol (purity, 99.9%) during a 3-week induction period and
    were challenged with 0.4 g of the compound 2 weeks after the last
    induction. The condition of the test sites was assessed approximately
    24 and 48 h after the challenge. No erythema or oedema was seen in any
    of the animals. The author concluded that 2-phenylphenol did not cause
    delayed contact hypersensitivity (Berdasco, 1991).

         Ten male Hartley albino guinea-pigs were clipped free of hair on
    the day before dosing and received three dermal applications of 0.4 g
    of 2-phenylphenol (purity, 99.9%) moistened with 0.20 ml of distilled
    water during a 3-week induction period. Two weeks after the last
    induction application, they were given a challenge application of
    0.4 ml of a 7.5% suspension of the compound in water on another site
    for 6 h. Five animals received no induction but a 0.4-ml aliquot of a
    7.5% suspension of 2-phenylphenol in water. The condition of the test
    sites was assessed approximately 24 and 48 h after challenge. No
    erythema was seen, and none of the uninduced animals showed
    irritation. The animals were in good health and gained weight during
    the study. The author concluded that 2-phenylphenol did not cause
    delayed contact hypersensitivity  (Gilbert, 1994b). 

          Rabbits 

         The ability of 2-phenylphenol (purity, 99.9%) to cause primary
    dermal irritation  was studied in three male and three female New
    Zealand white rabbits that received applications of aliquots of 0.5 ml
    of the substance moistened with 0.3 ml of distilled water for 4 h on
    intact skin on a clipped, 10 cm  10 cm area of the back. The

    application sites were graded for erythema and oedema within 30 min
    and 24, 48, and 72 h of removal of the patch and on days 7, 8, 9,
    10,11, 14, and 15. The animals were weighed on the day of treatment
    and at the end of the study. Very slight erythema was observed at the
    application site in one of the six rabbits within 30 min of removal of
    the test material and in two rabbits 24 h later. Severe to slight
    eschar formation was observed in four rabbits within 30 min of
    treatment which persisted throughout the remainder of the study. Four
    animals had burns at the site of application within 30 min, which
    resolved as scabs and then scars by the end of the study. Four rabbits
    had very slight-to-severe oedema 30 min and 24 h after removal of the
    test material, and slight-to-severe oedema was observed on three
    animals 48 and 72 h after removal. Body weight was not affected
    (Gilbert, 1994a).

         Instillation of 0.1 g of 2-phenylphenol (purity not given) into
    the right eye of six New Zealand white rabbits resulted in moderate
    corneal injury, iritis, and moderate-to-severe conjunctival redness
    and chemosis in all animals 24, 48, and 72 h and 7 days after dosing
    (Norris, 1971).

          Dogs 

         In studies to assess the palatability and toxicity of
    2-phenylphenol (purity, 99.8%) in beagle dogs, males and females were
    treated with several regimens. For palatability, one female was given
    feed containing 2-phenylphenol to give a dose of 300 mg/kg bw per day
    for 5 days. In a study to assess toxicity, groups of two males and
    three females were given 300-1000 mg/kg bw per day as a solution in
    peanut oil by gastric intubation for up to 9 days or 400-700 mg/kg bw
    per day by capsule for 1-2 days. In a 4-week study, groups of two
    males and two females were given 0, 100, 200, or 300 mg/kg bw per day
    as a solution in peanut oil by gastric intubation on 5 days per week.
    In a 1-year study, groups of four males and four females were given
    2-phenylphenol as a solution in peanut oil by gastric intubation at
    doses of 0, 30, 100, or 300 mg/kg bw per day for 5 days per week for
    1 year. The animals were examined daily for clinical signs, and body
    weight, feed consumption, haematological, urinary, and clinical
    chemical parameters, organ weights, ophthalmologic endpoints, and
    gross and histopathology were determined. 

         Administration of 300 mg/kg bw per day in feed or by intubation
    for 5 days resulted in decreased body weights and feed consumption
    that correlated with the unpalatability of 2-phenylphenol. Repeated
    emesis was seen at doses > 400 mg/kg bw per day administered in
    gelatin capsules or in peanut oil by intubation and at doses > 200
    mg/kg bw per day by intubation throughout the 4-week study.
    Dose-related emesis was also seen in animals given 2-phenylphenol for
    1 year. In general, more frequent emesis and ejection of greater
    volumes of gastric content was seen in dogs given 300 mg/kg bw per day
    than in those given lower doses. While emesis effectively limited the
    dose that could be retained, the degree of emesis did not appear to
    compromise the health of the animals over 1 year. The reaction was

    categorized as a local, transitory response of the mucosal lining of
    the upper alimentary tract rather than a reaction of the central
    nervous system. No adverse effects were seen on body weight, feed
    consumption, haematological, urinary, clinical chemical, or
    ophthalmological parameters, organ weights, or gross or histological
    appearance of a range of tissues from all dogs. The only deaths were
    of two males at the high dose in the 1-year study which died
    subsequent to inadvertent deposition of the test solution into the
    lungs after approximately 4.5 months of dosing. The NOAEL was 300
    mg/kg bw per day (Cosse  et al., 1990).

          Sodium 2-phenylphenol 

          Mice 

         Groups of 10 male and 10 female B6C3F1 mice were fed diets
    containing 0, 2500, 5000, 10 000, 20 000, or 40 000 ppm of sodium
    2-phenylphenol as the tetrahydrate (purity not given), equivalent to
    0, 270, 550, 1100, 2200, and 4400 mg/kg bw per day, for 13 weeks.
    Body-weight gain was significantly depressed in males fed 10 000 or
    20 000 ppm and in animals of each sex fed 40 000 ppm. Urinary analysis
    showed increased pH and decreased specific gravity at the highest
    dose. The relative weights of the livers of animals given 10 000,
    20 000, or 40 000 ppm were significantly greater than those of
    controls, but no treatment-related histopathological findings were
    made. Light and scanning electron microscope examination of the
    bladder epithelium at 4, 8, and 13 weeks in three males and three
    females in the control group and at 20 000 ppm showed no abnormaility
    in the appearance of the bladder epithelium of treated mice at any
    time. The NOAEL was 5000 ppm, equivalent to 550 mg/kg bw per day, on
    the basis of reduced body-weight gain and increased relative liver
    weight at 10 000 ppm (Shibata et al., 1985).

          Rats 

         Sodium 2-phenylphenol (purity, 98.7%) was administered in the
    diet to 30 male Fischer 344 rats at a concentration of 20 000 ppm for
    up to 90 days. Interim sacrifices were performed at 3, 7, 14, 30, and
    65 days. Only seven rats per group were permitted to live to 90 days,
    at which time they were killed. The lesions seen in the urinary
    bladder epithelium were increased mitosis beginning at 3 days and
    thickening (i.e. simple hyperplasia) beginning at 14 days. No tumours
    were observed in the bladder. A NOAEL could not be identified since
    lesions were observed in the bladder at 20 000 ppm, the only dose
    tested (Reitz et al., 1983).

         Groups of 10 male and 10 female Fischer 344 rats were fed diets
    containing 0, 1250, 2500, 5000, 10 000, 20 000, or 40 000 ppm of
    sodium 2-phenylphenol (purity, > 95%), equal to 0, 85, 180, 350, 710,
    1400, and 2500 for males and 0, 87, 180, 350, 690, 1300, and 2400
    mg/kg bw per day for females, for 13 weeks. The rats were observed
    daily for changes in general condition and were weighed weekly; the
    amounts of feed and water consumed were measured on 3 days every other

    week. No deaths occurred during the study. A 15-17% decrease in
    body-weight gain was seen in animals at doses > 5000 ppm. Urinary
    bladder tumours occurred in male rats at frequencies of 1/10 at 10 000
    ppm, 9/10 (five transitional-cell carcinomas) at 20 000 ppm, and 1/10
    at 40 000 ppm. Six rats at 40 000 ppm had pyelonephritis. In female
    rats, the frequencies of tumours were 0/10 at 20 000 ppm and 2/10
    (papillomas only) at 40 000 ppm. No bladder calculi were observed in
    this experiment. The NOAEL was 2500 ppm, equal to 180 mg/kg bw per
    day, on the basis of reduced body-weight gain at 5000 ppm (Iguchi et
    al., 1979; Hiraga & Fujii, 1981).

          Guinea-pigs 

         Sodium 2-phenylphenol (purity, 99.1%) was applied to the clipped
    skin of 10 male Hartley albino guinea-pigs as a 0.4-ml aliquot of a
    0.5% suspension in distilled water at 3-week intervals for induction.
    Two weeks after the last dose, a challenge of 0.4 ml of a 0.1%
    solution of the compound in distilled water was applied to the other
    side of the animals. No erythema occurred at the test site. The
    animals gained weight throughout the study. The author concluded that
    sodium 2-phenylphenol did not cause delayed contact hypersensitivity
    (Gilbert, 1994c).

          Rabbits 

         Sodium 2-phenylphenol diluted 1:200 with distilled water was
    instilled into the eyes of six rabbits (strain not given). Temporary,
    mild conjunctival reactions were observed in three animals. The
    authors concluded that the compound slightly irritated the eye (Davies
    & Liggett, 1973).

    (c)  Long-term studies of toxicity and carcinogenicity

          2-Phenylphenol 

          Mice 

         Groups of 50 male and 50 female B6C3F1 mice were fed diets
    (concentrations not given) supplying 2-phenylphenol (purity, 99.9%) at
    doses of 0, 250, 500, or 1000 mg/kg bw per day for 2 years. A
    satellite group of 10 male and 10 female mice at each dose was
    maintained on the diets for 12 months and then necropsied. All mice
    were observed at least once daily for overt signs of toxicity, and a
    thorough clinical examination was performed at least once a week
    throughout the study. Body weights and feed consumption were recorded
    weekly for the first 13 weeks and monthly thereafter. The diets were
    prepared weekly or every other week, with adjustments of the
    2-phenylphenol concentration according to group mean body weights and
    feed consumption to maintain the desired doses for each group.

         Clinical signs and mortality rates were unaffected by treatment,
    but decreased body weights (by 6-20%) and weight gain (by 10-38%) were
    seen in all treated groups except for males fed 250 mg/kg bw.

    Haematological, clinical chemical, and urinary parameters in mice
    necropsied at 12 and 24 months showed no consistent, toxicologically
    significant alterations indicative of target organ toxicity. Changes
    in the weights of the adrenal glands, brain, heart, kidneys, liver,
    testis, and spleen which were found to be statistically significant
    were confounded by the marked decrease in body weight. Nevertheless,
    the consistent increases in the absolute and/or relative weights of
    the liver at all doses suggests a treatment-related effect. Gross
    observations at necropsy in males at the high dose at 12 months and in
    males at the intermediate and high doses at 24 months showed a slight
    increase in the number of mice with liver masses or nodules.
    Microscopic examination of the livers of mice at 12 and 24 months
    revealed treatment-related effects at all doses. The cytoplasm of
    hepatocytes stained homogeneously, indicating liver enzyme induction,
    but there was no evidence of degeneration or necrosis. The microscopic
    changes were dose-related and resembled those associated with
    adaptation to metabolic demands. An increased incidence of
    eosinophilic hepatocellular foci was also observed in males at 100 and
    500 mg/kg bw per day.

         Males fed 1000 mg/kg bw per day and necropsied at 12 months had a
    slightly increased incidence of hepatocellular adenoma. At 24 months,
    a statistically significant increase in the number of males with
    hepatocellular adenoma was seen at 500 ( n = 40) and 1000 ( n = 41)
    mg/kg bw per day, the incidence in controls being 27/50. Low
    incidences of a variant form of hepatocellular carcinoma
    (hepatoblastoma) were observed in all treated groups of males (2/50 at
    250 mg/kg bw per day, 6/50 at 500 mg/kg bw per day, and 3/50 at 1000
    mg/kg bw per day  versus 0/50 in controls), but the incidence of
    hepatocellular carcinoma was not significantly increased at any dose.
    The combined incidence of hepatoblastoma and hepatocellular carcinoma
    was also not significantly increased in the male mice. The primary
    non-tumourous microscopic changes in the livers of male mice, which
    appeared to have been adaptive, ultimately resulted in the promotion
    of hepatocellular adenomas. The incidences of tumours in other tissues
    were not statistically significantly increased. The livers of female
    mice showed similar microscopic adaptive changes, but none of them had
    hepatoblastoma, and no statistically significant increase in the
    incidence of tumours in any tissues was found. Decreased incidences of
    microscopic lesions when compared with controls were found in the
    adrenals, kidneys, lungs, oral tissues, pancreas, peripheral nerve,
    spleen, and testis of males and in the kidneys, lungs, and nasal
    tissues of females. These findings were considered to reflect normal
    variation and the decreased body weights of the mice and not a primary
    response to 2-phenylphenol. A NOAEL for toxicity could not be
    identified. The NOAEL for carcinogenicity was 250 mg/kg bw per day on
    the basis of an increased incidence of hepatocellular adenomas at 500
    mg/kg bw per day (Quast & McGuirk, 1995). 

          Rats 

         2-Phenylphenol (purity, 98%) was administered in the diet at
    concentrations of 0, 6300, 13 000, or 25 000 ppm, equal to 0, 320,
    650, and 1300 mg/kg bw per day to groups of 20-24 male Fischer 344
    rats (Charles River) for 91 weeks. The percentage survival was 96, 90,
    71, and 65% in the four groups, respectively. In the rats that died
    during the study, the incidences of urinary bladder tumours were 0/1
    in controls, 0/2 at 6300 ppm, 7/7 at 13 000 ppm, and 0/8 at 25 000
    ppm. Bladder lesions were found in 10%, 96%, and 48% of rats at the
    three doses, respectively. The incidences of urinary bladder
    papillomas and transitional-cell carcinomas were 23/24 at 13 000 ppm
    and 4/23 at 25 000 ppm. A NOAEL could not be identified since bladder
    lesions were seen at all doses tested (Hiraga, 1983a; Hiraga & Fujii,
    1984). 

         Groups of 70-75 male and 70-75 female Fischer 344 rats were fed
    diets containing 2-phenylphenol (purity, 99.5%) at concentrations of
    0, 800, 4000, or 8000/10 000 ppm, equal to 0, 39, 200, and 400 mg/kg
    bw per day for males and 0, 49, 240, and 650 mg/kg bw per day for
    females, for 1 year before interim sacrifice of satellite groups of 20
    rats per dose and for 2 years for the remaining 50 rats of each sex.
    The animals were observed daily and were examined weekly for clinical
    signs of toxicity. Each animal was weighed once a week and also
    immediately before necropsy to allow calculation of organ:body weight
    ratios. Food consumption was measured weekly. Blood and overnight
    urine samples were collected at 3, 6, 12, 18, and 24 months from the
    first 20 surviving rats of each sex in the group scheduled for
    sacrifice at 2 years. Any dead or moribund animals were prepared for
    necropsy, and all surviving animals were killed at the end of the test
    periods.

         A 5% decrease in body-weight gain was seen in animals at 4000
    ppm, and a decrease of 11% was seen in males at 8000 ppm and in
    females at 10 000 ppm. Food consumption was unaffected in all groups.
    Minor clinical and gross observations included an increased incidence
    of abnormally coloured urine, urine stains, and red stains in male
    rats given 8000 ppm 2-phenylphenol and an increased incidence of urine
    and brown stains in female rats given 4000 or 10 000 ppm. There were
    no treatment-related changes in ophthalmological, haematological,
    clinical chemical, or urinary parameters, except for an increased
    incidence of blood in the urine of males at 8000 ppm. The mortality
    rate was slightly increased among males at 8000 ppm. Gross
    pathological examination showed increased incidences of urinary
    bladder masses in males fed 4000 ppm for 2 years or 8000 ppm for 1 or
    2 years and increased incidence of pitted zones and abnormal texture
    of the kidney in females fed 10 000 ppm for 2 years. Histopathological
    examination showed hyperplasia and transitional-cell carcinoma in the
    urinary bladders of males fed 4000 or 8000 ppm for 1 or 2 years, the
    increase being statistically significant at 8000 ppm and of borderline
    significance at 4000 ppm. The NOAEL for toxicity was 800 ppm, equal to
    39 mg/kg bw per day, on the basis of reduced body-weight gain and

    hyperplasia in the urinary bladder at all doses. The NOAEL for
    carcinogenicity was 800 ppm, equal to 39 mg/kg bw per day (Wahle &
    Christenson, 1996).

          Sodium 2-phenylphenol 

          Mice 

         Sodium 2-phenylphenol (purity, 97%) was administered in the diet
    to groups of 50 male and 50 female B6C3F1 mice (Charles River) at
    concentrations of 0, 5000, 10 000, or 20 000 ppm, equal to 0, 590,
    1400, and 3000 mg/kg bw per day for males and 0, 780, 1500, and 3100
    mg/kg bw per day for females, for 96 weeks. The mice were then given
    control diet for an additional 8 weeks. The survival rate of males at
    the high dose was slightly decreased. Decreased body weight was
    observed in males and females at 20 000 ppm and in females at 5000 and
    10 000 ppm. Alkaline phosphatase activity was increased in females at
    5000, 10 000, and 20 000 ppm. No urinary bladder stones, tumours, or
    extensive renal damage were observed in any of the mice. The NOAEL for
    carcinogenicity was 20 000 ppm, equal to 3000 mg/kg bw per day, the
    highest dose tested (Ito, 1983a; Hagiwara et al., 1984).

          Rats 

         Groups of 20-21 male and 20-21 female Fischer 344 rats were fed
    diets containing 0, 1250, 2500, 5000, 10 000, 20 000, or 40 000 ppm of
    sodium 2-phenylphenol as the tetrahydrate (purity, > 95%), equivalent
    to 0, 70, 140, 270, 550, 1100, or 2200 mg/kg bw per day, for 91 weeks.
    The rats were observed daily for changes in general condition. The
    survival rates were 90, 90, 95, 90, 90, 57, and 71% for the seven
    groups, respectively. Increased incidences of urinary bladder
    papil-lomas and transitional-cell carcinomas were seen, with 1/21 at
    5000 ppm, 7/21 at 10 000 ppm, 20/21 at 20 000 ppm, and 17/20 at 40 000
    ppm. Transitional-cell carcinomas of the kidney were also observed at
    doses > 5000 ppm. The NOAEL was 2500 ppm, equivalent to 270 mg/kg
    bw per day, on the basis of the increased incidence of urinary bladder
    tumours (Hiraga & Fujii, 1981).

         Groups of 50 male and 50 female Fischer 344/DuCrj rats were fed
    diets containing 0, 7000, or 20 000 ppm (males) or 0, 5000, or 10 000
    (females) of sodium 2-phenylphenol (purity, 95.5%) for 104 weeks
    followed by control diet for 2 weeks. In a second study, groups of 25
    male and 25 female rats were fed diets containing the compound at 0,
    2500, 7000, or 20 000 ppm, equal to 0, 95, 270, and 770 mg/kg bw per
    day, for males, and 0, 2500, 5000, or 10 000 ppm, equal to 0, 110,
    220, and 470 mg/kg bw per day, for females, for 104 weeks followed by
    control diet for life. 

         The survival rate at week 104 was 20% in males at 20 000 ppm in
    the first study and 24% in the second study, while those in the other
    groups were > 50%. Urinary bladder tumours were observed in the first
    study in 2/50 males at 7000 ppm, 47/50 males at 20 000 ppm, 1/50
    females at 5000 ppm, and 4/50 females at 10 000 ppm. In the second

    study, the bladder tumour incidence was 3/25 in males at 7000 ppm,
    23/25 in males at 20 000 ppm, and 2/25 in females at 10 000 ppm. In
    the first study, transitional-cell carcinomas were found in 2/2 males
    at 7000 ppm, 46/47 males at 20 000 ppm, and 1/4 females at 10 000 ppm.
    In the second study, carcinomas were found in 1/3 males at 7000 ppm,
    21/23 males at 20 000 ppm, and 1/2 females at 10 000 ppm. The
    incidence of bladder tumours was thus dose-dependent. The NOAEL was
    2500 ppm, equal to 95 mg/kg bw per day, on the basis of urinary
    bladder tumours at all doses (Hiraga, 1983b; Fujii & Hiraga, 1985).

         A working group convened by the International Agency for Research
    on Cancer (IARC) classified sodium 2-phenylphenol as possibly
    carcinogenic to humans and 2-phenylphenol as not classifiable as to
    its carcinogenicity to humans (IARC, 1987, 1999).

    (d)  Genotoxicity

         The results of tests for the genotoxicity of 2-phenylphenol,
    sodium 2-phenylphenol, and the metabolites phenylhydroquinone and
    phenylbenzoquinone are summarized in Table 2.

         Covalent binding to urinary bladder DNA was determined  in vivo 
    in pooled samples from eight male rats dosed with 500 mg/kg bw of
    [14C]2-phenylphenol (purity, 99.8%) or [14C]sodium 2-phenylphenol
    (purity, 98.7%). No radiolabel was detected in DNA from bladders
    excised 16 h after dosing with either compound. The detection limit
    was less than one alkylation per 106 nucleotides. Identical results
    were obtained in a second experiment (Reitz et al., 1983).

         The reactions of 2-phenylphenol and its metabolites
    phenylhydroquinone and phenylbenzo-quinone with DNA were investigated
    by a sequencing technique and by ultraviolet-visible and electron spin
    resonance spectroscopy. In the presence of Cu(II), phenylhydroquinone
    caused extensive DNA damage. Catalase, methionine, and methional
    inhibited the DNA damage completely, whereas mannitol, sodium formate,
    ethanol,  tert-butyl alcohol, and superoxide dismutase did not.
    Phenylhydroquinone plus Cu(II) frequently induced a piperidine-labile
    site at thymine and guanine residues. Addition of Fe(III), Mn(II),
    Co(II), Ni(II), Zn(II), Cd(II), or Pb(II) to phenylhydroquinone did
    not induce DNA damage. This metabolite also induced DNA damage in the
    presence of Cu(II) when peroxide was added, and Cu(II) accelerated the
    autoxidation of phenylhydroquinone to quinone. Electron spin resonance
    spectroscopy revealed that the semiquinone radical is an intermediate
    in the autoxidation. Catalase did not inhibit the acceleration by
    Cu(II). Superoxide dismutase promoted both the autoxidation of
    phenylhydroquinone and the initial rate of semiquinone radical
    production. Electron spin resonance trapping showed that addition of
    Fe(III) produced hydroxyl radicals during the autoxidation of
    phenylhydroquinone, whereas addition of Cu(II) did so sparingly. The
    results suggest that DNA damage induced by phenylhydroquinone plus
    Cu(II) is due to active species other than hydroxyl free radicals
    (Inoue et al., 1990).


        Table 2. Results of studies of the genotoxicity of 2-phenylphenol, sodium 2-phenylphenol, and the metabolites phenylhydroquinone 
             and phenylbenzoquinone

                                                                                                                                   
    End-point            Test object              Concentration          Purity    Result                   Reference
                                                                         (%)
                                                                                                                                   

     2-Phenylphenol 

     In vitro 

    DNA strand breaks    E. coli plasmid          10-6-10-2 mol/L        >  99     Negative - S9            Nagai et al. (1990)

    DNA                  Rat liver DNA            100 mol/L             NR        Positive + S9            Pathak & Roy (1992)
    32P-postlabelling                                                              Negative - S9

    DNA binding          Calf thymus DNA          40 mmol/L              >  99     Positive + S9            Ushiyama et al. (1992)
                                                                                   Negative - S9

    Gene mutation        B. subtilis H17, M45     NR                     NR        Negative                 Shirasu et al. (1978)

    Gene mutation        S. typhimurium           10 -1000 g/plate      NR        Negative + S9            Ishidate et al. (1983)
                         TA92, TA1535,                                             Negative - S9
                         TA100, TA1537, 
                         TA94, TA98

    Gene mutation        E. coli WP2 hcr          NR                     NR        Negative + S9            Shirasu et al. (1978)
                                                                                   Negative - S9

    Gene mutation        S.  typhimurium          3-200 g/plate         >  99     Negative + S9            National Toxicology
                         TA100, TA1535,                                            Weakly positive + S9     Program (1986)
                         TA1537, TA98

    Gene mutation        Mouse lymphoma           0.3-60 g/ml           >  99     Positive + S9            National Toxicology
                         (L5178Y) cells,                                           Positive - S9            Program (1986)
                         Tk locus
                                                                                                                                   

    Table 2. (continued)

                                                                                                                                   
    End-point            Test object              Concentration          Purity    Result                   Reference
                                                                         (%)
                                                                                                                                   

    Gene mutation        Human Rsa cells,         1-30 g/ml             NR        Positive                 Suzuki et al. (1985)
                         repair deficient,
                         HPRT locus

    Chromosomal          CHO-K1 cells             50-175 g/ml           >  99     Positive - S9            Tayama-Nawai et al. 
    aberration                                                                                              (1984)

    Chromosomal          CHO fibroblasts          12-125 g/ml           NR        Weakly positive + S9     Ishidate et al. (1983)
    aberration                                                                     Weakly positive - S9

    Chromosomal          CHO-K1 cells             60-90 g/ml            > 99      Negative + S9            National Toxicology
    aberration                                                                     Negative - S9            Program (1986)

    Chromosomal          CHO-K1 cells             25-175 g/ml           >  99     Positive + S9            Tayama et al. (1989)
    aberration                                                                     Negative - S9

    Chromosomal          CHO-K1 cells             100-200 g/ml          > 99      Positive + S9            Tayama & Nakagawa 
    aberration                                                                     inhibited by             (1991)
                                                                                   cysteine or
                                                                                   glutathione

    Host-mediated        S. typhimurium G46       200 or 600 mg/kg       NR        Negative                 Shirasu et al. (1978)
    gene mutation        in male JCL-ICR mice     bw orally for 5 days

     In vivo 

    Sex-linked           D. melanogaster          250 ppm in feed        >  99     Negative                 National Toxicology
    recessive lethal                              for 3 days or                                             Program (1986)
    mutation                                      injection of 500 ppm 

    DNA binding          Male rat urinary         500 mg/kg bw           >  99     Negative                 Reitz et al. (1983)
                         bladder                  orally
                                                                                                                                   

    Table 2. (continued)

                                                                                                                                   
    End-point            Test object              Concentration          Purity    Result                   Reference
                                                                         (%)
                                                                                                                                   

    DNA                  Rat urinary bladder      60-940 mg/kg           > 99      Positive at              Christenson et al. 
    32P-postlabelling                             bw per day                       570 and 940              (1996a)
                                                                                   mg/kg bw per day

    Chromosomal          Male rat bone            800 mg/kg bw           NR        Negative                 Shirasu et al. (1978)
    aberration           marrow                   over 5 days or
                                                  single doses 
                                                  < 4000 mg/kg 
                                                  bw orally

    Dominant lethal      Male mice                100 or 500 mg/kg       > 99      Negative                 Kaneda et al. (1978)
    mutation                                      bw per day for 5 days

    Dominant lethal      Male mice                100 or 500 mg/kg       NR        Negative                 Shirasu et al. (1978)
    mutation                                      bw per day for 5 
                                                  days

     Sodium 2-phenylphenol 

     In vitro 

    Gene mutation        S. typhimurium           50-5000 g/plate       NR        Negative + S9            Ishidate et al. (1983)
                         TA100, TA98                                               Negative - S9

    Gene mutation        S. typhimurium           0.025-250 g/plate     99        Negative + S9            Reitz et al. (1983)
                         TA100, TA98,                                              Negative - S9
                         TA1535, TA1537,
                         TA1538

    Uncheduled DNA       Male rat primary         10-7-10-4 mol/L        99        Negative                 Reitz et al. (1983)
    synthesis            hepatocytes

                                                                                                                                   

    Table 2. (continued)

                                                                                                                                   
    End-point            Test object              Concentration          Purity    Result                   Reference
                                                                         (%)
                                                                                                                                   

     In vivo 

    DNA                  Male rat urinary         2% in diet  for        >  99     Positive                 Ushiyama et al. (1992)
    32P-postlabelling    bladder                  13 weeks

    DNA                  Mouse skin               10 or 20 mg/animal     97        Positive                 Pathak & Roy (1993)
    32P-postlabelling                             topically

     Phenylhydroquinone 

     In vitro 

    DNA strand breaks    E. coli plasmid          10-6-10-2 mol/L        >  99     Positive                 Nagai et al. (1990)

    DNA                  Rat liver DNA            100 mol/L             NR        Positive + S9            Pathak & Roy (1992)
    32P-postlabelling                                                              Negative - S9

    DNA binding          Calf thymus DNA          40 mmol/L              >  99     Positive - S9            Ushiyama et al. (1992)

    Gene mutation        V79 CH  lung             6 -125 mol/L          NR        Negative                 Lambert & Eastmond 
                         fibroblast cells,                                                                  (1994)
                         Hprt locus,
                          arachidonic acid

    Chromosomal          CHO fibroblast           1-25 g/ml             NR        Negative + S9            Ishidate et al. (1983)
    aberration           cell line                                                 Negative - S9

    Chromosomal          CHO-K1 cells             5-150 g/ml            >  98     Positive + S9            Tayama et al. (1989)
    aberration                                                                     Negative - S9
                                                                                                                                   

    Table 2. (continued)

                                                                                                                                   
    End-point            Test object              Concentration          Purity    Result                   Reference
                                                                         (%)
                                                                                                                                   

    Chromosomal          CHO-K1 cells             0.3-30 mol/L          >  98     Positive,                Tayama & Nakagawa 
    aberration                                                                     inhibited by             (1991)
                                                                                   cysteine or
                                                                                   glutathione

    Sister chromatid     CHO-K1 cells             5-150 g/ml            >  98     Positive + S9            Tayama et al. (1989)
    exchange                                                                       Positive - S9

    Sister chromatid     CHO-K1 cells             0.3-30 mol/L          >  98     Positive,                Tayama & Nakagawa 
    exchange                                                                       inhibited by             (1991)
                                                                                   cysteine or
                                                                                   glutathione

    Micronucleus         V79 CH  lung             6-125 mol/L           NR        Positive + S9            Lambert & Eastmond 
    formation            fibroblast cells                                          Negative - S9            (1994)
                          arachidonic acid

     In vivo 

    DNA damage           Male rat urinary         0.0005-0.1%            99%       Negative                 Morimoto et al. (1989)
                         bladder                  by injection

    DNA                  Mouse skin               100 mol/L             NR        Positive + S9            Pathak & Roy (1992)
    32P-postlabelling                                                              Negative - S9

     Phenylbenzoquinone 

     In vitro 

    DNA strand breaks    E. coli plasmid          10-6-10-2 mol/L        >  99     Negative                 Nagai et al. (1990)

    DNA binding          Calf thymus DNA          40 mmol/L              >  99     Positive - S9            Ushiyama et al. (1992)
                                                                                                                                   

    Table 2. (continued)

                                                                                                                                   
    End-point            Test object              Concentration          Purity    Result                   Reference
                                                                         (%)
                                                                                                                                   

    Gene mutation        S. typhimurium           0.05-1000              NR        PBQ negative/            Ishidate et al. (1983)
                         TA100, TA2637,           g/plate                         PBQ negative
                         TA98 

    Gene mutation        V79 CH  lung             6 -125 mol/L          NR        Negative                 Lambert & Eastmond 
                         fibroblast cells,                                                                  (1994)
                         Hprt locus,
                          arachidonic acid

    Chromosomal          CHO fibroblast           1-25 g/ml             NR        Negative + S9            Ishidate et al. (1983)
    aberration           cell line                                                 Negative - S9

    Micronucleus         V79 CH lung              6-125 mol/L           NR        Negative                 Lambert & Eastmond 
    formation            fibroblast cells                                                                   (1994)
                          arachidonic acid

     In vivo 

    DNA damage           Male rat urinary         0.0005-0.1%            99%       Negative                 Morimoto et al.  (1989)
                         bladder                  by injection
                                                                                                                                   
    

         DNA adduct formation in HL-60 cells treated with the
    2-phenylphenol metabolites 2-phenylhydroquinone and
    2-phenylbenzoquinone was studied by 32P-postlabelling. Treatment
    with 25-500 mol/L of 2-phenylhydroquinone for 8 h produced one
    principal and three minor adducts, with a relative distribution of 80,
    10, 6, and 4%. The relative adduct frequencies were 0.26-2.3
    adducts/107 nucleotides. Treatment with 25-250 mol/L of
    2-phenylbenzoquinone for 2 h resulted in a similar level of DNA
    modification and adduct distribution. Reaction of purified calf thymus
    DNA with 2-phenylbenzo-quinone produced one DNA adduct, which did not
    correspond to the major adduct produced in HL-60 cells. These results
    show that both metabolites can form DNA adducts. Peroxidase activation
    of 2-phenylphenol may therefore play a role in its carcinogenic effect
    (Horvath  et al., 1992).

         In a similar study of covalent binding to DNA,
    32P-postlabelling analysis of the products of reaction of DNA with
    phenylbenzoquinone revealed four major and several minor adducts.
    Chemical reaction with deoxyguanosine 3'-phosphate also resulted in
    four major adducts, and their chromatographic mobility was identical
    to that of major adducts of phenylbenzoquinone-DNA, which were shown
    to be stable. More total covalent binding was found in deoxyguanosine
    3'-phosphate than in DNA. Reaction of DNA with 2-phenylphenol or
    phenylhydroquinone in the presence of microsomes and NADPH or cumene
    hydroperoxide also resulted in four major adducts, and their formation
    was drastically decreased by known inhibitors of cytochrome P450. The
    chromatographic mobility of these adducts matched that of the adducts
    observed in deoxyguanosine 3'-phosphate and DNA reacted with
    phenylbenzoquinone. Thus, both 2-phenyl-phenol and phenylhydroquinone
    can bind covalently to DNA in the presence of a microsomal cytochrome
    P450 activation system, and phenylbenzoquinone is one of the
    DNA-binding metabolites of 2-phenylphenol (Pathak & Roy, 1992).

         Covalent modification of skin DNA by sodium 2-phenylphenol
     in vivo was studied by the 32P-postlabelling method to elucidate
    the biochemical mechanism of promotion of chemically induced skin
    carcinogenesis by this compound. Topical application of sodium
    2-phenylphenol or phenylhydroquinone to the skin of CD-1 mice produced
    four distinct major and several minor adducts in skin DNA. Total
    covalent binding in skin DNA was 0.31 fmol/g DNA after treatment with
    10 mg of sodium 2-phenylphenol and 0.62 fmol/g DNA with 20 mg. The
    adducts were not observed in skin DNA of untreated animals.
    Pretreatment of the mice with a-naphthylisothiocyanate, an inhibitor
    of cytochrome P450, or indomethacin, an inhibitor of prostaglandin
    synthase, resulted in lower numbers of DNA adducts. Incubation of DNA
    with 2-phenylphenol or phenylhydroquinone  in vitro in the presence
    of cytochrome P450 or prostaglandin synthase activation systems
    resulted in four major adducts. The pattern of chromatographic
    mobility observed  in vitro in the presence of these enzymatic
    systems appeared to be similar to that of adducts  in vivo. The
    chemical reaction of DNA or deoxyguanosine monophosphate with
    phenylbenzoquinone also resulted in four major and several minor
    adducts. The four major adducts were identical in chromatographic

    mobility to the four major adducts produced  in vivo and  in vitro. 
    The results show that 2-phenylphenol and phenylhydroquinone can bind
    covalently to DNA and that one of the DNA-binding metabolites of
    2-phenylphenol may be phenylbenzoquinone (Pathak & Roy, 1993).

    (e)  Reproductive toxicity 

         (i)  Multigeneration reproductive toxicity

          2-Phenylphenol 

          Rats 

         Groups of 35 albino Sprague-Dawley rats, nine weeks old at the
    start of the study, were fed diets containing technical-grade
    2-phenylphenol (purity, 99.4-99.5%) at concentrations of
    200-10 000 ppm, equal to 0, 36, 120, and 460 mg/kg bw per day, for two
    generations. The dose was adjusted during the premating period
    according to changes in body weight, and adjustments were made during
    lactation to avoid overdosing the pups, although this was later
    considered unnecessary. Two F1 females at the high dose and 12 F2a
    pups were removed from the study for examination of the heritability
    of hypotrichosis. Parents (F0) for the F1 generation were assigned
    randomly to dose groups. The genealogy of the F1b pups was checked
    to prevent the mating of littermates. For production of the F2
    generation, 132 male and 134 female F1b pups were selected
    randomly,one or more pups of each sex per litter being used as parents
    (F1), divided into 35 pairs of rats per dose, except for the
    controls which consisted of 27 males and 29 females. Estrus cycles
    were studied by vaginal smears. The animals were examined daily, and
    routine observations of birth statistics and pup weights were made;
    the litters were culled to eight pups when necessary. Standard
    observations and extensive histological examination of the urinary
    tract were carried out at sacrifice.

         Treatment did not affect clinical signs, body-weight gain during
    gestation or lactation, or any of the reproductive variables examined.
    Histological examination of the adults and pups revealed no
    significant changes in the reproductive tracts. F0 and F1 adults
    at 460 mg/kg bw per day showed a treatment-related decrease in body
    weight which was not clearly related to a decrease in food
    consumption, and F1b, F2a, and F2b pups showed a statistically
    significant decrease in body weight on days 14 and/or 21 of lactation,
    an effect not seen during the first week of lactation, indicating that
    development had not been disturbed. The relative weight of the kidneys
    was increased in a dose-dependent manner, in the absence of changes in
    other organ weights, in males of the F0 and F1 generations. Male
    rats at 120 and 460 mg/kg bw per day had an increased incidence of
    calculi in the urinary tract. Transitional-cell hyperplasia was found
    in the urinary bladder, defined in the report as 'an area (focal or
    diffuse) of at least three to four cells thick (of cuboidal cells) in
    an inflated bladder', whereas normal bladders had a cell thickness of
    one or two flattened cells. Quantification by simple morphometry

    indicated a compound-related effect in the bladder in F0 males and
    females at 120 and 460 mg/kg bw per day and in F1 males at 457 mg/kg
    bw per day. Neoplasms of the urinary tract were found in four rats:
    one bladder and one ureteric tumour in rats at 125 mg/kg bw per day
    and two bladder tumours in rats at 457 mg/kg bw per day. The NOAEL for
    reproductive toxicity was 460 mg/kg bw per day and that for
    carcinogenicity was 36 mg/kg bw per day (Eigenberg, 1990).

         In a similar study, groups of 30 CD Sprague-Dawley rats were fed
    diets containing technical-grade 2-phenylphenol (purity, 99.5-100%) at
    concentrations of 200-10 000 ppm, equal to 0, 17, 92, and 460 mg/kg bw
    per day, for two generations. The dose was adjusted during the
    premating period according to changes in body weight. The F0 and
    F1 adults received the compound in the diet throughout the study,
    beginning at 7 weeks of age for the F0 adults and at weaning for the
    F1 adults. The animals received treated feed for 10 weeks before
    breeding, beginning approximately 2 weeks after weaning of the last
    F1b litter for F1 parents. F0 adults were mated to produce the
    F1a and F1b litters, and F1 adults (consisting of randomly
    selected F1b pups) were mated to produce the F2a and F2b
    litters.Adult animals were evaluated during the study for effects of
    2-phenylphenol on body weight, food consumption, clinical signs,
    estrous cycling, mating, fertility, length of gestation, and litter
    size. The offspring were evaluated for effects on sex ratio,
    viability, body-weight gain, and clinical signs. Gross necropsy was
    performed on all adults and pups, and the reproductive organs,
    pituitary, kidneys with ureter attached, urinary bladder, and gross
    lesions of all F0 and F1 adults were evaluated histologically.

         At 460 mg/kg bw per day, urine staining was observed in F0
    males and females and F1 males, urinary bladder calculi were found
    at necropsy in F1 adult males, and one F0 male died from renal
    failure. At this dose, there was an increase in food consumption by
    females during lactation, decreased pup weight, and a decrease in the
    terminal body weight of F0 and F1 adult males and females.
    Histopathological examination of the kidneys revealed debris in the
    renal pelvis, chronic active inflammation, and increased severity of
    background lesions in F0 and F1 males. Further, transitional-cell
    hyperplasia (simple, nodular, or papilary) of the bladder, calculi,
    chronic inflammation of the bladder, and dilatation and hyperplasia of
    the ureter were seen in F0 and F1 males. Two F1 males at this
    dose had malignant lymphomas in several tissues. One F1 female at 92
    mg/kg bw per day had a nephroblastoma, and one F0 male at the high
    dose and one control F1 female had a pituitary adenoma. All of these
    lesions were considered to be incidental to treatment.

         There were no treatment-related effects on adult reproductive
    parameters and no effect on litter size, sex ratio, the number of
    stillborn pups, pup viability, or clinical signs, no gross lesions in
    the pups, and no treatment-related effects on the organ weights of
    adults. The mean live birth indexes (with standard error) were 98
    (0.91) in the controls, 99 (0.77) at 17 mg/kg bw per day, 98 (1.3) at
    92 mg/kg bw per day, and 98 (0.85) at 460 mg/kg bw per day in the

    F1a generation; 98 (0.85) in the controls, 98 (1.5) at 17 mg/kg bw
    per day, 99 (0.71) at 92 mg/kg bw per day, and 99 (0.63) at 460 mg/kg
    bw per day in the F1b generation; 99 (0.84) in the controls, 98
    (1.0) at 17 mg/kg bw per day, 97 (1.4) at 92 mg/kg bw per day, and 100
    (0.38) at 460 mg/kg bw per day in the F2a generation; and 97 (1.2)
    in the controls, 99 (0.78) at 17 mg/kg bw per day, 96 (1.9) at 92
    mg/kg bw per day, and 99 (0.46) at 460 mg/kg bw per day in the F2b
    generation. The differences between the groups were not statistically
    significant. The NOAEL for reproductive toxicity was 460 mg/kg bw per
    day, the highest dose tested. The NOAEL for systemic and developmental
    toxicity was 92 mg/kg bw per day, on the basis of decreased body
    weight and morphological lesions in the kidneys, urinary bladder, and
    ureter and a decrease in pup body weight (Eigenberg, 1995). 

         (ii)  Developmental toxicity 

          2-Phenylphenol and sodium 2-phenylphenol 

          Mice 

         Groups of 20-21 pregnant JCL-ICR mice were given 0, 1500, 1700,
    or 2100 mg/kg bw per day of 2-phenylphenol (purity not given) or 100,
    200, or 400 mg/kg bw per day of sodium 2-phenylphenol (purity not
    given) by gavage on days 7-15 of gestation. The animals were weighed
    daily, and any change in their general condition was noted. On day 18
    of gestation, the dams were killed and their uteri opened, and the
    numbers of implantation scars, fetuses that died in early and late
    stages, and live fetuses were counted. The live fetuses were weighed
    and sexed and observed for external abnormalities. The number of
    corpora lutea in each ovary was counted, the major organs were
    weighed, and examinations were made to determine whether any
    macroscopic abnormalities were present.

         With 2-phenylphenol, body-weight gain was reduced at 1700 and
    2100 mg/kg bw per day. Four dams at 1500 mg/kg bw per day, seven at
    1700 mg/kg bw per day, and 16 at 2100 mg/kg bw per day died. Pregnancy
    was confirmed in 5/5 surviving dams at 2100 mg/kg bw per day, 14/14 at
    1700 mg/kg bw per day, 14/17 at 1500 mg/kg bw per day, and 20/21
    controls, in which implantation was confirmed at the time of sacrifice
    and laparotomy on day 18 of gestation. The only significant changes
    found at autopsy of these mice were reduced heart weights at 1700 and
    2100 mg/kg bw per day and significantly increased liver weights at
    1500 and 1700 mg/kg bw per day, a tendency that was also seen at 2100
    mg/kg bw per day. Live fetuses were found in all pregnant dams. The
    body weights of male and female fetuses at all three doses of
    2-phenylphenol were significantly reduced, and the decrease was
    dose-related in males. No unique external or internal deformities or
    abnormalities were found in the fetuses, and the skeletal
    abnormalities found were compatible with delayed development. No NOAEL
    could be identified for maternal or fetotoxicity. The NOAEL for
    developmental toxicity was 2100 mg/kg bw per day, the highest dose
    tested.

         With sodium 2-phenylphenol, body-weight gain was statistically
    significantly reduced in a dose-dependent manner in dams at all doses.
    Four animals at 200 mg/kg bw per day and 16 at 400 mg/kg bw per day
    died. No abnormalities were seen at autopsy of dams on day 18 of
    gestation. Significantly reduced liver, heart, and spleen weights were
    recorded in animals at 400 mg/kg bw per day, while the weight of the
    lungs was increased in dams at 200 mg/kg bw per day. Dams at 200 mg/kg
    bw per day had a low average number of implantations and a low average
    number of live fetuses. No NOAEL could be identified for maternal
    toxicity. The NOAEL was 100 mg/kg bw per day for fetotoxicity and 400
    mg/kg bw per day, the highest dose tested, for developmental toxicity
    (Ogata  et al., 1978). 

          2-Phenylphenol 

          Rats 

         Groups of 18-20 pregnant Wistar rats were given 0, 150, 300, or
    600 mg/kg bw per day of 2-phenylphenol (purity, 99.7%) by gavage on
    days 6-15 of gestation. An additional group of 11 rats was given 1200
    mg/kg bw per day, but this dose proved to be lethal. No untoward signs
    of toxicity were observed in the controls or at 150 mg/kg bw per day.
    At doses > 300 mg/kg bw per day, dose-related ataxia and decreased
    mean body-weight gains were observed. All surviving rats were killed
    on day 20 of gestation, and the uterine contents were examined.
    Fetuses were grossly examined; the skeletons were examined with
    Alizarin red S and the viscera by a modified Wilson method. The mean
    numbers of implantation sites, live fetuses, resorptions, and fetal
    weights in animals at 150 and 300 mg/kg bw per day were comparable to
    those of controls, but at 600 mg/kg bw per day the number of fetal
    resorptions was increased and fetal weight was decreased. Although a
    few fetal anomalies were observed in all groups, they did not appear
    to be related to treatment. The NOAEL was 150 mg/kg bw per day for
    maternal toxicity, 300 mg/kg bw per day for fetotoxicity, and 600
    mg/kg bw per day, the highest dose tested, for developmental toxicity
    (Kaneda et al., 1978).

         In a similar study, groups of 25-35 pregnant rats were given 0,
    100, 300, or 700 mg/kg bw per day of 2-phenylphenol (purity, 99.7%) by
    gavage on days 6-15 of gestation. They were killed on day 21, and the
    fetuses were removed surgically. All fetuses were weighed, sexed, and
    examined externally and skeletally, and the soft tissues of
    approximately one-third of the fetuses were examined. One rat at the
    high dose died as a result of a dosing accident. Pregnant rats given
    700 mg/kg bw per day gained significantly less body weight during the
    first 4 days of treatment (days 6-9 of gestation) than did controls,
    and their food consumption was significantly decreased on days 9-11 of
    gestation. At necropsy, the weights of the liver (but not the
    liver:body weight ratios) were significantly decreased. There was no
    effect on the number of implantation sites per dam, mean litter size,
    incidences of resorptions, or fetal body weight or crown-rump length.
    The only major malformation -- hypoplastic tail and missing sacral and
    caudal vertebrae -- was observed in a single fetus at 300 mg/kg bw per

    day. An increase in the incidence of delayed ossification of
    sternebrae and unossified sternebrae was observed at 700 mg/kg bw per
    day. The incidences of foramina and bony islands in the skull were
    also slightly increased in this group. No adverse effects on embryonic
    or fetal development were observed that were considered to be due to
    2-phenylphenol. The NOAEL was 300 mg/kg bw per day for maternal
    toxicity and 700 mg/kg bw per day, the highest dose tested, for
    fetotoxicity and developmental toxicity (John et al., 1981). 

          Rabbits 

         In a range-finding study, groups of two non-pregnant New Zealand
    white rabbits were given doses of 0, 100, 500, or 1000 mg/kg bw per
    day of 2-phenylphenol (purity, 99.8%) in corn oil for 13 consecutive
    days and were submitted to gross necropsy after the last day. The
    animals were examined for clinical signs, body weights, body-weight
    gain, kidney and liver weights, and gross appearance. The rabbits at
    1000 mg/kg bw per day appeared to have stopped eating and had lost 24%
    of their body weight by day 7. One rabbit at this dose died on day 8,
    and the second was killed in moribund condition on day 10 with
    nonspecific lesions or lesions secondary to anorexia. Rabbits given
    500 mg/kg per day showed a slight decrease in body-weight gain. All
    the other rabbits survived to the end of the study with no other
    treatment-related effects. The dose of 100 mg/kg bw per day was
    tolerated over the course of treatment.

         In the second study, groups of seven artificially inseminated
    females were given 0, 250, 500, or 750 mg/kg bw per day of
    2-phenylphenol (purity, 99.8%) in corn oil by gavage on days 7-19 of
    gestation. They were observed for clinical signs, body weight, and
    body-weight gain. On day 20 of gestation, all surviving animals were
    killed and examined for gross pathological alterations and changes in
    liver and kidney weights. The uteri and ovaries were examined for
    implantations, resorptions, and corpora lutea, and the liver, kidneys,
    and stomach were examined histologically. Dose-related signs of
    maternal toxicity were seen at all doses. One rabbit at 250 mg/kg bw
    per day, two at 500 mg/kg bw per day, and six at 750 mg/kg bw per day
    died. The dose-related effects observed included increased incidences
    of haemorrhage, gaseous distension, and erosions of the stomach,
    decreased or soft ingesta in the gastrointestinal tract, decreased
    body weight and body-weight gain, increased absolute and relative mean
    weights of the kidney, and increased incidence and/or severity of
    renal tubular degeneration and inflammation. Treatment-related effects
    were observed on reproductive, embryonal, or fetal parameters at 750
    mg/kg bw per day.

         In the third study, groups of 16-24 artificially inseminated
    adult female New Zealand white rabbits were given 0, 25, 100, or 250
    mg/kg bw per day of 2-phenylphenol (purity, 99.8%) in corn oil by
    gavage on days 7-19 of gestation. They were observed for clinical
    signs, body weight, and body-weight gain. On day 28 of gestation, all
    surviving rabbits were killed and necropsied, when the weights of the
    liver, kidney, and gravid uterus and the numbers of corpora lutea,

    implantations, resorptions, and live and dead fetuses were recorded.
    All fetuses were removed from the uterus, weighed, sexed, and examined
    for external, visceral, and skeletal alterations. The kidneys of all
    animals were examined histologically. Administration at 250 mg/kg bw
    per day resulted in maternal toxicity evidenced by treatment-related
    mortality (13%), gross pathological alterations (ulceration and
    haemorrhage of the gastric mucosa, haemolysed blood in the intestinal
    tract, and decreased ingesta), and histopathological alterations
    (renal tubular degeneration and inflammation). No significant maternal
    effects were observed at 25 or 100 mg/kg bw per day, and no adverse
    embryonal or fetal effects were observed at any dose. The overall
    NOAELs were 100 mg/kg bw per day for maternal toxicity, 500 mg/kg per
    day for fetotoxicity, and 750 mg/kg per day, the highest dose tested,
    for developmental toxicity (Zablotny et al., 1991). 

    (f)  Special studies: Mechanisms of carcinogenicity in rat urinary
         bladder

         In a study to determine whether 2-phenylphenol is a complete skin
    carcinogen or a promoter in a two-stage initiation and promotion
    process, the compound was applied to the interscapular area of the
    backs of 50 male and 50 female Swiss CD-1 mice at a dose of 55.5 mg in
    0.1 ml acetone, three times per week for 2 years. A second group of 50
    male and 50 female mice was treated identically except that their
    backs were pretreated once with 0.05 mg in 0.1 ml acetone of
    7,12-dimethylbenz[ a]anthracene (DMBA), a known initiator of skin
    cancer. Additional groups of 50 male and 50 female mice served as
    acetone vehicle controls, controls treated once with DMBA and
    thereafter only with acetone, and a positive control group treated
    once with DMBA and thereafter with 12- O-tetradecanylphorbol
    13-acetate (TPA), a known promoter of skin cancer, at a dose of 0.005
    mg in 0.1 ml acetone, three times per week for 2 years.

         The mean body weights and survival of the mice treated with
    2-phenylphenol or with DMBA plus 2-phenylphenol were generally similar
    to those of the respective negative control groups, but the survival
    of the group given DMBA plus TPA was substantially decreased. In this
    group, the incidences of squamous-cell papillomas and carcinomas,
    keratocanthomas, and basal-cell carcinomas at the site of application
    were clearly increased (52/100) over that in the group given DMBA plus
    acetone(15/100). The time to tumour was also substantially decreased
    in the group given DMBA plus TPA. Similar neoplastic skin lesions were
    observed with DMBA plus acetone (17/100), but at an incidence
    equivalent to that in the control group (15/100). No neoplastic skin
    lesions were observed in the group given 2-phenylphenol. The author
    concluded that 2-phenylphenol is not carcinogenic alone or as a
    promoter (Luster, 1986).

         The promoting effect of 2-phenylphenol (purity, 98%) and sodium
    2-phenylphenol (purity, 97%) in the urinary bladder was studied in
    male Fischer 344 rat initiated with
     N-nitrosobutyl- N-(4-hydroxybutyl)amine (NBHBA). Groups of 30 rats
    were given drinking-water containing 0.01% NBHBA for 4 weeks and then

    diets containing 20 000 ppm of sodium 2-phenylphenol (equivalent to
    1000 mg/kg bw per day) for 32 weeks, NBHBA for 4 weeks followed by
    untreated feed for 32 weeks, or drinking-water without NBHBA for 4
    weeks followed by diet containing 20 000 ppm of sodium 2-phenylphenol
    for 32 weeks. In another experiment, groups of 30 male rats were given
    0.05% NBHBA in drinking-water for 4 weeks followed by diets containing
    20 000 ppm of sodium 2-phenylphenol or 20 000 ppm of 2-phenylphenol
    for 32 weeks, no NBHBA for 4 weeks, and then 20 000 ppm of sodium
    2-phenylphenol (15 rats) or 20 000 ppm of 2-phenylphenol (15 rats) in
    the diet for 32 weeks. In a third experiment, groups of 15 rats were
    given diets containing 0, 20 000 ppm of sodium 2-phenylphenol, or
    20 000 ppm of 2-phenylphenol. Urine samples were obtained from these
    rats by forced urination on days 27, 29, and 32. 

         Administration of 20 000 ppm sodium 2-phenylphenol in the diet
    significantly increased the incidence and number of preneoplastic
    lesions (papillary or nodular hyperplasia) per 10 cm of basement
    membrane of the urinary bladder in male rats pretreated with 100 ppm
    NBHBA, and the incidence and number of papillomas and carcinomas of
    the urinary bladder in the group pretreated with 500 ppm NBHBA.
    Moreover, treatment with sodium 2-phenylphenol alone, without
    initiation, induced  papillary or nodular hyperplasia, papillomaa, and
    carcinoma. In contrast, administration of 2-phenylphenol in the diet
    after initiation only slightly increased the incidence of urinary
    bladder lesions over that with NBHBA alone, and its effect was not
    statistically significant. No tumours of the urinary bladder were
    induced by 2-phenylphenol alone. The authors concluded that sodium
    2-phenylphenol, and not 2-phenylphenol, has tumour promoting activity
    and might be a complete carcinogen in rat urinary bladder. Since the
    sodium salt increased the pH of the urine, the authors speculated that
    an active metabolite reaches the urinary bladder at a higher
    concentration than with 2-phenylphenol. They suggested that sodium
    2-phenylphenol is a carcinogens that acts by a non-genotoxic mechanism
    (Fukushima et al., 1983). 

         In a similar study, 2-phenylphenol or sodium 2-phenylphenol
    (purity of neither given) was administered in the diet at a
    concentration of 20 000 ppm to 28 male Fischer 344 rats for 64 weeks.
    One rat receiving sodium 2-phenylphenol had small stones in the
    urinary bladder, and this compound, but not 2-phenylphenol, induced
    papillary or nodular hyperplasia (19/28), papillomas (5/28), and
    carcinomas  (6/28) of the urinary bladder. Pretreatment of additional
    rats with NBHBA increased the incidence of papillary or nodular
    hyperplasia ( p < 0.05), papillomas (not significant), and
    carcinomas (not significant) over that in rats treated with NBHBA
    alone. In another experiment, 2-phenylphenol or sodium 2-phenylphenol
    was administered in the diet at concentrations of 2500, 5000, 10 000,
    or 20 000 ppm to groups of five to nine male Fischer 344 rats for up
    to 104 weeks. Animals from each group were killed and examined at 4,
    8, 12, 24, 36, and 104 weeks. No stone formation was observed in the
    urinary bladders of rats treated with sodium 2-phenylphenol. At 20 000
    ppm, simple hyperplasia of the urinary bladder was observed from 4
    weeks in 5/5 animals, papillary or nodular hyperplasia from 36 weeks

    in 5/5 animals, and papillomas in 2/5 and carcinomas in 2/5 at 104
    weeks. At 10 000 ppm of sodium 2-phenylphenol, only simple hyperplasia
    was observed from 36 weeks. 2-Phenylphenol alone did not cause bladder
    tumours and did not enhance the bladder lesions induced by NBHBA (Ito,
    1983b).

         In a short-term assay for bladder carcinogenicity in rats,
    increased agglutinability of bladder epithelial cells with
    concanavalin A was observed after a 1-week treatment with 10 000 or
    20 000 ppm of 2-phenylphenol or sodium 2-phenylphenol (purity of
    neither given), suggesting that these compounds cause bladder cancer.
    No such increase was observed in rats fed diets containing
    rho-phenylphenol or biphenyl derivatives at 20 000 ppm. In male Fisher
    rats fed diets containing 20 000 ppm of sodium 2-phenylphenol for 50
    weeks, bladder papillomas developed in 19 of 36 rats and bladder
    carcinomas in 14 of 36 rats (Honma et al., 1983).

         Groups of 20 male Fischer 344 rats were fed diets containing
    20 000 ppm of 2-phenylphenol, 20 000 ppm of sodium 2-phenylphenol
    (purity of neither given), or 5000 ppm of biphenyl for up to 24 weeks.
    Changes in the amounts of DNA synthesis and in the morphology of the
    renal papilla and renal pelvis  were recorded under light and scanning
    electron microscopes. Increased DNA synthesis in both renal papilla
    and pelvis and distinct morphological alterations in the cell surface
    were seen with 2-phenylphenol and its sodium salt by 4 weeks.
    Sequential light microscopy revealed renal papillary necrosis in
    animals fed 2-phenylphenol from week 4, followed by regenerative
    hyperplasia at weeks 16 (1/5) and 24 (3/5), but no changes in the
    renal pelvis. Feeding of sodium 2-phenylphenol caused similar changes
    in the renal papillae and also hyperplasia in the renal pelvis (2/5).
    No proliferative response of the kidney was apparent in rats fed
    biphenyl. The authors concluded that the proliferative responses
    caused by sodium 2-phenylphenol in the renal pelvic epithelium were
    similar to those induced by this compound in the urinary bladder
    (Shibata et al., 1989a).

         The interactive effects of ascorbic acid, saccharin, and hippuric
    acid on the carcinogenicity of 2-phenylphenol and sodium
    2-phenylphenol were studied in groups of 20 male Fischer 344 rats. The
    animals were given 2-phenylphenol (purity, 99.5%) or sodium
    2-phenylphenol in the diet at a concentration of 20 000 ppm
    (equivalent to 1000 mg/kg bw per day) for 24 weeks with or without
    ascorbic acid, sodium ascorbate, acid saccharin, sodium saccharin,
    hippuric acid, or sodium hippurate at 50 000 ppm. The urinary sodium
    concentration was increased in all animals receiving sodium salts
    and/or sodium 2-phenylphenol. The pH of the urine was increased in
    those given sodium 2-phenylphenol, sodium ascorbate, or sodium
    saccharin, and the osmolality was decreased in those given sodium
    2-phenylphenol, sodium ascorbate, or sodium hippurate. 2-Phenylphenol
    decreased the osmolality but did not affect the pH or sodium
    concentration of urine. Histopathologically, the bladders of rats
    given sodium 2-phenylphenol showed epithelial thickening (epithelial
    thickness, four to eight cells) at 8, 16, and 24 weeks and papillary

    and nodular changes at 16 and 24 weeks. Treatment with the other
    sodium salts provoked 'slight to moderate' hyperplasia at 8 and 16
    weeks but no papillary or nodular changes; the changes had regressed
    by 24 weeks. The combination of raised urinary pH and sodium promoted
    the effects of sodium 2-phenylphenol, while sodium hippurate raised
    urinary sodium but not pH and had no effect (Fukushima et al., 1989).

         In an essentially similar study, groups of 31 male Fischer 344
    rats received NaHCO3 to raise the urinary pH or NH4Cl to lower it.
    2-Phenylphenol was given at a dietary concentration of 12 500 ppm
    (equivalent to 625 mg/kg bw per day) and sodium 2-phenylphenol at 20
    000 ppm (equivalent to 1000 mg/kg bw per day or 625 mg/kg bw per day
    of 2-phenylphenol). Hyperplasia of the bladder epithelium was seen in
    animals given 2-phenylphenol, 2-phenylphenol plus NaHCO3, or sodium
    2phenylphenol. Administration of the sodium salt with NH4Cl had no
    significant effect. The incidence of tumours was significantly
    increased with 2-phenylphenol (12/31), sodium 2-phenylphenol (22/31),
    and 2-phenylphenol plus NaHCO3 (20/31), but only three tumours were
    seen in 31 rats given sodium 2-phenylphenol plus NH4Cl. Thus, the
    carcinogenic effects of 2-phenylphenol were promoted in alkaline
    urine, and those of sodium 2-phenylphenol were inhibited in acid urine
    (Fujii et al., 1987). 

         Changes in urinary parameters, particularly electrolyte levels
    and pH, DNA synthesis, and the morphology of the bladder epithelium
    were investigated in Fischer 344 rats fed diets containing various
    sodium, potassium, magnesium, and calcium carbonate salts at a
    concentration of 30 000 ppm, with or without L-ascorbic acid at 50 000
    ppm, for 4 or 8 weeks. The effects of treatment with NH4Cl at 10 000
    ppm (to acidify urine) and of combined treatment with sodium ascorbate
    at 50 000 ppm and NH4Cl were also investigated. Urinary pH was
    significantly raised in groups given NaHCO3, K2CO3, ascorbic
    acid plus NaHCO3, ascorbic acid plus K2CO3, or sodium ascorbate,
    whereas treatment with ascorbic acid or NH4Cl alone caused a
    significant decrease in urinary pH. Increases in urinary electrolyte
    or ascorbic acid contents were associated with the corresponding
    dosing regimen. DNA synthesis in the bladder epithelium was increased
    in groups given NaHCO3, K2CO3, ascorbic acid plus NaHCO3,
    ascorbic acid plus K2CO3, or sodium ascorbate. Furthermore, all
    treatments that increased DNA synthesis also induced some
    morphological alterations in the bladder epithelium. Administration of
    ascorbic acid in conjunction with NaHCO3 or K2CO3 induced more
    changes than those with either salt alone. In contrast, the degree of
    response of the bladder epithelium of rats given sodium ascorbate was
    reduced by simultaneous administration of NH4Cl. These results
    suggest that the degree of DNA synthesis and/or morphological
    alteration in rat bladder epithelium after treatment with various
    bases depends on changes in the urinary concentrations of Na+ or
    K+ and/or pH and the presence of ascorbic acid in the urine (Shibata
    et al., 1989b).

         The role of urinary pH and Na+ concentration on the
    carcinogenic effect of 2-phenylphenol and sodium 2-phenylphenol on rat
    urinary bladder was studied in two experiments. In the first, groups
    of 36 male Fischer 344 rats were fed diets containing 20 000 ppm of
    sodium 2-phenylphenol (equivalent to 1000 mg/kg bw per day), 12 500
    ppm of 2-phenylphenol (equivalent to 625 mg/kg bw per day), 6400 ppm
    of NaHCO3, 12 500 ppm of 2-phenylphenol plus 6400 ppm of NaHCO3,
    12 500 ppm of 2-phenylphenol plus 3200 ppm of NaHCO3, or 12 500 ppm
    of 2-phenylphenol plus 1600 ppm of NaHCO3 for 104 weeks. Body
    weights were measured weekly up to week 14 and monthly thereafter.
    Food consumption was measured on 2 consecutive days per week on a
    per-cage basis. Urine samples were obtained from four to six rats in
    each group by forced urination, and the urinary pH was determined 10
    times during the 2-year experiment. For measurement of urinary
    electrolytes, three or four rats in each group were housed
    individually in metal metabolic cages without food or water for 4 h in
    the morning during weeks 58, 80, and 96. All surviving animals were
    killed at the end of the experiment and were examined carefully for
    gross abnormalities at autopsy. The liver, kidney, and tissues with
    macroscopic lesions were removed and fixed for histological
    examination. Autopsies were also performed on all animals that died or
    became moribund and were killed during the experiment.

         Body-weight gain was reduced throughout the study in all treated
    groups, but the reduction was less and started later in animals given
    6400 ppm of NaHCO3 alone. The absolute and relative weights of the
    bladder were significantly higher in treated groups than in controls,
    especially in rats given 2-phenylphenol plus 6400 ppm of NaHCO3. The
    relative weights of the kidneys and liver in all treated groups were
    significantly higher than in controls. At week 104, 58% of rats given
    sodium 2-phenylphenol and 68-84% of those in other groups were still
    alive compared with 73% of controls. Macroscopically, more tumours
    were found in bladders of rats fed sodium 2-phenylphenol or
    2-phenylphenol plus 6400 ppm of NaHCO3 than in rats fed
    2-phenylphenol plus 3200 ppm or 1600 ppm NaHCO3, and no tumours were
    found in rats fed 2-phenylphenol alone or in controls. No stone
    formation related to tumours was seen in any group. The bladder
    lesions were classified as simple hyperplasia, papillary or nodular
    hyperplasia, papilloma, and carcinoma. The incidences of bladder
    carcinomas were significantly higher than controls in rats fed sodium
    2-phenylphenol or 2-phenylphenol plus 6400 ppm NaHCO3. 

         In the other experiment, groups of five rats were given diets
    supplemented with test chemicals as in the first experiment for only 8
    weeks before being killed. Urinary electrolytes and pH were determined
    at weeks 2, 4, 6, and 8, and osmolality was measured at weeks 4 and 8.
    When all animals were killed at week 8, no stone formation was
    observed macroscopically in any groups. Various changes in the luminal
    surface of the bladder, particularly in rats fed sodium 2-phenylphenol
    or 2-phenylphenol plus 6400 ppm of NaHCO3, were revealed by scanning
    electron microscopy. The authors concluded that sodium 2-phenylphenol
    is carcinogenic to the male rat bladder at 20 000 ppm in the diet.

    2-Phenylphenol was not carcinogenic, although it induced a low
    incidence of papillary or nodular hyperplasia (Fukushima et al.,
    1989).

         Species differences in the induction of urinary bladder lesions
    by sodium 2-phenylphenol were studied in groups of 30 male Fischer 344
    rats, B6C3F1 mice, Syrian golden hamsters, and Hartley guinea-pigs
    fed diets containing 20 000 ppm of sodium 2-phenylphenol (purity not
    given). Body weight and food consumption were determined periodically.
    Groups of five animals from each group were killed at weeks 4, 8, 12,
    24, 36, and 48, and five control animals were killed at weeks 12 and
    48. Urine was collected in metabolism cages for 4 h from all animals
    at weeks 12 and 48 for measurement of urine volume, pH, osmolality,
    and microscopic appearance. Although food consumption did not differ
    between the treated and untreated groups, retardation of growth was
    associated with administration of sodium 2-phenylphenol in the diet,
    especially during the first 8 weeks of the test. Although the absolute
    weights of the liver were similar in both treated and untreated
    groups, the relative weights were slightly increased in all species.
    Morphological changes in the urinary bladder were remarkable only in
    rats, which showed simple hyperplasia at week 4, increasing in
    incidence and density to week 48. Lesions classified as papillary
    nodular hyperplasia were observed in rats from week 36 of treatment,
    but no papillomas were found. Scanning electron microscopy revealed
    pleomorphic microvilli only in rats, which increased in grade with
    time. In the other species, no changes indicative of proliferation
    were observed, except for a slight effect in mice at weeks 24 and 48.
    The pH of the urine was slightly increased in rats, while the
    background pH in the other species was usually high, except in mice at
    48 weeks. Osmolality was not affected by administration of sodium
    2-phenylphenol, but crystal formation was seen in rats and
    guinea-pigs, which increased slightly in rats with time. The authors
    concluded that sodium 2-phenylphenol is likely to be a urinary bladder
    carcinogen in rats but not in mice, guinea-pigs, or hamsters (Hasegawa
    et al., 1990a).

         Sex differences in the carcinogenic effect in rat urinary bladder
    associated with administration of 2-phenylphenol (purity, > 99%) and
    sodium 2-phenylphenol (purity, > 99%) were investigated in groups of
    five or six male and five or six female Fischer 344 rats fed diets
    containing 12 500 ppm of 2-phenylphenol (equivalent to 625 mg/kg bw
    per day), 20 000 ppm of sodium 2-phenylphenol, equivalent to 1000
    mg/kg bw per day), 30 000 ppm of NaHCO3, 10 000 ppm of NH4Cl,
    2-phenylphenol plus NaHCO3, or sodium 2-phenylphenol plus NH4Cl
    for 8 weeks. Body weights and food and water consumption were
    determined weekly. Fresh urine specimens were obtained from all rats
    by forced urination at week 8 and examined for pH and osmolality.
    During the last week of the study, the rats were transferred to metal
    metabolism cages without food or water, and urine samples were
    collected from 07:00-13:00 h over 3 consecutive days to obtain enough
    pooled urine for analysis and determination of metabolites, although
    only pooled samples from the groups treated with 2-phenylphenol or its
    sodium salt were examined.

         No animals died before the end of the experiment, and food intake
    was not significantly different between groups. The body weights at
    week 8 were significantly lower in all treated groups of male rats and
    in female rats given 2-phenylphenol or sodium 2-phenylphenol. The
    urinary pH values for all groups were significantly different from the
    7.0 found in untreated males and the 6.8 found in untreated females,
    except in the groups given sodium 2-phenylphenol alone in which the pH
    values were comparable to those of controls. The pH values were
    highest in the groups given NaHCO3 alone, followed by the groups
    given 2-phenylphenol plus NaHCO3, while the pH was lower than in
    controls for groups given 2-phenylphenol alone, sodium 2-phenylphenol
    plus NH4Cl, or NH4Cl alone. The Na+ concentrations were higher
    in males than in females in all groups except controls and those given
    NH4Cl. In the groups given sodium 2-phenylphenol alone, the Na+
    concentration was slightly increased over control values in males but
    not in females. The depressive effect of NH4Cl on Na+
    concentration was also less pronounced in females. Only unconjugated
    urinary metabolites were identified. No urothelial hyperplastic
    changes were observed with 2-phenylphenol alone in either sex, while
    an equimolar dose of sodium 2-phenyl-phenol induced mild papillary and
    nodular hyperplasia or simple hyperplasia in male rats only. The
    possible mechanisms underlying the differences in response between the
    sexes might include excretion of other types of conjugated forms and
    the formation of microcrystals such as the silicate crystals found in
    the urine of rats fed sodium saccharin (Hasegawa et al., 1991).

         The physiological effects of 2-phenylphenol (purity, 99.5%) on
    urothelial cells and potential formation of DNA adducts were studied
    in male Fischer 344 rats. In an initial experiment, rats were fed
    dietary concentrations of 0, 1000, 4000, or 12 500 ppm for 13 weeks.
    There was no evidence of urinary calculi, microcrystalluria, or
    calcium phosphate-containing precipitate, but urothelial cytotoxicity
    and hyperplasia were seen at the highest dose. In a second experiment,
    rats were fed dietary concentrations of 0, 800, 4000, 8000, or 12 500
    ppm for 13 weeks. The urinary pH was > 7 in all groups. The urinary
    volume was increased at the highest dose, with consequent decreases in
    osmolality and the concentrations of creatinine and other solutes. The
    urinary excretion of total 2-phenylphenol metabolites was increased.
    Most of the metabolites were conjugates of 2-phenylphenol and of
    phenylhydroquinone, and free 2-phenylphenol and metabolites accounted
    for < 2% at each dose. Urothelial toxicity and hyperplasia occurred
    only at 8000 and 12 500 ppm. No 2-phenylphenol-DNA adducts were
    detected in the urothelium at any dose. The small percentage of
    unconjugated metabolites and the absence of DNA adducts suggest that
    2-phenylphenol acts as a bladder carcinogen in male rats by inducing
    cytotoxicity and hyperplasia without direct binding of the compound or
    its metabolites to DNA (Smith et al., 1998).

         The carcinogenic effect of sodium 2-phenylphenol and its
    metabolites on female rat urinary bladder after intravesicular
    instillation was studied in groups of nine 6-week-old Fischer 344 rats
    that received 0.2 ml of a saline solution of 0.1% sodium
    2-phenylphenol (purity, > 99%), phenylbenzoquinone (purity, > 99%),

    or phenylhydroquinone (purity, > 99%) through a catheter into the
    urethra once, twice, or four times. The pH values of the solutions
    were 11 for sodium 2-phenylphenol, 6.5 for phenylbenzoquinone, and 6.4
    for phenylhydroquinone. Saline or a solution of NaOH (pH 11) were
    given to controls. The animals were maintained under light ether
    anaesthesia during instillation and for a further 10 min thereafter to
    prevent spontaneous urination. Two or three animals from each group
    were killed under ether anaesthesia at 24 h and 4 and 7 days after the
    last instillation. The histopathological findings in rats killed 24 h
    after a single injection of saline, phenylbenzoquinone, or
    phenylhydroquinone included swelling and vacuolation of urothelial
    cells. The bladder epithelium of rats treated with alkaline solutions
    of sodium 2-phenylphenol or NaOH showed minimal hyperplasia associated
    with mild oedema and inflammatory-cell infiltration of the epithelial
    and submucosal tissues. Moderate epithelial hyperplasia was seen in
    rats killed 7 days after treatment with phenylbenzoquinone. In rats
    given two or four instillations of this metabolite, the grading of the
    hyperplastic changes was clearly dependent on the number of
    instillations and the time between the last treatment and death. The
    epithelial hyperplasia was marked and was classified as papillary
    and/or nodular in rats treated with four instillations of
    phenylbenzoquinone and killed 4 days after the last instillation. No
    carcinomas were induced.

         In another experiment, groups of 20 female rats were treated in
    the same way but twice a week for 5 weeks. From week 6, some rats were
    fed the basal diet supplemented with 5% sodium saccharin for 31 weeks
    as a promotion treatment, while the other rats were maintained on
    basal diet during this period. A separate group was given 500 ppm of
    NBHBA for 4 weeks, and then 50 000 ppm of sodium saccharin as a
    positive control. Body weights and food consumption were determined
    periodically. The histopathological findings in the positive control
    group included papillomas in two rats, papillary and/or nodular
    hyperplasia in nine rats, and simple hyperplasia in 11 rats. In
    contrast, no hyperplastic changes were seen in rats treated first with
    sodium 2-phenylphenol or its metabolites followed by promotion with
    sodium saccharin, except in nine rats given phenylbenzoquinone, which
    had papillary and/or nodular hyperplasia and/or simple hyperplasia.
    Formation of lymph follicles in the submucosa of the urinary bladder
    was seen in particular with phenylbenzoquinone and in the positive
    control group. The authors concluded that phenylbenzoquinone plays an
    essential role in the urinary bladder carcinogenesis induced by sodium
    2-phenylphenol (Hasegawa et al., 1990b).

         A series of studies was carried out on the carcinogenicity of
    2-phenylphenol and sodium 2-phenyl-phenol (purity of neither given) in
    rat urinary bladder. In the first study, groups of 10 male Fischer 344
    rats were fed diets containing sodium 2-phenylphenol at concentrations
    of 0, 2500, 5000, 10 000, or 20 000 ppm for 36 weeks. The rats were
    observed daily and were weighed periodically. Body-weight gain was
    suppressed at 20 000 ppm. No calculi or mucosal tumours were found
    grossly, but histological analysis revealed a statistically
    significant increase in the frequency of bladder lesions in rats at

    20 000 ppm, in which simple hyperplasia was seen in 10/10 and
    papillary or nodular hyperplasia in 4/10 animals. Simple hyperplasia
    was seen in 1/10 rats at 10 000 ppm.

         In the second study, groups of five male rats were fed diets
    containing 20 000 ppm of 2-phenylphenol or sodium 2-phenylphenol for 4
    weeks. The animals were weighed at the end of treatment, at which time
    the mean body weights of both treated groups were reduced, to 86% of
    the control value with 2-phenylphenol and to 96% with sodium
    2-phenylphenol. One hour before sacrifice, each rat was given an
    intraperitoneal injection of 100 mg/kg bw of 5-bromo-2'-deoxyuridine
    (BrdU), and the urinary bladders were stained immunohistochemically
    with anti-BrdU antibodies to investigate the capacity of the
    epithelial cells for proliferation. The number of cells that had taken
    up BrdU per 1000 urinary bladder epithelial cells was determined by
    light microscopy and expressed as per cent labelled cells. Rats fed
    sodium 2-phenylphenol showed increased urinary pH and extensive
    BrdU-labelling in urinary bladder epithelial cells, indicating
    increased DNA synthesis. Rats fed 2-phenylphenol also showed a
    tendency to increased BrdU-labelling, suggested that it also can
    cause, albeit weak, urinary bladder epithelium proliferation.

         In the third study, groups of five male Fischer 344 rats were
    given diets containing  6400 ppm of NaHCO3; 13 000 ppm of
    2-phenylphenol; 13 000 ppm of 2-phenylphenol plus NaHCO3 at 1600,
    3200, or 6400 ppm; or 20 000 ppm of sodium 2-phenylphenol for 8 weeks.
    The urinary pH at week 8 was significantly increased in animals fed
    NaHCO3 alone, 2-phenylphenol plus 3200 or 6400 ppm NaHCO3, or
    sodium 2-phenylphenol. The urinary concentrations of Na+ were
    significantly increased at 2, 4, 6, and 8 weeks in rats fed sodium
    2-phenylphenol and at 8 weeks in rats fed NaHCO3 alone or
    2-phenylphenol plus 3200 or 6400 ppm NaHCO3. At 8 weeks, a
    significant increase or a tendency to an increase in urine volume and
    significantly lower osmotic pressure were seen in pooled urine samples
    from all treated groups when compared with controls. When the bladders
    were examined by scanning electron microscopy, the surface of the
    epithelium appeared normal in the controls and in animals given only
    NaHCO3, and was made up of polygonal cells of uniform dimensions
    with reticular peaked microridges at the surface. In treated animals,
    the cells in the outermost layer of the bladder epithelium assumed a
    cobblestone configuration in pavement form; at high magnification,
    pleomorphic microvilli, short uniform microvilli, and ropy or leafy
    microridges were seen at the surface of these cells. These alterations
    were observed mainly in rats given sodium 2-phenylphenol or
    2-phenylphenol plus 6400 ppm NaHCO3. The extent of the changes and
    the frequency of their appearance was correlated with the
    concentration of NaHCO3 given with 2-phenylphenol.

         In the fourth study, groups of 30-31 male Fischer 344 rats were
    given 20 000 ppm of sodium 2-phenylphenol or 13 000 ppm of
    2-phenylphenol with or without NaHCO3 at 1600, 3200, or 6400 ppm for
    104 weeks. The animals were observed daily for deaths, and body weight
    and feed consumption were measured at regular intervals. Pooled 4-h

    urine samples were collected from three or four rats at each dose at
    weeks 58, 80, and 96 for measurement of electrolytes. The urinary pH
    was significantly increased in rats fed sodium 2-phenylphenol or
    2-phenylphenol alone or in combination with 3200 or 6400 ppm NaHCO3
    or 6400 ppm NaHCO3 alone. The Na+ concentrations were
    significantly increased in rats fed sodium 2-phenylphenol,
    2-phenylphenol plus 6400 ppm NaHCO3, or 6400 ppm NaHCO3 alone. No
    difference was seen in rats fed sodium 2-phenylphenol or
    2-phenylphenol plus 6400 ppm NaHCO3. Urinary bladder tumours were
    found in all groups except those given 2-phenylphenol alone, and the
    frequencies were highest with sodium 2-phenylphenol and with
    2-phenylphenol plus 6400 ppm NaHCO3. The presence of calculi could
    not be confirmed. Histological examination of the urinary bladder
    epithelium revealed simple hyperplasia, papillary or nodular
    hyperplasia, papillomas, and carcinomas. Carcinomas occurred in rats
    fed sodium 2-phenylphenol (41%), 2-phenylphenol plus 6400 ppm NaHCO3
    (31%), and 6400 ppm NaHCO3 alone. The results confirm that
    administration of 20 000 ppm of sodium 2-phenylphenol is carcinogenic
    in male rats, while an equimolar concentration of 2-phenylphenol
    causes only a low frequency of papillary or nodular hyperplasia and no
    papillomas or cancers. Administration of NaHCO3 in conjunction with
    2-phenylphenol caused carcinomas, correlated to the NaHCO3
    concentration, which also increased urinary pH and Na+ concentration
    (Inoue, 1993).

         The induction of DNA damage in the urinary bladder epithelium of
    male and female Fischer 344 rats by 2-phenylphenol and its metabolites
    was studied by the alkaline elution assay after an intravesicalar
    injection. Phenylbenzoquinone at 0.05-0.1% had weak DNA-damaging
    activity in animals of each sex, whereas 2-phenylphenol and
    phenylhydroquinone had no effect at the same dose. Histopathological
    examination revealed diffuse, moderate, simple hyperplasia 5 days
    after injection of 0.1% phenylbenzoquinone in male rats. The lesions
    were associated with submucosal infiltration, small round cells, and
    slight oedema. The only change in the bladders of rats injected with
    0.1% phenylhydroquinone was slight swelling and/or vacuolization of
    the epithelial cells, and the bladders of rats injected with 0.1%
    2-phenylphenol were normal (Morimoto et al., 1989).

         Groups of 5-10 Fischer 344 rats received diets containing sodium
    2-phenylphenol at concentrations of 0, 2500, 5000, 10 000, or 20 000
    ppm (equivalent to 0, 250, 500, 1000, and 2000 mg/kg bw per day) for
    up to 5 months to investigate the correlation between urinary
    phenylbenzoquinone and DNA damage in the bladder epithelium. Slight
    but dose-dependent DNA damage was observed in the epithelium of male
    rats fed 10 000 or 20 000 ppm for 3-5 months. A plot of the
    dose-response relationship for DNA damage at 3 months showed a
    threshold at 5000 ppm of sodium 2-phenylphenol. The amounts of
    unconjugated 2-phenylphenol, phenylhydroquinone, and
    phenylbenzoquinone in 24-h urine samples collected from males and
    females after 5 months correlated well with the dietary concentrations
    of sodium 2-phenylphenol. The total amounts of free metabolites in the
    urine of males given 5000 ppm were similar to those in the urine of

    females given 20 000 ppm. Free metabolites represented 0.3% of the
    total average intake of male rats fed 5000 ppm, 0.8% of the intake of
    10 000 ppm, and 1% of the intake of 20 000 ppm of sodium
    2-phenylphenol. The average concentrations of free phenylhydroquinone
    in the urine of males given 20 000 ppm of sodium 2-phenylphenol were
    significantly higher than those in males fed 5000 ppm or in females
    fed 20 000 ppm. The concentrations of phenylbenzoquinone were much
    lower than those of phenylhydroquinone. Only 10% of phenylbenzoquinone
    was recovered from spiked urine, indicating that this metabolite may
    react with urinary nucleophilic groups. The authors concluded that
    phenylbenzoquinone is the reactive species in the initiation of
    bladder tumours induced by 2-phenylphenol and sodium 2-phenylphenol
    (Morimoto et al., 1989).

         The interaction of 2-phenylphenol and its metabolites with pUC18
    DNA from  Escherichia coli plasmids was studied  in vitro. The major
    metabolite formed from 2-phenylphenol by mixed-function oxidases was
    phenylhydroquinone. This finding corroborates earlier reports that
    phenylhydroquinone in the form of a glucuronide conjugate is the main
    product in the bladders of rats fed 2-phenylphenol. When pUC18 DNA was
    incubated with phenylhydroquinone, DNA strand scission was observed,
    whereas barely detectable DNA cleavage was seen with 2-phenylphenol
    and phenylbenzoquinone. DNA cleavage by phenylhydroquinone was
    inhibited by superoxide dismutase, catalase, and several oxygen
    radical scavengers, indicating that the oxygen radicals generated in
    the process of oxidation of phenylhydroquinone in aqueous solution are
    responsible for the DNA cleavage. The attack seemed to occur at
    guanine residues in general and was not restricted to guanines with
    specific residues, indicating no hot spots (Nagai et al., 1990).

         The generation of 8-hydroxydeoxyguanosine in calf thymus DNA
    treated with 2-phenylphenol, phenylhydroquinone, or
    phenylbenzoquinone, was studied  in vitro. The content of
    8-hydroxydeoxy-guanosine residues was increased in DNA treated with
    phenylhydroquinone in a concentration-dependent manner, but
    phenylbenzoquinone had little effect, and 2-phenylphenol had no
    effect. The formation of 8-hydroxydeoxyguanosine by phenylhydroquinone
    was reduced by oxygen radical scavengers and accelerated by the
    addition of CuCl or CuCl2. Hydroxyl radicals generated during
    oxidation of phenylhydroquinone thus contribute to the formation of
    8-hydroxydeoxyguanosine in DNA, and copper ions facilitate the
    oxidative DNA damage. Copper ions greatly accelerated
    phenylhydroquinone-induced DNA cleavage  in vitro, although they had
    no effect on cleavage without phenylhydroquinone. In contrast, DNA
    cleavage occurred with the addition of FeCl2 in the absence and
    presence of phenylhydroquinone. The formation of
    8-hydroxydeoxyguanosine in bladder DNA is likely to be one of a series
    of events in the carcinogenesis induced by 2-phenylphenol (Nagai et
    al., 1995).

         The effect of the selective gamma-glutamylcysteine synthetase
    inhibitor, buthionine sulfoximine, on the hepatotoxic and nephrotoxic
    potential of 2-phenylphenol and its metabolites was studied in groups
    of four male Fischer 344/DuCrj rats. The animals were given an
    intraperitoneal injection of 0 or 900 mg/kg bw of buthionine
    sulfoximine and 1 h later received 2-phenylphenol, phenylhydroquinone,
    or phenylbenzoquinone at single oral doses of 0, 700, or 1400 mg/kg
    bw. The rats were killed 6 and 24 h later, and serum was collected for
    measurement of alanine and aspartate aminotransferase activities and
    urea nitrogen. The liver and kidneys were removed and weighed, and
    hepatic and renal glutathione were assayed.

         2-Phenylphenol caused acute hepatocellular damage, as shown by
    necrotic centrilobular hepatocytes accompanied by increased serum
    aminotransferase activity. Pretreatment with buthionine sulfoximine
    potentiated the hepatic and renal toxicity of 2-phenylphenol,
    indicating that the liver and kidneys are its target organs of at high
    doses. 2-Phenylphenol depleted hepatic and renal glutathione by 6 h
    after administration, and this effect was enhanced by pretreatment
    with buthionine sulfoximine. Recovery of glutathione concentrations in
    both organs was slower in rats given 1400 mg/kg bw of 2-phenylphenol
    than in those given 700 mg/kg bw, suggesting that the hepatic and
    renal damage caused by this compound is associated with prolonged
    depletion of glutathione and that it acts indirectly on the liver.
    Within 24 h, 75% of the rats treated with phenylbenzoquinone at 1400
    mg/kg bw had died. Administration of phenylbenzoquinone at 700 m/kg bw
    or phenylhydroquinone at 1400 mg/kg bw significantly increased
    aminotransferase activities. The activity of alanine aminotransferase
    in both groups was about twice that of rats given 1400 mg/kg bw
    2-phenylphenol. A slight decrease in liver weight, nuclear pyknosis,
    eosinophilic degeneration of periportal hepatocytes, increased
    relative kidney weight, slight renal papillary necrosis, dilatation of
    renal tubules, and increased serum urea nitrogen concentration were
    observed at 700 mg/kg bw of phenylbenzoquinone. The relative weight of
    the kidneys was increased at 1400 mg/kg bw of phenylhydroquinone.
    These results indicate that phenylbenzoquinone is more toxic to liver
    and kidney than phenylhydroquinone (Nakagawa & Tayama, 1988).

         In a study of the conjugation of 2-phenylphenol with glutathione
    in rat liver  in vitro and  in vivo, radiolabel derived from
    [14C]2-phenylphenol bound irreversibly to hepatic microsomal
    macromolecules in an NADPH-generating system, and the binding was
    inhibited by cysteine and glutathione. When [14C]2-phenylphenol and
    glutathione were incubated in a microsomal NADPH-generating system,
    the radiolabelled material derived from the aqueous phase of the
    incubation mixture was similar to a synthetic, water-soluble
    phenylhydroquinone-glutathione conjugate produced by a nonenzymic
    reaction between phenylbenzoquinone and glutathione.
    Phenylhydroquinone-glutathione was excreted as a minor conjugate in
    the bile after oral administration of 2-phenylphenol to rats at a dose
    of 1000 mg/kg bw. The cumulative biliary excretion of the conjugate
    over 6 h represented about 4% of the dose. The results show that a
    reactive intermediate of 2-phenylphenol can form adducts with

    glutathione to produce water-soluble conjugates. The reactive
    intermediate is probably phenylbenzoquinone derived from
    phenylhydroquinone. Since glutathione protects against cellular
    injury, the acute hepatic damage caused by high doses of
    2-phenylphenol is probably associated with the formation of an active
    intermediate (phenylbenzoquinone) which depletes cellular glutathione
    (Nakagawa & Tayama, 1989).

         The relationship between the metabolism and cytotoxicity of
    2-phenylphenol was studied in isolated rat hepatocytes. Addition of
    high concentrations of 2-phenylphenol to the cells caused
    dose-dependent toxicity, with death at the highest dose of 1.0 mmol/L.
    Pretreatment of the hepatocytes with a non-toxic dose of 5 mol/L of
    SKF-525A enhanced the cytotoxicity of 2-phenylphenol at 0.5-1.0 mmol/L
    and inhibited its metabolism. At lower concentrations (0.5 or 0.75
    mmol/L), 2-phenylphenol was converted sequentially to
    phenylhydroquinone and then to its glutathione conjugate. The
    concentrations of both metabolites and especially of the conjugate,
    were very low in hepatocytes exposed to 2-phenylphenol at 1.0 mmol/L
    alone or with SKF-525A. The cytotoxicity induced by 2-phenylphenol at
    0.5 mmol/L was enhanced by the addition of 1.25 mmol/L of
    diethylmaleate, which continuously depletes cellular glutathione. In
    contrast, the cytotoxicity induced by phenylhydroquinone at 0.5 mmol/L
    was significantly inhibited by addition to the hepatocytes of 5 mmol/L
    of dithiothreitol, cysteine,  N-acetyl-L-cysteine, or ascorbic acid.
    Loss of glutathione, protein thiols, and ATP was also prevented. These
    results indicate that the acute cytotoxicity of 2-phenylphenol at 1.0
    mmol/L is a direct action and that prolonged depletion of cellular
    glutathione enhances the cytotoxicity of low concentrations of
    2-phenylphenol metabolites. The cytotoxicity of phenylhydroquinone is
    prevented significantly by addition of cysteine, glutathione, or
    ascorbic acid (Nakagawa et al., 1992). 

         Groups of 22 male CDF (Fischer 344)/BR rats were given diets
    containing 2phenylphenol (purity, 99.5%) to provide concentrations of
    0, 800, 4000, 8000, or 12 500 ppm, equal to 0, 56, 280, 560, and 920
    mg/kg bw per day, for 13 weeks. During weeks 12-13 and 13-14 of the
    study, urine was collected for determination of metabolites and
    urinary characteristics, respectively. In addition, urinary bladders
    were collected from 12 animals per group during week 14 for analysisof
    the urothelium by 32P-postlabelling, while histopathological
    evaluation of 10 animals group included determination of a labelling
    index and light and scanning electron microscopy. The body-weight gain
    was reduced by about 10% at 8000 and 12 500 ppm, but food intake was
    unaffected at all doses tested. Histological examination showed simple
    hyperplasia of the urothelium at concentrations of 8000 and 12 500 ppm
    with significant changes in the bladder. The glucuronide and sulfate
    conjugates of 2-phenylphenol and the hydroxylated metabolite
    phenylhydroquinone were the major urinary metabolites, although the
    major conjugate at all doses was the sulfate. Minute levels of free
    2phenylphenol and phenylhydroquinone were found at all doses, free
    phenylhydroquinone comprising 0.6-1.5% of the total metabolites
    measured. An increase in the labelling index of the bladder epithelium

    was observed at 8000 and 12 500 ppm. 32P-Postlabelled urothelial DNA
    showed no evidence of formation of 2-phenylphenol-DNA adducts.

         The authors concluded that a hyperplastic response of the urinary
    bladder epithelium occurs after exposure to 2-phenylphenol at 8000 or
    12 500 ppm, which are unequivocal carcinogenic doses for the bladder
    of male rats, which is due to mild cytotoxicity with consequent
    regenerative hyperplasia. The increased mitotic activity (labelling
    index), the presence of very small amounts of free phenylhydroquinone
    in the urine, and the absence of DNA adducts in the bladder epithelium
    further suggest that the bladder carcinogenesis in male rats exposed
    to 2-phenylphenol is probably mediated by an indirect, dose-dependent
    cytotoxic effect on the bladder epithelium leading to regenerative
    hyperplasia and subsequent tumorigenesis of epigenetic origin, rather
    than to direct metabolic activation of 2-phenylphenol to reactive
    metabolites capable of forming 2-phenylphenol-DNA adducts (Christenson
    et al., 1996a).

         Groups of 20-30 male CDF (Fischer344)/BR rats were given diets
    containing 2-phenylphenol (purity, 99.9%) at concentrations of 0,
    1000, 4000, or 12 500 ppm, equal to 0, 54, 220, and 680 mg/kg bw per
    day, for 13 weeks. Animals from the control and high-dose groups were
    allowed to recover for 4 weeks. Urine was collected for chemical and
    and electron microscopic evaluation at various times, and urinary
    bladders were collected from animals in the recovery groups during
    weeks 4, 13, and 17 for histological evaluations which included
    determination of a labelling indexes and light and electron
    microscopy.

         Body-weight gain was reduced only in rats at 12 500 ppm, and food
    intake was unaffected at all doses. Weekly clinical examinations
    showed an increased incidence of urine staining at 4000 and 12 500
    ppm. No unusual precipitate or crystal was found in the urinary
    sediment of treated animals. Urothelial hyperplasia was observed only
    after 13 weeks at 12 500 ppm, and the effect was reversed by 4 weeks
    on control diet. After 4 and 13 weeks of exposure to 2-phenylphenol,
    necrotic foci were observed in the bladders of rats at 12 500 ppm, and
    at 13 weeks the bladders also showed evidence of regenerative
    hyperplasia. Increased labelling indexes were observed in the bladders
    of animals at the high dose at 4 and 13 weeks, but the index had
    returned to control values after 4 weeks' recovery, confirming the
    reversibility of the proliferative changes in the urothelium. The
    results of this study suggest that 2-phenylphenol acts by a mechanism
    involving a cytotoxic action on the urothelium leading to the
    formation of regenerative, reversible hyperplasia. The origin of the
    cytotoxicity remains unclear, however, as no evidence was found of
    either abnormal crystalluria or a calcium phosphate-containing
    amorphous precipitate (Christenson et al. 1996b).

         The relative importance of bladder distension, urinary pH, and
    Na+ concentration in the induction of cell proliferation in the
    bladder epithelium of rats fed various sodium salts was investigated.
    In male rats fed a diet containing 5% NaHCO3, the bladder epithelium

    showed an increased number of replicating cells, distension, increased
    urinary pH, and a high urinary Na+ concentration. Cell proliferation
    also occurred when the bladders were subjected to distension  in 
     vivo  by mechanical (female) or physiological (male) means.
    Inclusion of CaCO3 in the diet increased the urinary pH without
    altering other factors and did not induce cell proliferation, but
    proliferation was increased when CaCO3 was combined with the
    mechanical or physiological treatment. Thus, high urinary pH was of
    secondary importance to bladder distension as a causative factor but
    acted to enhance cell proliferation when distension occurred. Similar
    findings were obtained with regard to the Na+ concentration. The
    authors concluded that bladder distension is a prerequisite for
    proliferation of epithelial cells in the bladders of rats fed diets
    containing high concentrations of sodium salts and that changes in
    urinary pH and Na+ concentration also determine the degree of
    proliferation (Shioya et al., 1994).

    3.  Observations in humans

         In one of the earliest studies on the toxicity of 2-phenylphenol,
    skin irritation and sensitization due to exposure to this compound and
    its sodium salt were evaluated in 100 male and 100 female, unselected
    persons. A patch impregnated with the test material was placed in
    direct contact with the skin of the back of each person, covered with
    an impervious film, and taped securely in place. The first patch was
    kept in constant contact with the skin for 5 days, at which time the
    patch was removed and the reaction noted. A second patch was applied
    in the same way 3 weeks after removal of the first patch and was kept
    in direct contact with the skin for 48 h. Each subject was examined
    immediately and again 3 and 8 days after removal of the second patch.
    2-Phenylphenol as a 5.0% solution in sesame oil did not cause primary
    irritation or sensitization, but sodium 2-phenylphenol was
    significantly irritating when applied as a 5% or a 1% aqueous
    solution. A 0.5% solution caused very slight, simple irritation,
    whereas a 0.1% solution produced no irritation and no sensitization
    (Hodge et al., 1952).

    Comments

         After oral administration to mice and rats, 2-phenylphenol and
    its sodium salt are rapidly and extensively absorbed (95%) and
    distributed. Excretion is also rapid in these species, being almost
    complete within 48 h, and occurs mainly in urine (about 90%) and in
    faeces (about 5%). Little radiolabel (< 1%) is retained in organs and
    tissues, including the urinary bladder. After dermal application of
    2-phenylphenol to humans, about 43% of the applied dose was absorbed
    through the skin and about 58% was recovered in skin rinse and the
    protective enclosure. Most of the absorbed radiolabel was recovered in
    urine (99%, and only 1% was recovered in faeces. The absorption
    half-time was 10 h, and the elimination half-time was 0.8 h. The rapid
    excretion of the radiolabel into urine indicates that 2-phenylphenol
    is unlikely to accumulate in humans exposed repeatedly. The metabolic
    profiles of both compounds were similar in mice, rats, and humans at

    the various doses tested. The main metabolic pathways are conjugation
    of 2-phenylphenol or hydroxylation at the 5 position of the phenol
    ring, followed by conjugation with glucuronide or sulfate. The parent
    compound was detected in only very small amounts (0.4%) in urine. The
    metabolic profile in plants raised no toxicological concern, since
    about 90% of the residue found in oranges and pears is 2-phenylphenol
    or its conjugates.

         2-Phenylphenol and its sodium salt have low acute toxicity in
    mice and rats treated orally, the LD50 values ranging from 600 to
    3500 mg/kg bw. Neither 2-phenylphenol nor its sodium salt has been
    classified by WHO for acute toxicity.

         2-Phenylphenol and its sodium salt caused severe dermal
    irritation in rabbits, and the sodium salt caused severe dermal
    irritation in humans. 2-Phenylphenol irritated the eye of rabbits,
    whereas the sodium salt caused only moderate ocular irritation.
    Neither substance caused delayed contact hypersensitivity in
    guinea-pigs or humans.

         In medium- and long-term tests for toxicity, the urinary bladder
    was regarded as the main toxicological target organ of both
    2-phenylphenol and its sodium salt in male and female rats. At doses
    of 200 mg/kg bw per day and above, hyperplasia, papillomas, and
    transitional-cell carcinomas were seen with both compounds in male
    rats. Increased mitosis was observed in the bladder epithelium three
    days after the start of dosing, and thickening, i.e. simple
    hyperplasia, was seen at 14 days. In female rats, hyperplasia and
    papillomas were observed, but to a far lower degree than in males. In
    male and female mice, the liver was the primary target organ.
    Increased relative liver weights and an increased incidence of
    hepatocellular adenomas were seen with 2-phenylphenol at doses of 500
    mg/kg bw per day and above. Reduced body-weight gain was a common
    finding in mice and rats. In 90-day studies, the NOAELs for
    2-phenylphenol were 6300 ppm, equal to 760 mg/kg bw per day, in rats
    and 300 mg/kg bw per day (the highest dose tested for up to 1 year) in
    dogs. The NOAEL for the sodium salt was 5000 ppm, equivalent to 550
    mg/kg bw per day, in mice and 2500 ppm, equal to 180 mg/kg bw per day,
    in rats. In a 1-year study of toxicity, the NOAEL for 2-phenylphenol
    was 800 ppm, equal to 39 mg/kg bw per day, in rats. In 2-year studies
    of carcinogenicity, the NOAEL for 2-phenylphenol was 250 mg/kg bw per
    day in mice and 800 ppm, equal to 39 mg/kg bw per day, in rats. In
    2-year carcinogenicity studies with the sodium salt, the NOAEL for
    carcinogenicity was 20 000 ppm, equal to 3000 mg/kg bw per day, in
    mice and 2500 ppm, equivalent to 95 mg/kg bw per day, in rats. The
    Meeting concluded that both 2-phenylphenol and its sodium salt are
    carcinogenic in male rats and that 2-phenylphenol is carcinogenic in
    male mice.

         2-Phenylphenol has been more extensively tested for genotoxic
    activity than its sodium salt. Within that limitation, the results for
    the two compounds were similar. Data regarding covalent binding to DNA
    in the urinary bladder of rats dosed with either compound were

    conflicting. 2-Phenylphenol induced chromosomal aberrations in
    cultured mammalian cells, but negative results were obtained  in 
     vivo. The Meeting concluded that there are unresolved questions
    about the genotoxic potential of 2-phenylphenol. 

         Several studies have been conducted to elucidate the mechanism of
    the carcinogenic action of 2-phenylphenol and its sodium salt on the
    male rat urinary bladder, since neither compound has a carcinogenic
    effect on the urinary bladder of female rats or in mice, guinea-pigs,
    or hamsters of either sex. No clear mechanisms have been found,
    although raising the urinary pH or sodium concentration has a
    promoting effect. There was some evidence from studies with the sodium
    salt that initial irritation followed by hyperplasia might be involved
    in the bladder carcinogenicity in male rats. In addition,
    32P-postlabelling showed binding of 2-phenylphenol and its sodium
    salt to DNA in the male rat urinary bladder in some but not in other
    studies. The genotoxicity of the metabolites phenylhydroquinone and
    dihydroxybiphenyl appears to be similar to that of the parent
    molecules.

         The Meeting concluded that the urinary bladder tumours observed
    in male rats and the liver tumours observed in male mice exposed to
    2-phenylphenol are threshold phenomena that are species- and
    sex-specific, and that 2-phenylphenol is therefore unlikely to
    represent a carcinogenic risk to humans. In coming to this conclusion,
    the Meeting was aware that a working group convened by IARC had
    classified 2-phenylphenol, sodium salt, in Group 2B (possibly
    carcinogenic to humans) and 2-phenylphenol in Group 3 (not
    classifiable as to its carcinogenicity to humans). The Meeting noted,
    however, that the IARC classification is based on hazard
    identification, not on risk assessment, and is furthermore limited to
    published literature, with the exclusion of unpublished studies on
    toxicity and carcinogenicity.

         In two two-generation studies of reproductive toxicity in rats,
    2-phenylphenol had no reproductive toxicity, even at 460 mg/kg bw per
    day, the highest dose tested. The overall NOAEL for carcinogenicity
    was 92 mg/kg bw per day, since urinary bladder tumours were found in
    male rats at doses of 120 mg/bw per day and above. 

         In a study of developmental toxicity in mice with 2-phenylphenol
    and its sodium salt, the NOAELs for 2-phenylphenol were below 1500
    mg/kg bw per day (lowest dose tested) for maternal toxicity and
    fetotoxicity and 2100 mg/kg bw per day (highest dose tested) for
    teratogenicity. The NOAELs for the sodium salt were below 100 mg/kg bw
    per day (lowest dose tested) for maternal toxicity, 100 mg/kg bw per
    day for fetotoxicity, and 400 mg/kg bw per day (highest dose tested)
    for teratogenicity. In two studies of developmental toxicity in rats,
    the overall NOAELs for 2-phenylphenol were 150 mg/kg bw per day for
    maternal toxicity, 300 mg/kg bw per day for fetotoxicity, and 700
    mg/kg bw per day (highest dose tested) for teratogenicity. In two
    studies of developmental toxicity in rabbits, the overall NOAELs for
    2-phenylphenol were 100 mg/kg bw per day for maternal toxicity, 500

    mg/kg bw per day for fetotoxicity, and 750 mg/kg bw per day (highest
    dose tested) for teratogenicity.

         The Meeting established an ADI of 0-0.4 mg/kg bw for
    2-phenylphenol, on the basis of the NOAEL of 39 mg/kg per day in the
    2-year study of toxicity (based on decreased body-weight gain and
    hyperplasia of the urinary bladder) and carcinogenicity of the urinary
    bladder in male rats and a safety factor of 100.

         The Meeting determined that it was unnecessary to establish an
    acute reference dose because of the low acute toxicity of
    2-phenylphenol.

    Toxicological Evaluation

     Levels of 2-phenylphenol that cause no toxic effect 

    Mouse:    < 250 mg/kg bw per day for carcinogenicity (lowest dose
              tested; 2-year study of toxicity and carcinogenicity) 

              < 1500 mg/kg bw per day (lowest dose tested; study of
              developmental toxicity; maternal toxicity)

              2100 mg/kg bw per day (highest dose tested; study of
              developmental toxicity; not teratogenic)

    Rat:      800 ppm, equal to 39 mg/kg bw per day (2-year study of
              toxicity and carcinogenicity)

              460 mg/kg bw per day (two-generation study of reproductive
              toxicity; no reproductive toxicity; highest dose tested)

              92 mg/kg bw per day (two-generation study of reproductive
              toxicity; carcinogenicity)

              150 mg/kg bw per day (study of developmental toxicity;
              maternal toxicity)

              300 mg/kg bw per day (study of developmental toxicity;
              developmental toxicity)

              700 mg/kg bw per day (study of developmental toxicity;
              teratogenicity)

    Rabbit:   100 mg/kg bw per day (two studies of developmental toxicity;
              maternal toxicity)

              500 mg/kg bw per day (two studies of developmental toxicity;
              fetotoxicity)

              750 mg/kg bw per day (two studies of developmental toxicity;
              teratogenicity)

    Dog:      750 mg/bw per day (highest dose tested; 1-year study of
              toxicity)

     Estimate of acceptable daily intake for humans 

         0-0.4 mg/kg bw

     Estimate of acute reference dose 

         Unnecessary

     Studies that would provide information useful for continued 
     evaluation of the compound 

         1.   Mechanistic studies on urinary bladder tumours in male rats

         2.   Further observations in humans


        Toxicological end-points relevant for estimating guidance values for dietary and non-dietary exposure to 
    2-phenylphenol (unless otherwise specified)

     Absorption, distribution, excretion, and metabolism in mammals 

    Rate and extent of oral absorption           Rapid (24 h) and complete (95-100%), mice and rats 
    Dermal absorption                            Rapid and well absorbed (43%), humans
    Distribution                                 Small concentrations (< 1%) in tissues, mice and rats
    Potential for accumulation                   No accumulation, mice, rats, and humans
    Rate and extent of excretion                 Rapid and complete (95-100%), mice, rats, and humans
    Metabolism in animals                        Glucuronide and sulfate of 2-phenylphenol and phenylhydroquinone, 
                                                 mice and rats 
    Toxicologically significant compounds        2-Phenylphenol
    (animals, plants and environment)

     Acute toxicity 

    Rat, LD50, oral                              2800 mg/kg bw
    Rabbit, LD50, dermal                         > 5000 mg/kg bw
    Rabbit, LC50, inhalation (4 h)               > 36 mg/m3 air (aerosol)
    Dermal irritation                            2-Phenylphenol and its sodium salt: severe dermal irritation, 
                                                 rabbits
    Ocular irritation                            2-Phenylphenol: occular irritation, rabbits
                                                 Sodium salt: slight ocular irritation, rabbits
    Dermal sensitization                         2-Phenylphenol and its sodium salt: no dermal sensitization, 
                                                 guinea-pigs and humans

     Short-term toxicity 

    Target/critical effect                       Body-weight decrease, mice and rats, and urinary bladder tumours, 
                                                 male rats
    Lowest relevant oral NOAEL                   300 mg/kg bw per day, dogs
    Lowest relevant dermal NOAEL                 No NOAEL, 1000 mg/kg bw per day, highest dose tested, rats
    Lowest relevant inhalation NOAEL             Not investigated

    Genotoxicity                                 Unresolved questions 

     Long-term toxicity and carcinogenicity 

    Target/critical effect                       Urinary bladder, male rats
                                                 Liver, male and female mice
    Lowest relevant NOAEL                        39 mg/kg bw per day, male rats
    Carcinogenicity                              Urinary bladder tumours, male rats
                                                 Liver tumours, male and female mice

     Reproductive toxicity 

    Reproductive target/critical effect          No reproductive toxicity, rats
    Lowest relevant reproductive NOAEL           460 mg/kg bw per day, highest dose tested, rats
    Developmental target/critical effect         Developmental toxicity at maternally toxic doses, mice 
    Lowest relevant developmental NOAEL          300 mg/kg bw per day, rabbits 

    Neurotoxicity/Delayed neurotoxicity          No evidence of developmental neurobehavioural toxicity in rats. 
                                                 No evidence of neurotoxicity or neuropathology in medium- and 
                                                 long-term studies, mice, rats, dogs, or in developmental toxicity 
                                                 studies, mice, rats, and rabbits

     Other toxicological studies 

    Medical data                                 Dermal irritation with the sodium salt, not with 2-phenylphenol


                                                                                                           
    Summary                      Value                 Study                             Safety factor
                                                                                                           

    ADI                          0-0.4 mg/kg bw        Long-term study of toxicity       100
                                                       and carcinogenicity, rat 

    Acute reference dose         Unnecessary
                                                                                                           
    

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    See Also:
       Toxicological Abbreviations
       Phenylphenol, 2- and its sodium salt (Pesticide residues in food: 1985 evaluations Part II Toxicology)