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    PIPERONYL BUTOXIDE

     First draft prepared by
     A. Moretto
     Istituto di Medicina del Lavoro,
     Universita degli Studi di Padova, Padua, Italy

    Explanation
    Evaluation for acceptable daily intake
         Biochemical aspects
              Absorption, distribution, and excretion
              Effects on enzymes and other biochemical parameters
         Toxicological studies
              Acute toxicity
              Short-term toxicity
              Long-term toxicity and carcinogenicity
              Reproductive toxicity
              Developmental toxicity
              Genotoxicity
              Special studies
                   Dermal and ocular irritation and dermal sensitization
                   Studies with mixtures
         Observations in humans
         Comments
         Toxicological evaluation
    References

    Explanation

         Piperonyl butoxide was previously evaluated toxicologically by
    the JMPR in 1965, 1966, 1972, and 1992 (Annex I, references 3, 6, 18,
    and 65). An ADI of 0-0.03 mg/kg bw was allocated in 1972 on the basis
    of a one-year study in dogs. The 1992 JMPR confirmed the existing ADI
    and recommended that piperonyl butoxide be reviewed again in 1995
    after submission of studies on acute toxicity and teratogenicity in
    rats, appropriate studies of genotoxicity, the results of an on-going
    one-year study in dogs, an on-going study of carcinogenicity in mice,
    and studies of carcinogenicity in rats and mice performed within the
    US National Toxicology Program, and observations in humans.

         Piperonyl butoxide was re-evaluated at the present Meeting within
    the periodic review programme of the CCPR, and a monograph summarizing
    all of the available data was prepared.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution, and excretion

         Male Swiss-Webster mice (18-20 g) were given piperonyl butoxide
    labelled with 14C at either the alpha-methylene or the alpha-carbon
    of the 2-(2-butoxyethoxy)ethoxymethyl side-chain by gavage at 1.7 mg
    (5 mol)/kg bw. Radiocarbon was determined in expired carbon dioxide
    0.5, 1, 2, 4, and 6 h and every 6 h up to 48 h after treatment, and in
    urine and faeces 12, 24, and 48 h after treatment. Two days after
    administration of [methylene-14C]-piperonyl butoxide, 97.2% of the
    radiolabel was recovered, with 75.5% in carbon dioxide, 6.1% in urine,
    4% in faeces, and 6.8% in the carcass; after administration of
    piperonyl butoxide labelled in the 2(2-butoxyethoxy)ethoxymethyl
    side-chain, 75% of the radiolabel was recovered, with 65% in urine, 8%
    in faeces, and < 1% in carbon dioxide. In the latter case, the
    urinary metabolites were demethylated derivatives of the parent
    compound. Figure 1 shows the proposed metabolic pathway for piperonyl
    butoxide in mammals (Kamienski & Casida, 1970).

         In the same experiments, male Sprague-Dawley rats were given
    3.4 mg (10 mol)/kg bw labelled piperonyl butoxide by gavage. The
    total label (in expired carbon dioxide and urine) recovered at 48 h
    was 72-74%, and the pattern of excretion was similar to that observed
    in the mouse (Kamienski & Casida, 1970).

         Adult male Sprague-Dawley rats were given single intravenous
    doses of piperonyl butoxide labelled with 14C in the methylenedioxy
    or the alpha-methylene side-chain. Bile samples were collected from a
    fistula, and 10 urine samples were taken at various intervals before
    (one sample) and up to about 8 h after treatment; expired air was also
    collected. More than 10 metabolites were detected, but not identified,
    in urine and bile; the parent compound was identified only in fat
    (9-18% of the total radiolabel) and lung (15-25%) after about 8 h.
    After treatment with [14C-methylenedioxy]-piperonyl butoxide, about
    40% of the label was recovered as carbon dioxide, < 1% in urine, and
    3% in bile. After treatment with [14C-alpha-methylene]-piperonyl
    butoxide, 25-47% of the label was found in the bile and about 5% in
    urine; almost none was detected in expired air. The peak amount of
    label was detected in bile < 30 min after injection with either
    compound and in urine about 25 h later; significant amounts were
    present in both urine and bile about 8 h later (Fishbein  et al.,
    1969).

         A series of studies were performed on young male Charles River CD
    rats with piperonyl butoxide labelled with 14C on the alpha-carbon
    of the 2-(2-butoxyethoxy)ethoxymethyl side-chain. Four rats were given
    a single dose of about 500 mg/kg bw (15.6  0.4 Ci) by gavage. An
    average of 0.18% of the administered radiolabel was expired as carbon
    dioxide during the 24 h after treatment. Four rats were given a single
    dose of about 500 mg/kg bw (14.4  0.7 Ci) by gavage; blood samples
    were then collected from the tail vein for 24 h and analysed for
    radioactivity. Plasma radioactivity reached a peak 3-12 h after
    treatment and dropped to about half the peak level within 24 h. Four
    rats were given a single dose of about 500 mg/kg bw (14.1  1.3 Ci)
    by gavage; urine and faecal samples were collected and weighed 4, 8,
    12, and 24 h after treatment and then every 24 h for seven days. Most
    of the radiolabel was recovered in urine and faeces 12-2.4 h after
    treatment; by 168 h after treatment, an average of about 38% of the
    administered radioactivity was recovered in urine and 62% in faeces.

         Twenty rats were given a single dose of about 500 mg/kg bw by
    gavage; groups of five rats were killed 1, 6, 24, 48, and 168 h after
    treatment, and several tissues were examined for radioactivity. At
    each interval, the highest levels of radiolabel were found in the
    gastrointestinal tract and its contents; high levels were also found
    in lungs, liver, kidneys, fat, prostate, and seminal vescicles. At 1,
    6, 24, 48. and 168 h, 62, 67, 37, 13, and 1%, respectively, of the
    administered radiolabel was recovered. Five rats received piperonyl
    butoxide at about 500 mg/kg bw per day for 13 days and then a single
    dose of about 500 mg/kg bw of radiolabelled compound (9.6  0.2 Ci)
    by gavage. Most of the radiolabel was recovered in urine and faeces
    12-48 h after treatment. By 168 h after treatment, an average of about
    43% of the radiolabel was recovered in urine and 54% in faeces
    (Selim, 1985).

    (b)  Effects on enzymes and other biochemical parameters

         Many studies have been performed  in vitro and  in vivo to
    elucidate the mechanism of action of piperonyl butoxide. A single
    intraperitoneal injection of 450 mg/kg bw inhibited microsomal
    mixed-function oxidases in the livers of male Swiss-Webster mice; the
    duration and extent of inhibition depended on the substrate used
    (Skrinjaric-Spoljar  et al., (1971).

         In male Swiss mice given a single intraperitoneal dose of
    160 mg/kg bw of piperonyl butoxide, 50-60% inhibition of dimethyl-
    aminopyrine and hexobarbital hydroxylases was found 1 h after
    treatment (Jaffe  et al., 1968).

    CHEMICAL STRUCTURE

         A dose-dependent, biphasic effect was observed on the  ortho and
     para hydroxylations of biphenyls by liver microsomes of mice treated
    with 10-640 mg/kg bw of piperonyl butoxide intraperitoneally,
     para-Hydroxylation was inhibited by 80% at the highest dose 30 min
    after treatment but had recovered within 24 h;  ortho-hydroxylation
    was induced by up to 150% 1 h after treatment, and activity was still
    elevated after 96 h in the groups at the highest dose (Jaffe  et al.,
    1969).

         Piperonyl butoxide inhibits ethylmorphine  N-demethylation by
    liver microsomes  in vitro. The inhibition is proportional to the
    amount of cytochrome P450-piperonyl butoxide metabolite (unknown)
    complex formed. Differences between liver microsomes from rats and
    mice may account for the greater sensitivity of mice to inhibition of
    drug metabolism by piperonyl butoxide: the Ki for inhibition of
    ethylmorphine N-demethylase was found to be 19 mol/litre in rats and
    6 mol/litre in mice (Franklin, 1972).

         Mouse liver homogenates (20% w/v in 50 mmol/litre phosphate
    buffer, pH 7.4) were fractioned into nuclear, mitochondrial,
    microsomal, and soluble fractions, which were incubated with labelled
    piperonyl butoxide. NAD, NADP, NADH, or NADPH was sometimes added as a
    cofactor. Only the microsomal fraction had significant metabolic
    activity in the presence of NADPH and, to a lesser extent, with NADH
    (Kamienski & Casida, 1970).

         Male CF-1 mice were given a single intraperitoneal dose of
    piperonyl butoxide (purity, 87-89%) at 0.5-25 mg/kg bw 1 h before
    intraperitoneal administration of 40 mg/kg bw pentobarbital or
    100 mg/kg bw zoxazolamine. Pentobarbital-induced sleeping time was
    significantly increased by 10 or 25 mg/kg bw piperonyl butoxide, and
    zoxazolamine-induced paralysis time was significantly increased by 5,
    10, or 25 mg/kg bw (Conney  et al., 1972). Skinjaric-Spoljar  et al.
    (1971) found that a single intraperitoneal dose of 22 mg/kg bw
    piperonyl butoxide to mice increased the hexobarbital (62.5 mg/kg bw
    intraperitoneally) sleeping time from 28 to 100 min.

         Male Sprague-Dawley rats were given single intraperitoneal doses
    of piperonyl butoxide (purity, 87-89%) at 67-1000 mg/kg bw 1 h before
    intraperitoneal administration of pentobarbital (25 mg/kg bw) or
    zoxazolamine (70 mg/kg bw). Pentobarbital-induced sleeping time was
    significantly increased by 1000 mg/kg bw piperonyl butoxide, and
    zoxazolamine-induced paralysis time was significantly increased by 333
    or 1000 mg/kg bw (Conney  et al., 1972).

         Male mice and rats were given single intraperitoneal doses of
    piperonyl butoxide 1 h before injection of 200 mg/kg bw antipyrine.
    The NOAELs for effects on antipyrine metabolism were 100 mg/kg bw in
    rats and 0.5 mg/kg bw in mice (Conney  et al., 1972).

         Groups of six male weanling Sherman rats were fed technical-grade
    piperonylbutoxide (purity, 80%) at dietary concentrations of 0, 1000,
    5000, or 10 000 ppm for one, four, or eight weeks. The activities of
    hexobarbital oxidase, aniline hydroxylase,  para-nitroanisole
    demethylase, nitroreductase, and glucuronyltransferase and the P450
    content were increased two- to fourfold by 5000 or 10 000 ppm
    piperonyl butoxide. Liver weights and microsomal protein content were
    increased to a maximum of 50-70%. Electron microscopy showed
    enlargement and extensive proliferation of the smooth endoplasmic
    reticulum in liver parenchymal cells. The dose of 1000 ppm had minimal
    effects on liver weight and on P450 and glucuronyl transferase
    activity and no effect on proliferation of the smooth endoplasmic
    reticulum. Maximal effects on P450 content and on the activity of
    P450-related enzymes were observed after one week of treatment. The
    maximal effect on glucuronyltransferase activity was observed between
    four and eight weeks of treatment (Goldstein  et al., 1973).

         Piperonyl butoxide (purity unsepcified) dissolved in corn oil was
    administered to 10 male Sprague-Dawley rats at 400 mg/kg bw
    intraperitoneally; another group of animals received 100 mg/kg bw of
    piperine intraperitoneally. Control animals received 2 ml/kg bw corn
    oil. Groups of five animals were sacrificed 1 and 24 h after
    treatment, and hepatic levels of cytochromes P450 and b5, proteins,
    and the activities of NADPH cytochrome  c reductase, benzphetamine
     N-demethylase, aminopyrine  N-demethylase, and aniline hydroxylase
    were determined. The microsomal protein level was not affected by
    treatment. Piperonyl butoxide decreased the P450 but not the b5 level
    by about 30% after 1 h; however, after 24 h the contents were
    increased by about 100 and 30%, respectively. NADPH cytochrome  c
    reductase activity was increased by 50% 24 h after treatment. The
    activities of  N-demethylases and aniline hydroxylase were decreased
    by 20 and 50%, respectively, 1 h after administration of either
    piperine or piperonyl butoxide. By 24 h, the activities were still low
    in piperine-treated animals but had increased by 30-80% in those given
    piperonyl butoxide (Dalvi & Dalvi, 1991).

         Single intraperitoneal doses of 400 mg/kg bw piperonyl butoxide
    were given to male CD-1 mice 1 h before a single intraperitoneal dose
    of parathion-methyl, azinphos-methyl, parathion, or azinphos-ethyl, or
    their oxygen analogues. The LD50 of the oxygen analogues was not
    altered whereas that of the methyl homologues was reduced, with a
    40-fold increase in the LD50 for parathion-methyl and a threefold
    increase in that for azinphos-methyl; that of the ethyl homologues was
    increased, with a 50% decrease in the LD50 for parathion and an 85%
    decrease for azinphos ethyl. The plasma levels of all pesticides were
    increased by three- to sevenfold in piperonyl butoxide-pretreated
    animals 30 min after treatment (Levine & Murphy, 1977a). It has been
    suggested that detoxification of parathion-methyl, but not that of
    parathion- or azinphos-ethyl, continues through uninhibited
    glutathione-dependent pathways (Mirer  et al., 1977; Levine & Murphy,
    1977b).

         A single intraperitoneal dose of 600 mg/kg bw piperonyl butoxide
    to three-month-old CD-1 mice reduced the hepatotoxicity (as assessed
    by reduced glutathione content, plasma sorbitol dehydrogenase, and
    histopathological liver necrosis) caused by an oral dose of 600 mg/kg
    bw acetominophen given 2 h earlier or 1 h later. Since the hepatic
    mixed-function oxidase system generates a toxic, electrophilic
    metabolite of acetominophen, piperonyl butoxide probably acts by
    inhibiting: that enzymatic system (Brady  et al., 1988).

         Pretreatment of groups of 10 male Syrian golden hamsters with
    400 mg/kg bw piperonyl butoxide given subcutaneously 2 h before
    subcutaneous injection of 17.8 mg/kg bw  N-nitrosodiethylamine twice
    weekly for 20 weeks inhibited the development of pulmonary
    carcinogenesis by the nitrosamine. The numbers of animals with lung
    tumors were 0 controls, 6 treated only with  N-nitrosodiethylamine, 0
    treated with piperonyl butoxide and nitrosamine, and 0 given only
    piperonyl butoxide. Tracheal tumors were found in 0, 10, 5, and 0
    animals in the four groups, respectively. The protective effect was
    associated with reduced (by 50% or more) covalent binding of
     N-[ethyl-1-14C]-nitroso-diethylamine to macromolecules in trachea,
    lung, and liver (Schuller & McMahon, 1985).

         Eight-week-old male CD-1 mice were fed diets containing piperonyl
    butoxide at concentrations adjusted to provide doses of 0, 10, 30,
    100, or 300 mg/kg bw per day or sodium phenobarbital at 0.05% (w/w)
    for 42 days. Replicative DNA synthesis was studied by implanting
    seven-day osmotic pumps to administer 5-bromo-2'-deoxyuridine (BrDU)
    during days 0-7 and 35-42 of treatment; liver sections were
    immunostained with an antibody to BrDU, and the percentage of
    hepatocyte nuclei undergoing replicative DNA synthesis was calculated
    by microscopic examination of at least 1000 nuclei. Xenobiotic
    metabolism was assessed by measuring the liver P450 content and the
    activities of 7-ethoxyresorufin  O-deethylase, 7-pentoxyresorufin
     O-depentylase, and ethylmorphine  N-demethylase in liver
    microsomes. Sodium phenobarbital was used as a positive control. The
    relative liver weight was increased by about 15% after seven days of
    treatment with 300 mg/kg bw per day of piperonyl butoxide and by about
    20% with sodium phenobarbital; after 42 days, it was increased by
    about 10% after treatment with 100 and by about 20% after treatment
    with 300 mg/kg bw piperonyl butoxide. Midzonal liver-cell hypertrophy
    was observed in mice given 300 mg/kg bw per day for seven or 42 days
    or 100 mg/kg bw per day for 42 days. Sodium phenobarbital induced
    centrilobular hypertrophy after either seven or 42 days of treatment.
    Replicative DNA synthesis was induced to 350% by the high dose of
    piperonyl butoxide and to 825% by sodium phenobarbital for seven days;
    no significant increase was observed after 42 days at any dose. A
    dose-related increase in microsomal protein and P450 content was
    observed on day 42, and the increases were statistically significant

    at doses > 100 mg/kg bw. Inconsistent increases were observed in
    the activities of 7-ethoxyresorufin  O-deethylase and 7-pento-
    xyresorufin  O-depentylase in piperonyl butoxide-treated mice, but a
    dose-related increase from 20 to 50% in the activity of ethylmorphine
     N-demethylase was seen on day 42. Treatment with sodium pheno-
    barbital for 42 days resulted in increases in all of the measured
    parameters. Piperonyl butoxide (and sodium phenobarbital) thus caused
    a transient stimulation of liver-cell replication and a permanent
    effect on liver weight, morphology, and enzyme induction at doses that
    induced liver nodules in long-term studies (Phillips  el al., 1995).

    2.  Toxicological studies

    (a)  Acute toxicity

         Groups of five Sprague-Dawley CD rats of each sex were exposed by
    inhalation for 4 h to a mean analytical concentration of 5.9 mg/litre
    piperonyl butoxide (purity, 90.78%). The particle median aerodynamic
    diameter was 2.6 m; about 15% of the aerosol was < 1 m in diameter
    and about 95% < 10 m. All animals survived the exposure and the
    15-day observation period after treatment. Exposure caused excessive
    lacrimation and salivation, nasal discharge and laboured breathing.
    All animals recovered. No remarkable signs were seen  post mortem
    (Hoffman, 1991). Other data are reported in Table 1.

    (b)  Short-term toxicity

    Mice

         Groups of 10 male and 10 female ICR (Crj:CD-1) mice were fed
    diets containing 0, 1000, 3000, or 9000 ppm piperonyl butoxide (purity
    unspecified) for 20 days. Animals were observed daily for mortality;
    body weight was measured and clinical observations were made on days
    1, 2, 3, 7, 14, and 20; the food consumption of five animals per cage
    was measured on days 0-3, 3-7, 7-14, and 14-20. At the end of
    treatment, blood clinical chemistry was assessed, liver, kidneys, and
    spleen were weighed, and livers and kidneys were examined
    histologically. No deaths occurred. The body weights of animals at the
    high dose were about 15% lower than those of controls at the end of
    the study, and those of females at the middle dose were about 10%
    lower on day 2 and about 8% lower at termination (not statistically
    significant). The lowered body weights were apparently associated with
    decreased food intake. The weights of livers showed a dose-related
    increase in both males (by 22% at the low dose, 63% at the middle
    dose, and 79% at the high dose) and females (62% at the middle dose
    and 78% at the high dose); at the high dose, the weights of the
    kidneys were reduced by 29% in males and 18% in females, and the
    weights of the spleen by 25% in males and 33%, in females. In animals
    at the high dose, a 31% increase in serum cholesterol was seen in
    males and a 67% increase in females, a 28% increase in serum
    phospholipids was seen in males and a 38% increase in females, a 19%

        Table 1.  Acute toxicity of piperonyl butoxide in animals
                                                                                                             

    Species     Sex               Route                LD50 or LC50        Purity      Reference
                                                       (mg/kg bw or        (%)
                                                       mg/litre air)
                                                                                                             

    Mouse       NR                Oral                        4.0          NR          Negherbon (1959)
    Rat         Male              Oral                       4.57          90.78       Gabriel (1991a)
                Female                                       7.22
                NR                Oral                   8.0-10.6          NR          Sarles et al. (1949)
                NR                Oral                       13.5          NR          Lehman (1948)
                NR                Oral                       11.5          NR          Lehman (1951)
                Male, female      Inhalation (4 h)         > 5900          90.78       Hoffman (1991)
                NR                Subcutaneous             > 15.9          NR          Sarles et al. (1949)
    Rabbit      Male, female      Dermal                      > 2          90.78       Gabriel (1991b)
                NR                Oral                    2.7-5.3          NR          Sarles et al. (1949)
    Cat         NR                Oral                     > 10.6          NR          Sarles et al. (1949)
    Dog         NR                Oral                      > 8.0          NR          Sarles et al. (1949)
                                                                                                             

    NR, Not reported
    
    increase in total serum proteins occurred in males and an 18% increase
    in females, and a 235% increase in gamma-glutamyl transpeptidase was
    seen in females. Some of these levels were also increased in the
    animals at the middle dose. Microscopic examination of the liver
    showed hypertrophic hepatocytes, single-cell necrosis, and cell
    infiltration in the centrilobular area in all mice at the high dose.
    The kidneys were unremarkable. The NOAEL for effects on the liver was
    1000 ppm, equivalent to 150 mg/kg bw per day (Fujitani  et al.,
    1993a).

         Groups of 20 Crj:CD-1 male mice were fed diets containing 0,
    1500, 3000, or 6000 ppm piperonyl butoxide (purity unspecified) for
    seven weeks. Food intake was measured every week and behavioural tests
    were performed at weeks 4 (exploratory behaviour), 6 (multiple water
    T-maze), and 7 (exploratory behaviour) of treatment. A treatment-
    related reduction in food intake was observed in animals at the middle
    and high doses during the first week of treatment. No consistent,
    significant effect was observed in the T-maze test or in the
    exploratory behaviour test at week 4. At week 7, some parameters in
    the exploratory behaviour test were altered (Tanaka, 1993).

    Rats

         Hypertrophy of periportal cells with slight fatty changes were
    observed in rats fed a diet containing 5000 ppm piperonyl butoxide for
    17 weeks (Lehman, 1952a,b).

         Feeding of six weekly doses of up to 224 mg/kg bw of active
    ingredient to rats caused no observable effects at autopsy three weeks
    after the final dose, but few details were given (Sarles  et al.,
    1949).

         In a range-finding study, groups of 10 male and 10 female
    Sprague-Dawley rats were fed diets containing concentrations of
    piperonyl butoxide adjusted to obtain doses of 0, 62.5, 125, 250, 500,
    1000, or 2000 mg/kg bw per day for four weeks. All animals were
    observed for mortality and morbidity twice daily, and body weight and
    food consumption were measured weekly. Haematological and biochemical
    tests were performed on day 24 or 25 of treatment. At termination,
    survivors were necropsied, certain organs were weighed and microscopic
    examinations were performed on several organs. Six animals died before
    termination, but the deaths were considered to be due to anaesthesia
    for bleeding. General signs of toxicity, such as prominent backbone,
    thinness, poor fur condition, brown staining and piloerection, were
    observed in animals at the highest dose. Body-weight gain was reduced
    by about 20% in females at 500 mg/kg bw per day, by 27% in males and
    37% in females at 1000 mg/kg bw per day, and by 66% in males and 92%
    in females at 2000 mg/kg bw per day. These reductions were only
    partially associated with reduced food intake. Haematology and
    clinical biochemistry showed no significant alterations. Absolute and
    relative liver weights were increased in animals given doses >
    500 mg/kg bw per day; the relative liver weights were also increased
    in males at 250 mg/kg bw per day. The relative weights of the
    adrenals, kidneys, and brain were slightly increased in the group at
    the highest dose and in some animals at 1000 mg/kg bw per day.
    Eosinophilia and loss of vacuolation in hepatocytes were noted in all
    treated animals, and the severity of these lesions was dose-related.
    These may be adaptive changes. Necrosis of hepatocytes and cytoplasmic
    inclusions were observed in animals at 1000 and 2000 mg/kg bw per day.
    The NOAEL was 125 mg/kg bw per day on the basis of effects on the
    liver (Modeweg-Hansen  et al., 1984).

         In a study preliminary to the study of carcinogenicity by the
    same authors summarized below, groups of 10 Fischer 344/DuCrj rats of
    each sex were fed diets containing 0, 2500, 5000, 10 000, 20 000, or
    30 000 ppm piperonyl butoxide (purity, 89%) for 13 weeks. At
    termination, survivors were necropsied, and limited microscopic
    examination was performed. One male at the high dose died before
    termination. The final body weights and body-weight gains were reduced
    in a dose-related manner in all treated males; in females, a
    significant effect was seen only at the two highest doses. Liver
    weights were increased in all treated animals, by up to 50% in males
    and 108% in females. Absolute kidney weights were increased in animals
    at the high dose, and the relative weight was also increased in groups
    at lower doses. Hepatocyte hypertrophy and focal necrosis were seen in
    animals at higher doses (data not shown). No relevant macro- or
    microscopic lesions were observed in the digestive tract, but the
    caecum was not examined histologically. There was no NOAEL because of
    effects on the liver at all doses (Maekawa  et al., 1985).

         Groups of 15 Charles River CD rats of each sex were exposed by
    inhalation for 6 h per day, five days per week, for 13 weeks to
    piperonyl butoxide (purity, 90.78%) at mean analytical concentrations
    of 15, 74, 155, or 512 mg/m3. The highest concentration was the
    maximum that could be generated at the appropriate particle size.
    Fifteen controls were exposed to conditioned room air only. The
    concentrations in chamber air were monitored gravimetrically four
    times per day and by gas chromatography once a day. Particle size
    distribution was measured once during each exposure. The test aerosol
    had a mass median aerodynamic diameter of 1.7 m with a geometric
    standard deviation of 2.6. On average, 37% of the particles were <
    10 m in diameter and 29% < 1 m. Abnormal signs were looked for once
    during each exposure, and detailed physical examinations were
    conducted on all animals once before treatment and weekly during
    treatment. Ophthalmoscopic examinations were performed on all animals
    before treatment and on the day before sacrifice. Body weight was
    recorded before exposure, immediately before exposure on test day 1,
    weekly thereafter, and just before sacrifice. At termination,
    haematological and blood chemical parameters were evaluated in all
    animals, selected organs were weighed, complete gross post-mortem
    examinations were conducted, and selected tissues from all animals
    were examined microscopically.

         Body-weight gain and food consumption were not affected by
    exposure, and all animals survived to the end of treatment.
    Ophthalmoscopic examinations showed no indication of exposure-related
    ocular effects. There was a dose-related increase in nasal discharge,
    dried material on the facial area and extremities, and anogenital
    staining in animals at 155 and 512 mg/m3. The serum levels of
    aspartate and alanine aminotransferases and of glucose were slightly
    decreased (10-28%), while blood urea nitrogen, total protein, and
    albumin levels were slightly increased (4-10%) in animals of each sex
    at the high dose. Not all of these differences were statistically
    significant, and a dose-response relationship was not seen
    consistently; however, a similar trend was seen in animals of each
    sex. Statistically significant increases in the absolute and relative
    weights of the livers and kidneys were seen in animals at the high
    dose; the relative kidney weights were increased by 10-12%, and the
    absolute and relative liver weights were both increased by 20-29%.
    Relative liver weights were also significantly elevated (8-9%) in
    males and females at 155 mg/m3. Vesiculation and vacuolation of the
    hepatocellular cytoplasm was seen in almost all treated animals but
    tended to be more pronounced in those at the highest level. No clear
    dose-related increase in either incidence or severity was observed.
    Squamous or squamoid metaplasia of the pseudostratified columnar
    epithelium of the larynx was seen in several exposed animals and one
    control female, the severity being greater in animals at the high
    dose. Similar metaplastic changes were seen in the columnar epithelium
    lining the ventral diverticulum in several animals at 512 mg/m3 and

    in one female at 15 mg/m3. Hyperplasia and hyperkeratosis of the
    squamous epithelium normally found in the larynx were seen in a small
    number of animals at 512 mg/m3. Laryngeal mucosal inflammation was
    seen in all treated animals and was slightly more severe in animals at
    the highest dose. All of the changes were considered to be localized
    responses indicative of irritation rather then systemic toxicity.
    Other findings in tissues and organs examined macroscopically and
    microscopically  post mortem were unremarkable. No systemic toxicity
    was seen at 155 mg/m3; effects on the liver and kidney were seen at
    512 mg/m3 (Newton, 1992).

         Groups of 10 male and 10 female Fischer 344/DuCrj rats were fed
    diets containing 0, 6000, 12 000, or 24 000 ppm piperonyl butoxide
    (technical grade of unspecified purity) for 13 weeks. Animals were
    observed daily for mortality; body weight and clinical signs were
    monitored daily for the first five days and then twice weekly. Food
    and water consumption was measured on days 4, 11, 18, and 39 for about
    six rats in each group. At the end of treatment, haematological and
    blood chemical parameters were determined, animals were necropsied,
    and selected organs were weighed; the liver and kidneys were examined
    histologically. There were no deaths. Clinical signs consisted of nose
    bleeds from about day 2 to day 20 and dose-dependent abdominal
    distension. In animals at the high dose, food consumption was reduced
    by about 46% and water consumption by 28% on day 4 but not at other
    times. The body weights of animals at the high dose were significantly
    reduced, by 36% in males and 24% in females. Blood haemoglobin levels
    were decreased in a dose-dependent manner, but the decrease was
    statistically significant only in animals at the high dose (by 10-11%)
    and females at the middle dose (by 7%). A reduced mean corpuscular
    haemoglobin content was also observed in females at the high dose.
    Serum albumin was increased by 16-23%, cholesterol by 88-93%,
    gamma-glutamyl transpetidase activity to five to six times the control
    values in males at the high dose, and urea nitrogen by 24% in males at
    that dose. Serum protein was increased in all treated females and
    serum phospholipid in females at the high dose; decreases were seen in
    serum levels of triglyceride in all males and bilirubin and glucose in
    males at the high dose. Absolute and relative liver weights were
    increased in a dose-related manner, but were significantly increased
    only in males at the middle dose (by 47%) and males and females at the
    high dose (by 57-94%). Relative kidney weights were increased in a
    dose-related manner to up to 1.42% of control values. Hypertrophic
    hepatocytes containing a basophilic, fine granular substance were seen
    in animals at the high dose. In the periportal area, marked
    vacuolation of hepatocytes was observed, with occasional coagulative
    necrosis and oval-cell proliferation. In male rats, atrophy of the
    epithelium of the proximal convoluted tubules was observed in the
    renal cortex. There was no NOAEL because of effects on the liver and
    kidney (Fujitani  et al., 1992).

         In a subsequent study performed under the same experimental
    conditions, haematological, clinical chemical, and morphological
    observations after 1, 2, 4, or 12 weeks of treatment showed that all
    of the alterations occurred in a time-dependent manner, and that some
    of them (increased liver and kidney weights, morphological alterations
    in the liver) were already present after one week (Fujitani  et al.,
    1993b).

    Rabbits

         Three weekly doses of up to 108 mg/kg bw active ingredient of a
    5% emulsion of piperonyl butoxide showed no effects at autopsy three
    weeks after the final dose, but few details were given (Sarles
     et al., 1949).

    Dogs

         Groups of one to three dogs of each sex were given piperonyl
    butoxide at 0, 3, 32, 106, or 320 mg/kg per day orally in capsules on
    six days per week for one year. All dogs at the two highest doses lost
    weight, but large variations in body-weight gain and the small number
    of animals prevented meaningful comparisons between the controls and
    animals at the lower doses. A dose-related increase in relative liver
    weight (no statistical analysis reported) was observed, which was
    associated with unspecified histopathological changes. Increased
    kidney and adrenal weights were observed at 100 and 320 mg/kg bw per
    day (Sarles & Vandergrift, 1952).

         Groups of two beagle dogs of each sex were fed diets containing
    piperonyl butoxide (purity, 90.78%) for eight weeks at concentrations
    corresponding to 500, 1000, 2000, or 3000 ppm active ingredient. The
    diets were prepared weekly; the actual concentrations and the
    homogeneity of the mixture were measured at three intervals and found
    to be satisfactory. Animals were observed at least twice daily for
    mortality and signs of toxicity; detailed observations were recorded
    at least once weekly, and body weight and food consumption were
    recorded weekly. Physical examinations and haematological and
    biochemical tests were conducted on all animals before exposure and at
    termination. All major tissues and organs were examined
    microscopically  post mortem. Selected organs were weighed. All
    animals survived to study termination. Reduced body-weight gains were
    seen in males and females receiving 1000 ppm (14 and 9% of the initial
    body weight, respectively), 2000 ppm (6 and 7%), and 3000 ppm (7 and
    4%) ppm in comparison with the control group (17% and 14% increases).
    Decreased food consumption (by 19 and 23%) was observed animals at
    3000 ppm. There were no treatment-related changes in haematological
    parameters. Males and females at 2000 and 3000 ppm had alkaline
    phosphatase activities that were about 1.5 times the control value and
    increased absolute and relative liver and gall-bladder weights. No

    macroscopic changes were observed that could be attributed to
    treatment, and treatment-related microscopic changes were limited to
    diffuse, mild hypertrophy of hepatocytes in all treated males and in
    females at the two highest doses. There was no NOAEL because of
    effects on the liver (Goldenthal, 1993a).

         Groups of four beagle dogs of each sex were fed diets containing
    piperonyl butoxide (purity, 90.78%) for one year at concentrations
    corresponding to 100, 600, and 2000 ppm active ingredient. The diets
    were prepared weekly and stored at room temperature. The stability of
    the compound was assessed twice: an initial test showed a loss of
    about 10% over 10 days, possibly due to technical problems; a second
    test showed 97-101% of the initial concentration on day 10. The actual
    concentrations were measured 16 times and were found to be 87-110% of
    the nominal concentrations. The homogeneity of the mixture was also
    found to be satisfactory. The dogs were observed at least twice daily
    for mortality and signs of toxicity; detailed observations were
    recorded weekly. Body weight and food consumption were recorded weekly
    for the first 14 weeks and every two weeks thereafter. Ophthalmoscopic
    examinations were conducted before the beginning of treatment and at
    termination. Physical examinations were conducted on all animals
    before exposure and after 3, 6, 9, and 12 months of treatment.
    Haematological and biochemical tests and urinalyses were conducted on
    all animals after 6 and 12 months. All animals were examined
     post mortem, and all major tissues and organs were examined
    microscopically. Selected organs were weighed. All animals survived to
    the end of the study, with no relevant clinical or ophthalmological
    signs. The body weights of males and females receiving 2000 ppm
    piperonyl butoxide were 2% lower to 3% higher than the initial
    weights, whereas those of controls were 22-25% higher. Treatment-
    related decreases in food consumption were observed in males at 600
    (by 15%) and 2000 (by 20%) ppm. Small, not statistically significant,
    not dose-related decreases in food consumption were observed in
    treated females.

         No treatment-related change in haematological parameters was
    observed. The activity of alkaline phosphatase was increased to three
    to five times the control values in animals at 2000 ppm after 6 and 12
    months, and cholesterol levels were decreased (not statistically
    significant) at these intervals in females. Males and females at this
    dose had increased absolute (by 22 and 36%) and relative (by 52 and
    86%) liver and gall-bladder weights, and small increases in thyroid
    and parathyroid weights (average, 34%) were observed in females. These
    changes were not associated with microscopic changes in the thyroid
    and were considered to be of questionable biological significance. No
    macroscopic pathological changes were observed that could be

    attributed to treatment. Treatment-related histopathological changes
    were limited to diffuse, mild hypertrophy of the hepatocytes in males
    and females at 2000 ppm. The NOAEL was 100 ppm, equal to 16 mg/kg bw
    per day, on the basis of effects on the liver, some clinical chemical
    alterations, and reduced body-weight gain at 2000 ppm (Goldenthal,
    1993b).

    Monkeys

         Two African green monkeys were given 32 or 106 mg/kg bw per day
    piperonyl butoxide orally for six days per week for four weeks.
    Minimal microscopic changes were observed in the livers (Sarles &
    Vandergrift, 1952).

    (c)  Long-term toxicity and carcinogenicity

    Mice

         Groups of 18 (C57Bl/6  C3H/Anf)F1 and (C57Bl/6  AKR)F1 mice of
    each sex received piperonyl butoxide (purity unspecified) at 100 mg/kg
    bw or 'Butacide' at 464 mg/kg bw in 0..5% gelatin at seven days of age
    by stomach tube and the same amount (not adjusted for increasing body
    weight) daily up to four weeks of age; subsequently, the mice were fed
    diets containing 300 ppm. The dose was reported to be the maximal
    tolerated dose for infant and young mice. All animals were killed when
    they were about 70 weeks of age, and their tumour incidences were
    compared with those of 79-90 necropsied mice of each sex and strain,
    which had either been untreated or had received gelatine only. No
    significant difference in the incidence of tumours was found between
    treated and control mice, although the authors concluded that
    additional evaluation was required (Innes  et al., 1969). This study
    was not considered to be adequate by the Meeting.

         Groups of 50 B6C3F1 mice of each sex were fed diets containing
    5000 or 10 000 ppm piperonyl butoxide (purity, 88.4%) for 30 weeks;
    the doses were decreased to 500 or 2000 ppm for the following 82
    weeks, due apparently to a greater than expected decrease in
    body-weight gain. The control group consisted of 20 males and 20
    females. Animals were observed twice daily for signs of toxicity; body
    weight was recorded at least once per month. Necroscopic examination
    was performed at termination on all moribund animals and on those
    found dead, unless precluded by autolysis. Major tissues and organs
    and all gross lesions were examined microscopically. At termination,
    85% of control males and 75% of control females, 82% and 68% at the
    low dose, and 84% and 70% at the high dose, respectively, were still
    alive. Body weight was reduced in a dose-related manner, by up to
    about 15% in females at the high dose at termination. No
    treatment-related increase in tumour incidence was found, but
    hepatocellular carcinomas occurred frequently, especially in males,

    with incidences of 50% in controls, 34%, at the low dose, and 40% at
    the high dose. No statistically significant differences were found in
    the incidences of neoplastic and non-neoplastic lesions (US National
    Cancer Institute, 1979)

         Groups of 60 CD-1 mice of each sex were fed diets containing
    piperonyl butoxide (purity, 90.78%) at concentrations adjusted to give
    doses of 0 (two control groups), 30,100, or 300 mg/kg bw per day, for
    78 weeks. The diets were prepared weekly and the concentrations
    adjusted on the basis of body weights and food consumption. The diets
    were analysed for the actual concentration, homogeneity, and stability
    of piperonyl butoxide. The average actual concentrations at the low
    and high doses were 99 and 95%. The homogeneity and stability (no
    decrease in concentration after 14-21 days) of piperonyl butoxide in
    the diet were considered to be satisfactory. Animals were observed
    twice daily for signs of toxicity. Food consumption and body weight
    were determined weekly for the first week and then every other week.
    Haematological measurements were made for 10 rats of each sex in the
    control and high-dose groups at week 52 and for all groups at
    termination. All surviving animals were necropsied; major tissues and
    organs from controls and animals at the high dose and the lungs,
    liver, kidneys, and gross lesions from other groups were examined
    microscopically. All slides from the livers underwent an independent
    pathological review; the results reported here are the consensus or
    majority opinions of all the pathologists involved.

         No treatment-related clinical signs or palpable masses were
    observed. The mortality rates were (males/females) 27/32, 32/27,
    27/38, 22/33, and 40/18% in the first control group, animals at 30,
    100, and 300 mg/kg bw per day, and the second control groups,
    respectively. Body weights and body-weight gains of animals at the
    high dose were slightly (occasionally statistically significantly)
    decreased. No significant effects on food consumption were observed.
    Haematological parameters were also unaffected by treatment. A
    dose-related increase in absolute and relative liver weights was
    observed in mice of each sex at the middle and high doses.
    Hepatocellular hypertrophy was more frequent in males at the high
    (72%) and middle doses (27%) than in controls (10 and 18%) and in
    females at the high dose (15%; 0 and 7% in controls). Hepatocellular
    hyperplasia (also called eosinophilic/clear-cell focus), characterized
    by aggregates of hepatocytes with a swollen eosinophilic cytoplasm,
    was present in 8% of males at the high dose, 2% of females at the
    middle dose, and 7% at the high dose. The incidences of hepatocellular
    hyperplasia were 0 and 3% in control males and 0% in both female
    control groups. The incidence of hepatocellular adenomas was higher in
    male mice at the middle (37%) and high (47%) doses than in either
    control group (15 and 13%) and in females at the high dose (17%; 3% in
    controls). The hepatocellular adenomas observed in many treated
    animals were composed of large, polyhedral, densely-packed cells with
    abundant granular eosinophilic cytoplasm. This appearance is different
    from that of spontaneous adenomas found in CD-1 mice, where small to

    medium, well-differentiated, basophilic cells, distributed in solid to
    normal sinusoidal patterns, are found. Only the incidence of the
    former type of adenoma was increased in piperonyl butoxide-treated
    mice. A nonsignificantly increased incidence of hepatocellular
    carcinomas was observed in males at the high dose (12%; 3% in
    controls). No other treatment-related neoplastic or non-neoplastic
    lesion was found. The NOAEL was 30 mg/kg bw per day on the basis of
    effects on the liver (Hermanski & Wagner, 1993).

         A 12-month study of carcinogenicity was conducted in male
    Crj:CD-1 mice fed diets containing 0 ( n = 52), 6000 ( n = 53), or
    12 000 ( n = 100) ppm piperonyl butoxide (purity, 94.3%; no
    detectable safrole or isosafrole). Animals were observed daily for
    mortality and morbidity; those that died or were sacrificed at
    termination were necropsied, and gross lesions and the liver were
    examined histologically. The mortality rates were 6% of controls, 2%
    at the low dose, and 19% at the high dose. Terminal body weight was
    found to have been reduced by 17% at the low dose and 29% at the high
    dose. Hepatocellular hyperplasia was seen in 38% of animals at the low
    dose and 8% at the high dose, hepatocellular adenomas in 13 and 22%,
    hepatocellular carcinomas in 11 and 52%, and haemangioendothelial
    sarcoma in 2 and 42%. It should be noted that the hepatocellular
    adenomas and carcinomas may have obscured the true dose-response
    relationship for hepatocellular hyperplasia. A condition termed
    'post-necrotic peliosis', consisting of multifocal necrosis with blood
    cysts, was also found in 17% of animals at the high dose. One control
    animal showed hepatocellular hyperplasia and one had a hepatocellular
    adenoma (Takahashi  et al., 1994a).

    Rats

         Groups of 12 Wistar rats of each sex were fed diets containing 0,
    100, 1000, 10 000, or 25 000 ppm piperonyl butoxide for two years, and
    an additional group was fed a diet containing 1000 ppm piperonyl
    butoxide plus 167 ppm pyrethrins. Animals were observed daily for
    clinical signs of toxicity, and food consumption and body weight were
    determined weekly. Autopsies were performed on all animals. Males and
    females were paired for the duration of the study, except during
    nursing (see section ( d)). Body-weight gain was reduced in animals
    at 10 000 (by 10-20%) and 25 000 ppm (by about 90%), in association
    with reduced food intake. All animals at the high dose were dead by
    week 68; the mortality rates in the other groups were (males/females):
    30/20%, 40/30%, 40/50%, and 60/60% for controls and animals at 100,
    1000, and 10 000 ppm, respectively. The relative weights of livers and
    kidneys were higher than those of controls in animals at 10 000 (by
    40% for liver and kidney) and 25 000 ppm (by 275% for liver and 150%
    for kidney). These effects were not associated with morphological
    alterations. The incidence of tumours was not increased (Sarles &
    Vandergrift, 1952).

         Sixty Sprague-Dawley Crl:CDR (SD)BR rats of each sex were fed
    diets containing piperonyl butoxide (purity, 89%) at concentrations
    adjusted weekly (every two weeks after week 15) to achieve daily
    intakes of 0 (two groups of controls), 30, 100, or 500 mg/kg bw per
    day for 104-105 weeks. The stability and homogeneity of the diets and
    the correspondence of actual to nominal concentrations were checked
    before and throughout the study and found to be acceptable. The
    animals were observed for mortality, clinical signs, food consumption,
    body weight, and ophthalmoscopic, haematological, clinical
    biochemical, urinary, and pathological parameters. Complete
    histological examinations were made of animals in the two control
    groups and at the high dose and of those animals at the low and
    intermediate doses which died during the study. Histological
    examination of animals at these doses killed at the end of the study
    was limited to the liver, kidney, lung, thyroid, testis, epididymides,
    ovary, and any observed abnormalities.

         No clinical signs related to treatment were observed.
    Sialodacryoadenitis was diagnosed in a large percentage of treated
    animals in weeks 25-28 and weeks 63-67. Body weights were lower in
    rats of each sex at 500 mg/kg bw per day than in controls throughout
    the study. At week 104, the mean body weight was 20-30% lower than the
    control value. A trivial reduction in food consumption was observed in
    animals of each sex at the highest dose. Ophthalmoscopic examination
    in week 99 revealed no changes attributable to treatment, and
    haematological tests and urinalysis revealed no adverse effects.
    Female rats receiving 500 mg/kg bw per day had higher cholesterol
    levels than controls throughout the study and increased blood urea
    nitrogen at 98 weeks. Other statistically significant differences in
    biochemical parameters were of no biological relevance. At the end of
    the study, the mortality rates were 82, 78, 87, 82, and 78% in males
    and 55, 68, 63, 43, and 50% in females at 0 (two groups), 30, 100, and
    500 mg/kg bw per day, respectively.

         Increased liver weights were seen in animals of each sex at 100
    and 500 mg/kg bw per day, corresponding to a higher incidence of
    macroscopic enlargement associated with hyperplasia and hypertrophy of
    centrilobular hepatocytes and enlarged eosinophilic cells, which
    occasionally contained a brownish cytoplasmic pigment. Increased
    kidney weights were observed in female rats at the two highest doses,
    corresponding histologically to a higher incidence of chronic
    interstitial glomerulonephritis. Other histological changes were
    confined to the endocrine organs: Enlarged thyroid glands were
    observed in animals of each sex at 500 mg/kg per day, corresponding
    histologically to a higher incidence of generalized and focal
    hyperplasia of follicles and increased pigment deposition in the
    colloid. A slightly higher incidence of adrenal and ovarian
    enlargement seen among females receiving 500 mg/kg per day was not
    associated with histological changes. In males, there was a negative
    trend in the presence of enlarged, focal, coarsely vacuolated cortical

    cells in the adrenals. The combined incidence of bilateral and
    unilateral testicular atrophy was comparable between groups, but there
    were significant differences from controls in the incidences of
    bilateral testicular atrophy at all doses, with 17, 33, 47, and 43% at
    0, 30, 100, and 500 mg/kg bw per day, respectively. After an
    additional, full review of the relevant data, this finding was
    considered unlikely to be related to treatment because the atrophy was
    not associated with degeneration of seminiferous tubules or
    aspermatogenesis, and testicular weights were not decreased.
    Morphometric analysis of the pituitary gland area showed reduced size
    in males at the highest dose. A trend analysis of tumour incidence
    with dose showed both increases (in the lymphoid system and thyroid)
    and decreases (in mammary glands and pituitary). These differences
    were not statistically significant in pairwise comparisons between
    groups, and the incidences were within the range of those of
    historical controls of this strain of rats. These observations provide
    no evidence that the treatment had carcinogenic potential, but the
    wide range of gross alterations, effects on organ weight, and
    histological changes observed reflect the biological activity of the
    test compound. The enlargement of the liver, the primary target organ,
    is consistent with the activity of the compound as a hepatic enzyme
    inducer. The wide differences in the incidences of morphological
    changes and lesions in endocrine and hormone-sensitive organs between
    controls and treated groups strongly support the interpretation that
    they are the results of changes in hormonal levels brought about by
    hepatic enzyme induction. The NOAEL was 30 mg/kg bw per day on the
    basis of effects on the liver (Graham, 1987).

         Groups of 50 Fischer 344/Du Crj rats of each sex were fed diets
    containing 0, 5000, or 10 000 ppm piperonyl butoxide (purity, about
    89%) for two years at doses selected on the basis of the results of
    the 13-week study by the same authors, summarized above. Animals were
    observed daily for clinical signs and mortality; food consumption was
    measured monthly, but the frequency of body-weight measurement was not
    stated. Treatment was stopped after 104 weeks, and surviving animals
    were sacrificed after six more weeks on the basal diet. All animals,
    including those sacrificed when moribund or found dead, were
    necropsied and a complete histological examination was performed. The
    mortality rates were (males/females) 16/14, 38/22, and 42/34% in
    controls and animals at 5000 and 10 000 ppm; the increased mortality
    was statistically significant for animals at the high dose. Just
    before death, many animals showed signs of anaemia (not specified) and
    had blood in their faeces. A dose-related reduction in body weight was
    seen in animals of each sex (statistical analysis not reported), with
    no concomitant decrease in food intake. No statistically significant
    difference in the incidence of malignant or benign neoplasms was
    found, except a decreased incidence of thyroid C-cell adenomas in
    males at the high dose (9%; 44% in controls). Dose-related increases
    were seen in the incidences of ulcers (0/0%, 35/2%, and 52/45% in male

    and female controls and those at the low and high doses,
    respectively), regenerative hyperplasia (0/0%, 13/0%, and 22/4%),
    ossification in the ileocaecal mucosa (0/0%, 15/0%, and 20/0%), and
    haemorrhage of the caecum and colon (0/0%, 10/0%, and 17/12%). The
    main histological lesions were chronic ulcers with inflammatory-cell
    infiltration and granulation, sometimes with ossification of the
    surrounding mucosa. Most of deaths in treated animals before the end
    of the study were attributed by the authors to lesions leading to
    anaemia. Piperonyl butoxide was not found to be carcinogenic
    (Maekawa  et al., 1985).

         Groups of 50 Fischer 344 rats of each sex were fed diets
    containing 5000 or 10 000 ppm piperonyl butoxide (purity, 88.4%) for
    24 months; the control group consisted of 20 animals. The diets were
    prepared freshly each week and stored at 7C. Animals were observed
    twice daily for mortality and underwent a complete clinical
    examination monthly. The food consumption of 10 animals in each group
    was measured during the first week of each month; body weights were
    measured about twice monthly. Moribund animals were sacrificed and
    necropsied and their organs were examined histologically. All
    surviving animals were sacrificed and submitted to a complete
    histopathological examination. All control females and about 80% of
    treated females survived until termination, whereas no difference in
    survival was found for males (70-75% survival in all groups). A
    dose-related reduction in body weight was found for both females (16%
    at the low dose and 24% at the high dose) and males (7% at the low and
    16% at the high dose). No difference was found in the incidence of
    malignant or benign neoplasms, except for that of lymphoreticular
    malignant lymphoma which was significantly increased in treated
    females (14% at the low and 30% at the high dose; 5% in controls) and
    nonsignificantly decreased in males (30% at the low and 26% at the
    high dose; 35% in controls). The author concluded that the results
    were equivocal because of the variability in the historical control
    data and the difficult diagnosis of these neoplasms (Cardy  et al.,
    1979; US National Cancer Institute, 1979).

         Groups of 30-33 Fischer 344/DuCrj rats of each sex were fed diets
    containing 0, 6000, 12 000, or 24 000 ppm piperonyl butoxide (two
    lots: purity, 94.5 and 94.3%; no detectable safrole or isosafrole) for
    95 (males) or 96 (females) weeks. The study was ended before 104 weeks
    because of the high mortality of males at 12 000 ppm (see below).
    Animals were observed daily for clinical signs and mortality; body
    weights were measured monthly. Food consumption was determined in a
    preliminary experiment involving six rats of each sex per group
    (duration not reported), which resulted in calculated compound intakes
    of 537, 1061, and 2002 mg/kg per day by females and 547, 1052, and
    1877 mg/kg per day by males at 6000, 12 000 and 24 000 ppm,
    respectively. Rats that died were necropsied and observed for tumors
    and non-neoplastic lesions, which were then examined microscopically.

    At termination, surviving rats were necropsied; hepatic nodules (when
    multiple nodules were present, three major ones were taken) and major
    organs were weighed and examined histologically. Blood was collected
    for haematological tests (including prothrombin and partial
    thromboplastic times) and for standard clinical chemistry. The
    mortality rates were 17, 23, 50, and 24% in males and 20, 10, 17, and
    21% in females at 0, 6000, 12 000, and 24 000 ppm, respectively. Males
    at 12 000 ppm began to die as early as week 40 and at a statistically
    significantly different rate from other groups from week 45. Deaths
    were most frequently associated with haemorrhage in the caecum. Body
    weights were reduced in a dose-related manner in all treated animals
    but statistically significantly only in animals at the middle and high
    doses. Males and females at the high dose lost weight from week 60,
    and at termination their body weights were about 50% of those of
    controls. The weights of all organs except the liver were reduced in
    animals at the high dose. The absolute liver weights were increased in
    animals at 12 000 ppm (in males by 22% and in females by 51%) and in
    females at 6000 ppm (by 29%). During the first month of treatment,
    rough hair, lethargy, epistaxis, and a fall in food consumption (data
    not reported) were observed in animals of each sex at 24 000 ppm.
    Haematological tests showed a significant increase in hypochromic,
    microcytic anaemia, the severity of which was dose-related, in all
    treated animals. The most significant findings in plasma were
    decreased cholinesterase activity and triglyceride contents in animals
    at all doses and an increased urea nitrogen level in animals at the
    high dose and in all treated females. Hepatic tumours were observed
    from week 74 of treatment. Nodular lesions of the liver were observed
    only in treated animals, and their incidence, numbers per rat, and
    size were dose-related. Hepatocellular adenomas and carcinomas were
    found in animals at the middle and high doses: the incidences of
    adenomas were 27% in males and 13% in females at 12 000 ppm and 15% in
    males and 30% in females at 24 000 ppm; the incidences of carcinoma
    were 13% in males and 0% in females at 12 000 ppm and 73% in males and
    46% in females at 24 000 ppm. The nodules in animals at the low dose
    were classified as foci or hepatocellular focal hyperplasia. The only
    other difference between groups was found for interstitial-cell
    tumours of the testis, with incidences of 23/25 in controls, 19/23 at
    6000 ppm, 13/15 at 12 000 ppm, and 15/25 at 24 000 ppm. Essential
    (haemorrhagic) thrombocytaemia (defined as > 3  106 platelets per
    microlitre, with a bimodal platelet distribution curve) was found in
    0/24 male controls and in 6/23 males at 6000 ppm, 3/15 at 12 000 ppm,
    and 9/24 at 24 000 ppm and in only 1/25 females at the high dose. Of
    the non-neoplastic lesions, the following are significant: an
    increased incidence of stomach haemorrhage (males: 9/33; 5/30 in
    controls; females: 19/33; 2/30 in controls) at the high dose, an
    increased incidence of haemorrhage and/or oedema of the caecum in all
    treated males and in females at the middle dose, an increased

    incidence of black kidneys in males at the middle and high doses and
    all treated females, and an increased incidence of whitish spotting in
    the lungs of males at the middle and high doses. Tubular dilatation,
    distension of Bowman's space and interstitial fibrosis were found in
    the kidneys of animals at the high dose (actual numbers not reported)
    (Takahashi  et al., 1994b).

    (d)  Reproductive toxicity

    Mice

         In a two-generation study, with one litter per generation, groups
    of 10 male and 10 female Crj:CD-1 mice were fed diets containing 0,
    1000, 2000, 4000, or 8000 ppm piperonyl butoxide (purity unspecified).
    Animals of the F0 generation, five weeks old at the start of the
    study, were mated for five days at nine weeks of age. Animals of the
    F1 generation was removed from their dams at four weeks of age and
    allocated randomly to continue treatment; the F2 generation was
    produced similarly to the F1 generation. Individual food intake was
    measured before mating and during mating, gestation, and lactation for
    all generations; litter size and weight and sex ratio were measured at
    birth, and pups were weighed 0, 4, 7, 14, and 21 days after birth.
    Some neurobehavioural tests were performed during lactation of the
    F2 generation and the results analysed on a litter basis. The tests
    included surface righting and negative geotaxis on postnatal days 4
    and 7, cliff avoidance on postnatal day 7, swimming behaviour on
    postnatal days 4 and 14, and olfactory orientation on postnatal day
    14. Food consumption was reduced by 4-44% in F0 animals at 8000 ppm
    except during mating, by 47% in F1 animals at 8000 ppm during
    lactation, and by 16-22% in F0 and F1 animals at 4000 ppm during
    lactation. The mean litter size of F1 animals at 8000 ppm was not
    significantly reduced (7.7; 10.6 in controls), but the mean F1
    litter weight was reduced by 38% (statistically significant) at
    8000 ppm and by 18% (not statistically significant) at 4000 ppm. F1
    pups at 8000 ppm had a lower survival index at postnatal day 21 (63%;
    91% in controls for males, statistically significant; 79%; 89% in
    controls for females, not statistically significant). The weights of
    all F1 pups were reduced, but there was no dose-response
    relationship at 1000-4000 ppm. The results of neurobehavioural tests
    for treated F1 animals were different from those of controls in some
    instances, but no dose-related response was evident. The mean F2
    litter size was significantly reduced at 4000 ppm (10.3; 13.1 in
    controls) and 8000 ppm (7.0), and the mean F2 litter weights were
    reduced in all treated groups by 13-57%. F2 pups at 8000 ppm had a
    lower survival index than controls at postnatal day 21 (59% in males,
    statistically significant; 79% in females, not statistically

    significant), and F2 pup weights were reduced at 2000, 4000, and
    8000 ppm. At 2000 and 4000 ppm, the effect was evident only from
    postnatal day 4; at 1000 ppm, reductions in pup weights were observed
    on postnatal day 4 in males and postnatal day 7 in animals of each
    sex. There was no NOAEL because of effects on pup weights at all doses
    (Tanaka  et al., 1992).

         Groups of 10 male and 10 female Crj:CD-1 mice were fed diets
    containing 0,1500, 3000, or 6000 ppm piperonyl butoxide (purity
    unspecified) for four weeks before mating (F0 generation), during
    gestation, and until the F1 generation was eight weeks old.
    Open-field activity was measured in the F0 generation after three
    weeks of treatment. Litter size, pup weight, and some developmental
    behavioural signs (surface righting, negative geotaxis, cliff
    avoidance, swimming, and olfactory orientation) were measured during
    lactation of the F1 generation; open-field activity was measured at
    three and eight weeks of age and multiple water T-maze activity at six
    weeks of age. A dose-related decrease in ambulation and rearing was
    seen in the open-field test in males of the F0 generation, but only
    the former was statistically significant at the high dose. Pup body
    weight at birth was reduced in all groups; on postnatal day 21, the
    body weight of animals at the middle dose was 7% lower than that of
    controls, and that of animals at the high dose was 41% lower. The
    survival indexes at postnatal day 21 were 79, 93, 80, and 52 in
    controls and in mice at the low, middle, and high doses, respectively.
    The results of the behavioural test during lactation were not
    significant, except for reduced olfactory orientation in animals at
    the middle and high doses. The results of the open-field test and the
    multiple water T-maze test were not altered by treatment, except for a
    few, not dose-related differences in some parameters (Tanaka, 1992).

    Rats

         In a two-litter, two-generation study of reproductive toxicity,
    groups of 26 Sprague-Dawley CD rats of each sex, seven weeks of age,
    received piperonyl butoxide in the diet at 0, 300, 1000, or 5000 ppm,
    equal to 0, 20, 68, and 350 mg/kg bw per day for males (average food
    intake calculated in weeks 1-28 of the study) and to 0, 29, 94, and
    480 mg/kg bw per day for females (calculated in weeks 1-12 of the
    study). The stability, homogeneity, and correspondence of the actual
    concentrations in the diets to the nominal concentrations were checked
    several times before and during the study and found to be acceptable.
    Rats were maintained on their respective diets for 85 days before
    mating, throughout the two mating periods, and until scheduled
    sacrifice. The F1b generation litters were weaned on day 21
     post partum, and groups of 26 rats of each sex were selected to form
    the F1b adult generation. These animals were maintained on their
    respective diets for 83 days before mating. Animals were observed for
    signs of toxicity, and body weight and food consumption were recorded.
    External and internal gross examinations were performed on adult rats

    and on a selected number of weanlings; histopathological examination
    was undertaken for the reproductive tracts of control animals, those
    at the high dose, and those of rats at the low and middle doses that
    failed to mate successfully in either mating period.

         Clinical and pathological examinations showed no
    treatment-related toxic effects in adult F0 or F1b animals. The
    body weights of adult animals of each sex at 5000 ppm were lower than
    those of controls from a few weeks after the beginning to the end of
    the study. This effect occasionally corresponded to reduced food
    intake. Mating performance, fertility indexes, gestation indexes,
    length of gestation, and numbers of live and dead pups at birth were
    not affected by treatment. The viability and lactation indexes,
    clinical conditions, and pathological appearance of pups of all
    generations were also unaffected. The body weights of male and female
    pups of all generations at 5000 ppm were lower than those of control
    pups. This effect was not detectable at birth but was seen as early as
    day 4  post partum. The NOAEL for parental toxicity and pup development
    was 1000 ppm, equal to 68 mg/kg bw per day (Robinson  et al., 1986).

         During the study of carcinogenicity reviewed above (Sarles &
    Vandergrift, 1952), rats of each sex were pair-caged throughout the
    experiment, except during nursing. No effect on reproductive
    efficiency was seen in the first, second, or third generations (no
    details given).

    (e)  Developmental toxicity

    Mice

         Groups of 20 pregnant Crj:CD-1 mice were given piperonyl butoxide
    (purity, > 95%) in olive oil by gavage on day 9 of gestation at doses
    of 0, 1065, 1385, or 1800 mg/kg bw. Dams were weighed on gestation
    days 9 and 18 and then sacrificed, and the uteri were examined for the
    presence and position of resorption sites, fetuses, and implantation
    sites. Viable fetuses were weighed and examined for external
    malformations and variations, and then fixed and stained for
    visualization of the skeleton. No abnormal behaviour or mortality was
    observed in dams. One dam at the middle dose and two at the high dose
    aborted; the litters of one dam at the middle dose and three at the
    high dose were resorbed. Maternal body-weight gain was comparable in
    all groups. The total resorption rates were significantly greater at
    the middle (26%) and high (32%) doses than in controls (6%), probably
    due to total litter resorptions, since the number of viable fetuses
    per dam was comparable in all groups. (The raw data were not available
    to confirm the reviewer's hypothesis.) The average fetal body weight
    was significantly reduced in males, by 3% at the middle dose and 7% at
    the high dose, and in females, by 4% at the low dose, 5% at the middle
    dose, and 6% at the high dose. Exencephaly, craniochisis, open

    eyelids, omphalocele, kinky tail, and talipes varus were observed in
    all groups. Oligodactyly in the forelimbs was found in none of the
    controls but in one fetus at the low dose, four (2%) at the middle
    dose, and 27 (6%) at the high dose. A single oral dose > 1065 mg/kg
    bw per day of piperonyl butoxide to dams on day 9 of gestation was
    thus embryo- and fetotoxic (Tanaka  et al., 1994).

    Rats

         Groups of 20 pregnant COBS random-bred albino rats were given 0,
    300, or 1000 mg/kg bw per day piperonyl butoxide (technical-grade;
    purity unspecified) dissolved in corn oil by gavage on days 6-15 of
    gestation. The doses were chosen after a pilot study in which six
    animals per group were treated with 0, 100, 300, 1000, or 3000 mg/kg
    bw per day; reduced body-weight gain was observed during gestation
    days 6-15 at the highest dose. Pregnant animals were shipped from the
    breeder to the test laboratory on day 1 of gestation and were observed
    and weighed daily. On day 20 of gestation, they were sacrificed and
    the usual parameters were determined. No signs of toxicity were
    observed. Body-weight gain was reduced by 10% in animals at the low
    dose and by 15% in those at the high dose, mainly between days 15 and
    20 of gestation. Reproductive parameters were not significantly
    affected by treatment. One female at 300 mg/kg bw resorbed 8 of 11
    fetuses, and one at the high dose resorbed the entire litter. Fetuses
    of treated dams had no internal, external, or skeletal malformations
    that could be related to treatment. There was no NOAEL for maternal
    toxicity because of reduced body-weight gain at both doses. The NOAEL
    for embryo- and fetotoxicity was 1000 mg/kg bw per day on the basis of
    the absence of any significant finding (Kennedy  et al., 1977). This
    report from Industrial Biotest Laboratories has not been validated by
    a governmental agency and was therefore not taken into account by the
    Meeting.

         Groups of 17-20 pregnant Wistar rats were given piperonyl
    butoxide (purity, 80%) at 0, 62.5, 125, 250, or 500 mg/kg bw dissolved
    in corn oil by gavage on days 6-15 of gestation. They were sacrificed
    on day 22 of gestation and necropsied, and their fetuses were removed
    and examined for external, visceral, and skeletal changes. There were
    no deaths or signs of toxicity in dams and no effect on the numbers of
    corpora lutea, live fetuses, resorption sites plus dead fetuses,
    anomalous fetuses, anomalous litters, or fetal body weight. The types
    and incidences of anomalies in the treated groups were comparable to
    those of the control group (data not shown). There was no evidence of
    embryotoxicity, fetotoxicity, or teratogenicity, and no maternal
    toxicity was elicited at the doses used (Khera  et al., 1979).

         Groups of 20 timed-pregnant Sprague-Dawley CD rats were given
    undiluted piperonyl butoxide (purity, 90.78%) at 0, 200, 500, or
    1000 mg/kg bw per day by gavage on days 6-15 of gestation. The doses
    were chosen in a preliminary pilot study in which five animals per
    group were treated with 0, 250, 500, 1000, 2000, or 4000 mg/kg bw per
    day, and in which all animals at 4000 and four-fifths of those at
    2000 mg/kg bw per day groups died or became moribund. Animals were
    observed and weighed daily. On day 20 of gestation, they were
    sacrificed and the usual parameters plus liver weight were determined.
    No females aborted or delivered early, but wetness in the urogenital
    area was observed in 13/24 dams at the highest dose, and stains of
    urine, red urogenital discharge, and perinasal encrustation were seen
    at lower incidences. Body-weight gain was reduced between days 6 and 9
    of gestation in animals at the low dose (an effect considered to be
    nonsignificant because of the low weight of non-pregnant animals) and
    between days 6 and 15 in animals at the middle and high doses; these
    reductions were associated with reduced food intake, resulting in a
    significantly lower body weight (by about 5%) in animals at 500 and
    1000 mg/kg bw per day on days 15 and 18 of gestation. Increased
    absolute (by 8%) and relative (by 11%) weights were observed at the
    high dose. Reproductive parameters were not significantly affected by
    treatment. Fetuses of treated dams had no internal, external, or
    skeletal malformations that could be related to treatment. The NOAEL
    for maternal toxicity was 200 mg/kg bw per day on the basis of reduced
    body-weight gain and food consumption, increased liver weight, and
    signs of toxicity at higher doses. The NOAEL for embryo- and
    fetotoxicity was 1000 mg/kg bw per day on the basis of the absence of
    any significant finding (Chun & Neeper-Bradley, 1991).

    Rabbits

         In a range-finding study, five inseminated New Zealand white
    rabbits were treated by gavage with piperonyl butoxide during
    gestation days 7-19 at doses of 0, 50,100, 200, 300, or 400 mg/kg bw
    per day in corn oil. All animals except one control survived until the
    end of the study. Two animals each at 300 and 400 mg/kg bw per day and
    one at 100 mg/kg bw per day group aborted all or part of their litters
    between gestation days 22 and 26. Decreased defaecation was observed
    at the highest dose, and animals at the two highest doses were thinner
    than normal at the end of the treatment period. Significant reductions
    in body-weight gain and some body-weight loss were observed in rabbits
    at 300 and 400 mg/kg bw per day; a trivial reduction in body-weight
    gain was observed in animals at 200 mg/kg bw per day. No consistent
    effects were seen on uterine parameters. Doses of 50, 100, and
    200 mg/kg bw per day were therefore chosen for the definitive study of
    teratogenicity.

         Groups of 16 inseminated New Zealand white rabbits were treated
    by gavage with piperonyl butoxide (purity, 100%) during gestation days
    7-19 at doses of 0, 50, 100, or 200 mg/kg bw per day in 0.5 ml/kg bw
    corn oil. Caesarean sections were performed on gestation day 29, and
    the fetuses were removed for teratological evaluation. All animals
    survived until the end of the study. Decreased defaecation was
    observed in animals at 100 and 200 mg/kg bw per day. Slight
    body-weight loss was observed at the middle and high doses during the
    treatment period, but there was substantial recovery of body weight
    during the post-treatment period, so that the weight gain during the
    overall gestation period was comparable to that of controls. The mean
    post-implantation losses of treated does were slightly greater than
    those of controls but were not dose-related. Malformations were
    observed incidentally and were considered to be unrelated to
    treatment. The numbers of fetuses with more full ribs than normal (45,
    58, 59, and 60% at 0, 50, 100, and 200 mg/kg bw, respectively) or with
    27 presacral vertebrae (20, 32, 27, and 40%, respectively) were
    greater than those in controls, but the number of litters was not
    different. There was no clear dose-effect relationship, and the
    relevance of this finding is dubious. The NOAEL for maternal toxicity
    was 50 mg/kg bw per day on the basis of decreased defaecation and
    dose-related body-weight losses during the treatment period. The NOAEL
    for teratogenicity was 200 mg/kg bw per day (Leng  et al., 1986). The
    1992 JMPR wrongly reported an NOAEL of 100 mg/kg bw per day.

    (f)  Genotoxicity

         The results of studies of the genotoxicity of piperonyl butoxide
    are reported in Table 2.

    (g)  Special studies

    (i)  Dermal and ocular irritation and dermal sensitization

         Piperonyl butoxide was applied at 0.5 ml to intact, clipped skin
    of six New Zealand white rabbits for 4 h under a semi-occlusive
    dressing, and the treated areas were examined for erythema and oedema
    up to 78 h after the contact period. An adjacent area of untreated
    skin served as the control. Piperonyl butoxide was very mildly
    irritating (Romanelli, 1991a). An older study reported similar results
    (Sarles  et al., 1949).

         Piperonyl butoxide at 0.1 ml was instilled into the conjuntival
    sac of one eye of six New Zealand white rabbits, and the treated eyes
    were examined up 72 h after instillation. Conjunctival irritation was
    observed, which fully recovered within 72 h. No corneal or iridal
    lesions were observed (Romanelli, 1991b). An older study reported
    similar results in rabbits, cats, and dogs (Sarles  et al., 1949).

        Table 2.  Results of tests for the genotoxicity of piperonyl butoxide
                                                                                                                                              

    End-point            Test system                      Concentration            Purity          Results          Reference
                                                          or dose                  (%)
                                                                                                                                              

    In vitro

    Reverse mutation     S. typhimurium TA98, TA100,      100-5000 g/plate        90.78           Negativea        Lawlor (1991)
                         TA1535, TA1537, TA1538
    Reverse mutation     S. typhimurium TA98,             NR                       NR              Negative         White et al. (1977)
                         TA 100, TA1537
    Reverse mutation     E. coli WP2                      1 mg/disc                80              Negative         Ashwood-Smith
                                                                                                                    et al. (1972)
    Gene mutation        Mouse lymphoma L5178Y            6.3-100 g/mlb           NR              Positive         McGregor et al.
                         cells                                                                                      (1988)
    Gene mutation        Chinese hamster ovary, cells     10-100 g/mlc            NR              Equivocal        Tu et al. (1985)
                                                          25-500 g/mld                            Negative
    Unscheduled DNA      Rat hepatocytes                  1-100 g/mle             90.78           Negative         McKeon & Phil
    synthesis                                                                                                       (1991)
    Unscheduled DNA      Human liver slices               0.05-2.5 mmol/litre      90.78           Negative         Lake (1995)
    synthesis
    Chromosomal          Chinese hamster ovary cells      25-99.9 g/ml            90.78           Negativea        Murli (1991)
    aberration                                            62.6-251 g/mla
                                                                                                                                              

    Table 2.  (cont'd).
                                                                                                                                              

    End-point            Test system                      Concentration            Purity          Results          Reference
                                                          or dose                  (%)
                                                                                                                                              

    In vivo

    Dominant lethal      ICR/Ha Swiss mice                0, 200, or 1000 mg       Commercial      Equivocal        Epstein et al. (1972)
    mutation                                              intraperitoneally or     formulation
                                                          1000 mg/kg bw orally
                                                           5
                                                                                                                                              

    NR, not reported
    a With and without metabolic activation
    b Lethal at 100 g/ml
    c Without metabolic activation; cytotoxic at > 30 g/ml
    d With metabolic activation; cytotoxic at > 300 g/ml
    e Cytotoxic at > 49.9 g/ml
             Skin sensitization was studied in 10 male Hartley guinea-pigs by
    a modified Buehler method. A gauze patch containing 0.4 ml piperonyl
    butoxide was applied for 6 h onto clipped skin, three times a week for
    three weeks. After a two-week rest period, the animals were challenged
    on another site with a single 6-h application. Chlorodinitrobenzene
    (0.1% w/v in a 50% ethanol:0.9% saline solution) was used as the
    positive control. No evidence of contact sensitization was observed
    (Romanelli, 1991c). An older study also reported no response in
    rabbits (Sarles  et al., 1949)

    (ii)  Studies with mixtures

         Neonatal ICR/Ha Swiss mice (n = 91) were given subcutaneous
    injections of 5 mg piperonyl butoxide alone at the ages of one and
    seven days and 10 mg at the ages of 14 and 21 days. Further groups
    were given the same doses in combination with Freon-112 (n = 137) or
    -113 (n = 94) at doses of 10 mg at one and seven days and 20 mg at 14
    and 21 days. Control animals were treated with the solvent,
    tricaprylin. All surviving animals were killed after 52 weeks. No
    tumours were found in animals treated with piperonyl butoxide, but the
    incidence of hepatomas in male mice given piperonyl butoxide plus
    Freon was significantly increased when compared with that in groups
    receiving solvent, piperonyl butoxide, or Freon alone (Epstein
     et al., 1967).

         Groups of 45 Sprague-Dawley CD rats of each sex were fed either
    standard diets or diets containing 400 ppm pyrethrins plus 2000 ppm
    piperonyl butoxide for 104 weeks. The diets were prepared daily from a
    10-fold concentrated pre-mix prepared freshly once a week. The purity
    of piperonyl butoxide was 90.5%, and that of pyrethrins was 53.1%
    (batch used until week 55) or 52.4% (batch used from week 56 until
    termination); the diets were prepared taking into account the purity
    of the compounds. The actual concentrations and the stability of the
    diet were not tested. Body weight and food consumption were determined
    once a week. The average daily intakes were 79 and 101 mg/kg bw
    piperonyl butoxide and 16 and 20 mg/kg bw pyrethrins for males and
    females, respectively. Animals were observed for clinical signs, skin
    lesions, and palpable masses. All animals found dead or moribund were
    inspected, and macroscopic lesions were examined microscopically.
    During week 101, urinalysis and haematological and clinical chemical
    tests were performed in 10 animals per group. At termination, all
    animals were observed grossly and selected organs were examined
    microscopically.

         No treatment-related clinical signs of toxicity were observed.
    Mortality was 84 and 76% in control males and females and 80 and 58%
    in treated males and females, respectively. The body weights of
    treated females were about 20% lower than those of controls, mainly

    during the first 78 weeks, while at termination treated animals were
    about 10% lighter than controls; the difference was less evident
    (about 5%) in males. Slightly reduced food intake was also observed
    until week 27 of treatment. Haematological, clinical chemical, and
    urinary parameters were not significantly altered in treated animals,
    and macroscopic and microscopic examinations revealed no significant
    treatment-related neoplastic or non-neoplastic effects (Hunter
     et al., 1977).

    3.  Observations in humans

         In nine men, antipyrine metabolism was not affected by a single
    oral dose of 50 mg (0.71 mg/kg bw) piperonyl butoxide (Conney  et al.,
    1972).

         Two male infants, whose mothers were sisters and who were born
    within two weeks of each other, had coarctation of the aorta. Exposure
    to insect repellents and insecticides, including piperonyl butoxide,
    during week 8 of gestation was reported (Hall  et al., 1975).

         Percutaneous absorption of pyrethrin and piperonyl butoxide was
    studied in six male volunteers. A formulation containing 0.3% pyrethin
    and 3.0% piperonyl butoxide was spread on the ventral forearm for 30
    min. This formulation was chosen because it corresponds to that of a
    commercially available product used for the treatment of head lice.
    The formulation contained either 14C-pyrethrin or 14C-piperonyl
    butoxide. After application, urine was collected for up to seven days,
    and percutaneous absorption was determined from total urinary
    excretion of radioabel, assuming that 22.5% of pyrethin and 51.3% of
    piperonyl butoxide are excreted in the urine after parenteral
    injection, as was shown in monkeys. Absorption of pyrethrin was 1.9 
    1.2% and that of piperonyl butoxide 2.1  0.6% of the administered
    dose. No radiolabel was found in blood taken 1 h after application.
    The extrapolated absorption from human scalp was 7.5  4.7% of
    pyrethin and 8.3  2.4% of piperonyl butoxide (Wester  et al., 1994).

    Comments

         Piperonyl butoxide is metabolized by oxidation of the methylene
    group of the methylenedioxyphenyl moiety to yield carbon dioxide. The
    remainder of the molecule undergoes further degradation and is
    excreted mainly in the urine. As it is an alternative substrate (and
    therefore a competitive inhibitor) for the microsomal cytochrome P450
    system, piperonyl butoxide inhibits the metabolism of several drugs
    and pesticides. The mechanism of action of this inhibition has been
    elucidated in several studies.

         In male rats given single oral doses of about 500 mg/kg bw of
    [methylene-alpha 14C]-piperonyl butoxide, the label reached a peak
    in blood after 3-12 h and dropped by about 50% within 24 h. The
    highest levels of radiolabel were found in the gastrointestinal tract
    and its, contents, suggesting that enterohepatic circulation occurs.
    High levels of radioactivity were also found in the lung, liver,
    kidney, fat, prostate, and seminal vesicles. The excretion pattern was
    unchanged after 14 repeated doses of piperonyl butoxide.

         Piperonyl butoxide has negligible acute toxicity, and it has been
    classified by WHO as unlikely to present an acute hazard in normal
    use.

         Both short-term and long-term studies show that the target organ
    of the toxicity of piperonyl butoxide is the liver. Male animals were
    slightly more sensitive than females. In a number of short-term
    studies in rodents, hepatic toxicity was characterized by liver
    enlargement with associated hypertrophic hepatocytes, focal necrosis,
    and, at times, alteration of some clinical chemical parameters. The
    NOAEL for liver toxicity was about 100 mg/kg bw per day.

         In rats exposed to piperonyl butoxide by inhalation at 0, 15, 74,
    155, or 512 mg/m3 for 6 h per day on five days a week for 13 weeks,
    effects were seen on the liver at the highest dose. Irritation of the
    upper airways was seen at all doses, with squamous metaplasia of the
    larynx.

         In a one-year study in dogs fed diets containing 0, 100, 600, or
    2000 ppm piperonyl butoxide, reduced body-weight gain, increased liver
    weight with hypertrophic hepatocytes, and alteration of some clinical
    chemical parameters were observed at 2000 ppm. The NOAEL in this study
    was 600 ppm, equal to 16 mg/kg bw per day.

         Several studies have been conducted of carcinogenicity in mice
    and rats, some of which were considered to be inadequate. In a
    112-week study, mice were given diets containing 5000 or 10 000 ppm
    piperonyl butoxide during weeks 1-30 and 500 or 2000 ppm during weeks
    31-112. No increase in tumour incidence was observed. Mice were fed

    diets that gave a daily intake of 0, 30, 100, or 300 mg/kg bw for 78
    weeks. Eosinophilic foci and adenomas with eosinophilic cells were
    observed more frequently in the livers of males at the middle dose and
    males and females at the high dose. The NOAEL in this study was
    30 mg/kg bw per day on the basis of effects on the liver.

         In a 12-month study of carcinogenicity in the liver, male mice
    were fed diets containing 0, 6000, or 12 000 ppm piperonyl butoxide.
    Body weights were reduced in a dose-related manner, and increased
    mortality was observed in animals at the high dose. Hepatocellular
    adenomas and carcinomas were observed in treated groups. Piperonyl
    butoxide was carcinogenic at doses that were toxic to the liver and
    caused general toxicity.

         In a two-year study in rats at dietary concentrations adjusted to
    achieve doses of 0, 30, 100, or 500 mg/kg bw per day, increased liver
    weights, with corresponding hyperplasia and hypertrophy of
    hepatocytes, morphological changes, and lesions in the endocrine and
    hormone-sensitive organs, were observed at 100 and 500 mg/kg bw per
    day. These effects were considered to be secondary to the ability of
    piperonyl butoxide to induce hepatic cytochrome P450 enzymes.
    Piperonyl butoxide was not found to be carcinogenic in this study.
    After reconsideration of the data on testes in this study, previously
    evaluated by the 1992 JMPR, and taking into account the results of
    other long-term studies, the Meeting concluded that the NOAEL was
    30 mg/kg bw per day on the basis of effects on the liver.

         Two studies of carcinogenicity were conducted in rats fed diets
    containing 0, 5000, or 10 000 ppm piperonyl butoxide. No significantly
    increased incidence of neoplasia was found in treated rats. Effects on
    body weight and mortality were observed at the highest dose. In one
    study, ileocaecal lesions were observed in both groups. Another study
    of carcinogenicity was conducted in rats fed diets containing 0, 6000,
    12 000, or 24 000 ppm. Increased mortality was observed in females at
    the middle dose. Absolute liver weights were increased in females at
    the low dose and in animals of each sex at the high dose. Nodular
    lesions of the liver were observed in treated animals, and their
    incidence and severity were related to the dose. Hepatocellular
    adenomas and carcinomas were observed in animals at the middle and
    high doses. Gastric and caecal haemorrhages, renal lesions, anaemia,
    and platelet alteration were observed in treated animals. Piperonyl
    butoxide was carcinogenic at doses that caused general toxicity.

         In a 104-week study in rats, piperonyl butoxide was given at
    2000 ppm in the diet in combination with pyrethrins at 400 ppm. A
    slight reduction in the body weights of treated females was the only
    adverse effect observed.

         A two-generation study of reproductive toxicity, with one litter
    per generation, was conducted in mice fed diets containing 0, 1000,
    2000, 4000, or 8000 ppm piperonyl butoxide. There was no NOAEL because
    of reduced pup weight at all doses. Pup viability was reduced at the
    highest dose. In a two-litter, two-generation study of reproductive
    toxicity in rats at dietary levels of 0, 300, 1000, or 5000 ppm, the
    NOAEL for parental toxicity and pup development was 1000 ppm (equal to
    68 mg/kg bw per day) on the basis of lowered body weight at 5000 ppm.
    Embryo- and fetotoxicity were observed when mice were given single
    doses of piperonyl butoxide at 0, 1070, 1390, or 1800 mg/kg bw by
    gavage on day 9 of gestation.

         Piperonyl butoxide was not embryotoxic or teratogenic in rats or
    rabbits. Maternal toxicity was found in rats at doses > 500 mg/kg
    bw per day. The NOAEL was 200 mg/kg bw per day in a study of
    developmental toxicity in rabbits given 0, 50, 100, or 200 mg/kg bw
    per day by gavage. The incidence of common developmental variations,
    such as more full ribs and more than 27 presacral vertebrae, was
    increased in all treated groups. Since a clear dose-effect
    relationship was lacking, the association of this finding with
    treatment was considered dubious. The NOAEL for maternal toxicity was
    50 mg/kg bw per day.

         Piperonyl butoxide was a mild dermal and ocular irritant but not
    a dermal sensitizer in rabbits.

         Piperonyl butoxide was adequately tested for genotoxicity in a
    range of assays  in vivo and  in vitro. The Meeting concluded that
    it is not genotoxic.

         A single dose of 0.71 mg/kg bw piperonyl butoxide did not alter
    antipyrine metabolism in humans. A study in which a formulation
    containing 3% piperonyl butoxide was spread onto the ventral forearm
    of adult male volunteers indicated that about 8% of the applied dose
    would be absorbed through the human scalp. These data did not
    contribute directly to the establishment of the ADI.

         An ADI of 0-0.2 mg/kg bw was established on the basis of the
    NOAEL of 600 ppm (equal to 16 mg/kg bw per day) in the one-year study
    in dogs, with a 100-fold safety factor.

    Toxicological evaluation

     Levels that cause no toxic effect

    Mouse:    30 mg/kg bw per day (78-week dietary study of toxicity and
              carcinogenicity)

    Rat:      30 mg/kg bw per day (two-year dietary study of
              carcinogenicity)
              200 mg/kg bw per day (maternal toxicity in study of
              developmental toxicity)
              500 mg/kg bw per day (embryo- and fetotoxicity in study of
              developmental toxicity)
              1000 ppm, equal to 68 mg/kg bw per day (study of
              reproductive toxicity)

    Rabbit:   50 mg/kg bw per day (study of developmental toxicity)
              200 mg/kg bw per day (embryo- and fetotoxicity and
              teratogenicity in study of developmental toxicity)

    Dog:      600 ppm, equal to 16 mg/kg bw per day (one-year study of
              toxicity)

     Estimate of acceptable daily intake for humans

         0-0.2 mg/kg bw

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

    1.   Further observations in humans

    2.   Short-term studies with mixtures of piperonyl butoxide and
         other active ingredients, in ratios relevant to human
         exposure

        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to piperonyl butoxide
                                                                                                                                              

    Exposure                    Relevant route, study type, species              Results, remarks
                                                                                                                                              

    Short-term (1-7 days)       Skin, irritation, rabbit                         Mildly irritating
                                Eye, irritation, rabbit                          Irritating
                                Skin, sensitization, guinea-pig                  Not sensitizing
                                Inhalation, lethality, rat                       Lacrimation, salivation, nasal
                                                                                 discharge, and laboured breathing at
                                                                                 5.9 mg/litre for 4 h. No mortality
                                Oral, lethality, mouse, rat, cat, dog            LD50 = 4-14 g/kg bw
                                Dermal, lethality, rabbit                        LD50 > 2 g/kg bw

    Medium-term (1-26 weeks)    Repeated oral, toxicity, mice, rats,             NOAEL = 100 mg/kg bw per day; effects on
                                3-13 weeks                                       liver
                                Repeated inhalation, toxicity, rats, 13 weeks    Irritating to upper airways at 15 mg/m3 for
                                                                                 6 h per day, 5 days per week, with squamous
                                                                                 metaplasia of the larynx; effects on the liver at
                                                                                 512 mg/m3
                                Oral, developmental toxicity, rabbit             NOAEL = 50 mg/kg bw per day for maternal
                                                                                 toxicity; no fetotoxicity or teratogenidty
                                Oral, reproductive toxicity, rat                 NOAEL = 68 mg/kg bw per day; maternal
                                                                                 and pup toxicity

    Long-term (> one year)      Repeated oral, toxicity, dog, one year           NOAEL = 16 mg/kg bw per day
                                                                                                                                              
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    See Also:
       Toxicological Abbreviations
       Piperonyl butoxide (ICSC)
       Piperonyl Butoxide (FAO Meeting Report PL/1965/10/1)
       Piperonyl butoxide (FAO/PL:CP/15)
       Piperonyl butoxide (FAO/PL:1967/M/11/1)
       Piperonyl Butoxide (FAO/PL:1969/M/17/1)
       Piperonyl butoxide (WHO Pesticide Residues Series 2)
       Piperonyl butoxide (Pesticide residues in food: 1992 evaluations Part II Toxicology)
       Piperonyl Butoxide (IARC Summary & Evaluation, Volume 30, 1983)