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    PIPERONYL BUTOXIDE

    First draft prepared by S. Caroldi
    Istituto di Medicina del Lavoro
    Padova, Italy

    EXPLANATION

         Piperonyl butoxide was previously evaluated by JMPR in 1965,
    1966, and 1972 (Annex 1, references 3, 6, and 18). An ADI of 0-0.03
    mg/kg bw was allocated in 1972 based on a short-term study in dogs.
    In 1972 JMPR stated that a reproduction study in a second species
    and studies on the effects of piperonyl butoxide on the liver of
    dogs as requested by the 1966 Meeting were not available. Pharmaco-
    kinetic studies in rats, a long-term study in rats, a reproduction
    study in rats and a teratogenicity study in rabbits have been
    submitted for evaluation at the present Meeting.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution, and excretion

    Rats

         The following studies were performed on young male Charles
    River CD rats using 14C]piperonyl butoxide (labelled on the alpha
    carbon of the 2-(2-butoxyethoxy) ethoxymethyl side chain).

         Four rats were administered a single dose of approximately 500
    mg [14C]piperonyl butoxide/kg bw (15.6 ▒ 0.4 ÁCi) by gavage.
    Animals were individually housed in Roth metabolism cages for the
    collection of volatile labelled materials. The rats expired an
    average of 0.18% of the dosed radioactivity as 14CO2 during the
    24 h following dosing.

         Four rats were administered a single dose of approximately 500
    mg/kg bw of [14C]piperonyl butoxide (14.4 ▒ 0.7 ÁCi) by gavage.
    Blood samples were collected from the tail vein for a 24-h period
    after dosing and samples were analyzed for radioactivity. Plasma
    radioactivity reached a peak between 3-12 h after dosing and dropped
    to about half the peak level within 24 h.

         Four rats were administered a single dose of approximately 500
    mg [14C]piperonyl butoxide/kg bw (14.1 ▒ 1.3 ÁCi) by gavage. Urine
    and faecal samples were collected and weighed at 4, 8, 12 and 24 h
    after dosing and then every 24 h for 7 days. Most radioactivity was
    recovered in urine and faeces between 12 and 24 h after dosing. At
    168 h after dosing the average percentage of dosed radioactivity
    recovered was approximately 38% and 62% in urine and faeces,
    respectively.

         Twenty rats were administered a single dose of approximately
    500 mg [14C]piperonyl butoxide/kg bw by gavage. Groups of 5 rats
    were killed at 1, 6, 24, 48 and 168 h after dosing and radioactivity
    was analyzed in several tissues. At each time interval, the highest
    levels of radioactivity was always found in the GIT and in its
    content. High levels of radioactivity 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 radioactivity was recovered from analyzed tissues.

         Five rats received approximately 500 mg piperonyl butoxide/kg
    bw/day for 13 days followed by a single dose of approximately 500 mg
    radiolabelled piperonyl butoxide/kg bw (9.6 ▒ 0.2 ÁCi) by gavage.
    Most radioactivity was recovered in urine and faeces between 12 and

    48 h after dosing. At 168 h after radioactive dosing, the average
    percentage of radioactivity recovered was approximately 43% and 54%
    in urine and faeces, respectively (Selim, 1985).

    Effects on enzymes and other biochemical parameters

         CF-1 male mice were treated i.p. with single doses of piperonyl
    butoxide (87-89% pure; range of doses 0.5-25 mg/kg bw) one hour
    prior to the i.p. administration of pentobarbital (40 mg/kg bw) or
    zoxazolamine (100 mg/kg bw). Pentobarbital-induced sleeping time was
    significantly increased by 10 or 25 mg/kg bw of piperonyl butoxide
    and zoxazolamine-induced paralysis time was significantly increased
    by 5, 10 or 25 mg/kg bw piperonyl butoxide.

         Sprague-Dawley male rats were treated i.p. with single doses of
    piperonyl butoxide (87-89% pure; range of doses 67-1000 mg/kg bw)
    one hour prior to the i.p. 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 of piperonyl
    butoxide and zoxazolamine-induced paralysis time was significantly
    increased by 333 or 1000 mg/kg bw piperonyl butoxide. 

         Both male mice and male rats were treated i.p. with single
    doses of piperonyl butoxide one hour prior to i.p. injection of 200
    mg/kg bw of antipyrine. The NOAEL of piperonyl butoxide on
    antipyrine metabolism was 100 mg/kg bw in rats and 0.5 mg/kg bw in
    mice.

         Weanling male Sherman rats (6 animals/group) were fed technical
    grade piperonyl butoxide (80% purity) at dietary concentrations of
    0, 1000, 5000 and 10 000 ppm for 1, 4 and 8 weeks. Activities of
    hexobarbital oxidase, aniline hydroxylase, p-nitroanisole-
    demethylase, nitroreductase, glucuronyltransferase and P-450 content
    were increased 2- to 4-fold by 5000 or 10 000 ppm piperonyl
    butoxide. Liver weight and microsomal protein were increased a
    maximum of 50-70%. Electron microscopy showed enlargement and
    extensive proliferation of the SER in liver parenchimal cells. A
    dose of 1000 ppm produced minimal effects on liver weight, P-450 and
    glucuronyltransferase activity, but no effects of this dose could be
    detected on proliferation of the SER. Maximum effect on P-450
    content and on activity of P-450 related enzymes was observed after
    one week of treatment with piperonyl butoxide. Maximum effect on
    glucuronyltransferase activity was observed between 4 and 8 weeks of
    treatment ( Goldstein et al., 1973).

         A study performed on nine men showed that antipyrine metabolism
    was not influenced by a single oral administration of 50 mg (0.71
    mg/kg bw) of piperonyl butoxide (Conney  et al., 1972).

    Toxicological studies

    Long-term toxicity/carcinogenicity studies

    Rats

         Sixty Sprague-Dawley Crl:CDR (SD)BR rats/sex were treated with
    piperonyl butoxide (89% purity grade) at dietary concentrations
    adjusted weekly (every two weeks after week 15) to achieve daily
    intakes of piperonyl butoxide equal to 0 (control rats, two groups),
    30, 100 or 500 mg/kg bw/day for 104/105 weeks. Stability and
    homogeneity of diet preparations and correspondence of actual
    concentrations of piperonyl butoxide in diets to nominal
    concentrations were checked before and throughout the duration of
    the study and found acceptable. Monitored parameters were:
    observations for viability/mortality, clinical signs, food
    consumption, body-weight, ophthalmoscopic examinations, haematology,
    clinical biochemistry, urinalysis and pathology. Complete
    histological examination was undertaken on animals in the two
    control groups and high-dose group and on those animals in the low-
    and intermediate-dose groups which died during the study.
    Histological examination was limited to liver, kidney, lung,
    thyroid, testis, epididymis, ovary and any observed abnormalities
    for animals in the low- and intermediate-dose groups killed at the
    end of the study.

         No clinical signs related to piperonyl butoxide administration
    were observed. Sialodacryoadenitis affecting a great percentage of
    animals in all groups was diagnosed in two occasions (between weeks
    25-28 and 63-67). Body-weight was lower in rats of both sexes at 500
    mg/kg bw/day than in controls throughout the duration of the study.
    At week 104 mean bodyweight was 20-30% lower than control values.
    Trivial reduction in food consumption was observed in both sexes at
    the highest dose level. Ophthalmoscopic examination performed on
    week 99 revealed no changes attributable to piperonyl butoxide
    treatment. Haematology and urinalysis did not reveal adverse effects
    related to the administration of the test substance. Female rats
    receiving 500 mg/kg bw/day of piperonyl butoxide showed increased
    cholesterol levels as compared to controls throughout the duration
    of the study. Moreover increased BUN was observed in this group at
    the 98 week sampling. Other statistical difference in biochemical
    parameters were of no biological relevance. At the end of the study,
    mortality rate was 82%, 78%, 87%, 82% and 78% (male), and 55%, 68%,
    63%, 43% and 50% (female), at 0 (two groups), 30, 100 and 500 mg/kg
    bw/day dose levels, respectively.

         Increased liver weight was present in both sexes at 100 and 500
    mg/kg bw/day dose levels which corresponded to a higher incidence of
    macroscopic enlargment, and histologically to hyperplasia and
    hypertrophy of centrilobular hepatocytes, and of enlarged
    eosinophilic cells which occasionally contained brownish cytoplasmic

    pigment. Increased kidney weights were observed in female rats at
    the two highest dose levels which corresponded histologically to a
    higher incidence of chronic interstitial gremerulonephritis. Other
    histological changes were confined to the endocrin organs.
    Enlargment of thyroid glands was observed in both sexes at 500 mg/kg
    bw/day which corresponded histologically to a higher incidence of
    generalized and focal hyperplasia of follicles and increased pigment
    deposit in the colloid. A slightly higher incidence of adrenal and
    ovarian enlargments among females receiving 500 mg/kg/day was not
    associated with histological changes. In male adrenals there was a
    negative trend in the presence of enlarged focal, coarsely
    vacuolated cortical cells. The combined incidence of bilateral and
    unilateral testicular atrophy was comparable between groups. There
    was a significant difference between controls and all dose levels in
    the incidence of bilateral testicular atrophy. Moreover there was a
    positive dose-related trend towards increased severity of lesions
    and in the incidence of interstitial cell hyperplasia. The incidence
    of bilateral testicular atrophy was 17%, 33%, 47% and 43% at 0, 30,
    100 and 500 mg piperonyl butoxide/kg bw/day, respectively. The
    morphometric analysis of pituitary gland area showed reduced values
    in the highest dose group in comparison with controls in males only.
    Trend analysis over dose levels showed both increases (lymphoid
    system, thyroid) as well as decreases (mammary glands, pituitary) in
    the number of tumours. Such differences were not statistically
    significant in intergroup pairwise comparisons and incidences were
    within the range of the inhouse historical controls for this strain
    of rats. Based on these observations there was no evidence of
    carcinogenic potential associated with piperonyl butoxide treatment
    under the conditions of the present study.

         The wide range of gross, organ weight and histological changes
    observed reflected the biological activity of the test compound. The
    enlargment of the liver, the primary target organ, is consistent
    with the biological activity of the compound as a hepatic enzyme
    inducer. The widespread differences in incidences of morpho-logical
    changes and lesions between controls and treatment groups in the
    endocrine and hormone sensitive organs strongly supported the
    interpretation that these were the results of changes in hormonal
    levels brought about by the hepatic enzyme induction. A NOAEL was
    not determined in this study owing to an increased incidence of
    bilateral testicular atrophy observed at all dose levels (Graham,
    1987).

    Reproduction studies

    Rats

         In a two-litter, two-generation reproduction study, groups of
    26 (F0) CD rats/sex (Sprague-Dawley) 7 weeks old received
    piperonyl butoxide incorporated into the diet at 0, 300, 1000 and
    5000 ppm equal to 0, 20, 68 and 350 mg/kg bw/day for males (average

    food intake calculated between weeks 1-28 of the study) and to 0,
    29, 94 and 480 mg/kg bw/day for females (calculated between weeks 1-
    12 of the study). Stability, homogeneity and correspondence of
    actual concentrations in the diets to nominal concentrations were
    checked several times before and during the study and found
    acceptable. Rats were mantained on their respective diets for 85
    days prior to 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/sex were selected to form the
    F1b adult generation. These animals were maintained on their
    respective diets for 83 days prior to mating. Animals were observed
    for signs of toxicity, and body-weight and food consumption were
    recorded. Pathology was mostly external and internal gross
    examination of adult rats and a selected number of weanlings.
    Histopathology was performed on reproductive tracts of animals of
    the control and high-dose groups and of rats in the low- and mid-
    dose groups failing to mate successfully in both mating periods.

         Clinical examination and pathology did not show treatment-
    related toxic effects in the F0 and F1b adult generations. Body-
    weights were lower in the 5000 ppm groups than in control groups for
    both sexes in both adult generations starting a few weeks from the
    beginning of the study throughout the duration of the study. This
    effect occasionally corresponded to reduced food intake. Mating
    performance, fertility index, gestation index, length of gestation,
    numbers of live and dead pups at birth were not affected by
    piperonyl butoxide administration. Both viability and lactation
    indices, clinical conditions and pathology of all generation pups
    were unaffected by the test substance. Body-weights of male and
    female pups of all generations at 5000 ppm were lower than those of
    control pups. This effect on body-weight was not detectable at birth
    but was seen as early as day 4 post-partum.

         The NOAEL for parental toxicity and pup developement was 1000
    ppm of piperonyl butoxide equal to 68 and 94 mg/kg bw/day in males
    and females, respectively, and for reproductive toxicity 5000 ppm of
    piperonyl butoxide, equal to 350 and 480 mg/kg bw/day in males and
    females, respectively (Robinson  et al., 1986).

    Special studies on embryotoxicity and teratogenicity

    Rabbits

         In a range-finding study, five inseminated New Zeeland 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/day (vehicle corn oil). All animals except one control survived
    until the end of the study. Two animals each in the 300 and 400
    mg/kg bw/day groups and one in the 100 mg/kg bw/day group aborted
    all or part of their litters between gestation days 22 and 26.
    Decreased defaecation was observed at the highest dose. Animals of

    the two highest dose groups appeared thinner than normal at the end
    of the treatment period. Significant reduction of body-weight gain
    and sometimes body-weight losses were observed in rabbits of 300 and
    400 mg/kg bw/day groups. Trivial reduction of body-weight gain was
    observed in animals of the 200 mg/kg bw/day dose. No consistent
    treatment effects were observed in the uterine examination
    parameters of all groups. Based on these results, doses of 50, 100
    or 200 mg/kg bw/day were chosen for a definitive teratology study in
    New Zeeland white rabbits with piperonyl butoxide.

         Sixteen inseminated New Zeeland white rabbits were treated by
    gavage with piperonyl butoxide (100% purity grade) during gestation
    days 7-19 at doses of 0, 50, 100 or 200 mg/kg bw/day (vehicle corn
    oil, 0.5 ml/kg). Caesarean sections were performed on gestation day
    29 and the fetuses were removed for teratologic evaluation. All
    animals survived until the end of the study. Decreased defecation
    was observed in animals at 100 and 200 mg/kg bw/day. Slight body-
    weight losses were observed at mid- and high-dose during the
    treatment period. There was a substantial recovery of body-weight
    during the post-treatment period so that the weight gain values
    during the overall gestation period were comparable to that of
    controls. Mean post-implantation losses for all treated groups were
    slightly increased as compared with controls without following a
    dose-related pattern. Malformations observed in treated groups
    occurred incidentally and were considered unrelated to treatment.
    The number of fetuses having a greater number of full ribs than
    normal and the number of fetuses having 27 presacral vertebrae were
    increased in all treated groups when compared with control. The
    former developmental variation was observed in 45%, 58%, 59% and 60%
    of fetuses and the latter variation in 20%, 32%, 27% and 40% of
    fetuses at 0, 50, 100 and 200 mg/kg bw, respectively. The number of
    litters in the treated groups with this observation was not
    different from the control. A clear dose-effect relationship was
    lacking and the relationship of this finding to treatment is
    dubious. Based on decreased defaecation and dose-related body-weight
    losses during the treatment period, the NOAEL for maternal toxicity
    was 50 mg piperonyl butoxide/kg bw/day. The NOAEL for teratogenicity
    was 100 mg/kg bw/day (Leng  et al., 1986)

    COMMENTS

         Pharmacokinetic studies performed on male rats with single oral
    doses of approximately 500 mg/kg bw of [14C]piperonyl butoxide
    showed that peak blood radioactivity was reached between 3 and 12 h
    after dosing and that peak values dropped to about 50% within 24 h.
    Most of the radioactivity was eliminated via urine and faeces
    between 12 and 24 h after dosing. At 168 h after dosing the
    proportion of radioactivity recovered was approximately 38% in urine
    and 62% in faeces. Tissue distribution showed that the highest
    levels of radioactivity were persistently in the gastrointestinal
    tract and its contents, which suggested that enterohepatic
    circulation occurs after piperonyl butoxide administration. High
    levels of radioactivity were also found in lungs, liver, kidneys,
    fat, prostate and seminal vesicles. The excretion pattern was
    unchanged after 14 repeated doses of piperonyl butoxide.

         A number of studies served to elucidate the mechanism of action
    as an inhibitor and inducer of MFOs in rodents.

         In a two-year study in rats at dietary concentrations adjusted
    to achieve doses of 0, 30, 100 or 500 mg/kg bw/day, a NOAEL was not
    determined owing to an increased incidence of bilateral atrophy of
    the testes in all dose groups. Other effects were increased liver
    weights with corresponding hyperplasia and hypertrophy of
    hepatocytes at 100 and 500 mg/kg bw/day and widespread morphological
    changes and lesions in the endocrine and hormone-sensitive organs.
    These effects were considered to be secondary to the ability of
    piperonyl butoxide to induce hepatic MFOs. Piperonyl butoxide was
    not carcinogenic in rats.

         In a two-litter, two-generation reproduction study in rats at
    dietary levels of 0, 300, 1000 or 5000 ppm, the NOAEL for
    reproductive toxicity was 5000 ppm equal to 350 mg/kg bw/day and 480
    mg/kg bw/day in males and females, respectively. The NOAEL for
    parental toxicity and pup development was 1000 ppm equal to 68 and
    94 mg/kg bw/day for males and females, respectively based on lower
    body-weight at 5000 ppm in comparison with controls.

         In a teratogenicity study in rabbits at 0, 50, 100 or 200 mg/kg
    bw of piperonyl butoxide given by gavage on days 7-19 of gestation,
    the incidence of common developmental variations such as a greater
    number of full ribs and more than 27 presacral vertebrae was
    increased in all dosed groups. A clear dose-effect relationship was
    lacking and the relationship of this finding to treatment was
    considered dubious. The NOAEL for maternal toxicity was 50 mg/kg
    bw/day and the NOAEL for embryofoetal toxicity was 100 mg/kg bw/day.

         Toxicological data reviewed by the present Meeting did not give
    rise to particular concern. The Meeting was informed that additional
    toxicological data on piperonyl butoxide existed, that more were

    being generated and that these data will be provided to the Joint
    Meeting for evaluation. Accordingly, the previously determined ADI
    of 0-0.03 mg/kg bw was maintained, pending future review.

         The Meeting recommended that piperonyl butoxide should be
    reviewed again in 1995 following submission of the following studies
    to WHO by 1994:

              1)   acute toxicity studies
              2)   teratology studies in rats
              3)   appropriate genotoxicity studies
              4)   ongoing one-year study in dogs
              5)   ongoing carcinogenicity study in mice.
              6)   carcinogenicity studies in rats and mice performed by
                   the US National Toxicology Program (1979)
              7)   observations in humans.

    REFERENCES

    Conney, A.H., Chang, R., Levin, W.M., Garbut, A., Munro-Faure, A.D.,
    Peck, A.W., & Bye, A. (1972) Effects of piperonyl butoxide on drug
    metabolism in rodents and man.  Arch. Environ. Health., 24: 7-106.

    Goldstein, J.A., Hickman, P., & Kimbrough R.D. (1973) Effects of
    purified and technical piperonyl butoxide on drug metabolizing
    enzymes and ultrastructure of rat liver.  Tox. Appl. Pharmacol.,
    26: 444-458.

    Graham, C. (1987) 24-Month dietary toxicity and carcinogenicity
    study of piperonyl butoxide in the albino rat. Unpublished report
    No. 81690 from Bio-Research Ltd. Laboratory, Seneville, Quebec,
    Canada. Submitted to WHO by Piperonyl Butoxide Task Force. 

    Leng, J.M., Schwartz, C.A., & Schardein, J.L. (1986) Teratology
    study in rabbits. Unpublished report No. 542-002 from IRDC,
    Mattawan, Michigan, USA. Submitted to WHO by Piperonyl Butoxide Task
    Force.

    Robinson, K., Pinsonneault, L., & Procter, B.G. (1986) A two-
    generation (two-litter) reproduction study of piperonyl butoxide
    administered in the diet to the rat. Unpublished report No. 81689
    from Bio-Research laboratories LTD.,Montreal, Canada. Submitted to
    WHO by Piperonyl Butoxide Task Force.

    Selim, S. (1985) Single and repeated oral dose pharmacokinetic and
    distribution studies of piperonyl butoxide. Unpublished report No.
    WP509070/1 from Biological Test Center, Irvine, CA 92714, USA.
    Submitted to WHO by Piperonyl Butoxide Task Force, Minneapolis USA.


    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: 1995 evaluations Part II Toxicological & Environmental)
       Piperonyl Butoxide (IARC Summary & Evaluation, Volume 30, 1983)