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    PIPERONYL BUTOXIDE                              JMPR 1972

    Explanation

    Piperonyl butoxide was evaluated by the 1966 Joint Meeting (FAO/WHO,
    1967) and also considered in 1965 (FAO/WHO, 1965), 1967 (FAO/WHO,
    1968) and together with pyrethrins in 1969 (FAO/WHO, 1970). New data
    relating to the evaluation of the acceptable daily intake and further
    information regarding methods of analysis are now available.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

    In an experiment in which 87.6% of a large dose of piperonyl butoxide
    given to a dog was recovered (chiefly from the faeces), only 0.09% was
    found in the urine (Sarles and Vandergrift, 1952).

    Biotransformation

    Studies on the metabolism of piperonyl butoxide in rat indicate that
    breakdown is rapid, although clearance from the body is relatively
    slow. Fishbein et al., (1969) recorded a considerable number of
    metabolites in bile and urine following its i.v. administration to
    rats. However, no metabolites were identified. The presence of
    piperonyl butoxide in bile or urine was not observed, although it was
    observed unchanged in lungs and fat following i.v. dosing. Casida et
    al. (1966) administered piperonyl butoxide to rats and mice and
    observed an oxidative reaction of the methylene dioxy carbon to
    formate and CO2.

    Breakdown by photolytic mechanisms is extremely slow (Fishbein and
    Gaibel, 1970), and exposure to sunlight and normal lighting conditions
    does not degrade piperonyl butoxide. Under extreme conditions of
    intense light, small (<3%) quantities of unknown products were
    produced.

    Effect on enzymes and other biochemical parameters

    Chamberlain (1950) explored the hypothesis that, in insects, piperonyl
    butoxide synergizes pyrethrins by inhibiting lipase (esterase), but
    his results were inconclusive.

    In vitro experiments using purified erythrocyte acetyl
    cholinesterase showed that malathion had decreased anticholinesterase
    activity in the presence of piperonyl butoxide (Rai and Roan, 1956).

    Piperonyl butoxide at dose levels of 0.1 - 1.0 ml per rat given by the
    oral, intraperitoneal or intravenous routes retarded the elimination
    of intravenously administered 3,4-benzpyrene. Detoxification and
    biliary excretion of this carcinogen were also decreased. It was
    suggested that the induced hepatic damage may have increased the
    retention of the carcinogen (Falk et al., 1965).

    However, piperonyl butoxide inhibits microsomal oxidation of a wide
    variety of compounds which are detoxified by hydroxylation reactions.
    This effect can explain the ability of piperonyl butoxide to prolong
    the action of barbiturates and zoxazolamine, slow the metabolism of
    benzpyrene and enhance the toxicity of pyrethrins. In addition,
    piperonyl butoxide has been shown to induce glucuronyl transferase
    following prolonged exposure (Lucier et al., 1971). Treatment of
    mice by intraperitoneal injection resulted in a biphasic action on
    microsomal enzyme activities (Skrinjaric-Spoljar et al., 1971;
    Mathews et al., 1970; Kamienski and Murphy, 1971); activity returned
    to levels that were higher than normal after 24 to 72 hours. These
    in vitro studies were substantiated by in vivo tests on
    hexabarbital sleeping time. In addition to affecting microsomal
    enzymes, oral administration of piperonyl butoxide at 1 gm/kg to rats
    resulted in an increased level of neutral lipid in blood, several
    other tissues and organs. No fat deposition was noted in liver,
    kidneys, thymus or testis, but an increased level was observed in
    blood, heart, spleen, pancreas, lungs and adipose tissue (Albro and
    Fishbein, 1970).

    TOXICOLOGICAL STUDIES

    Results of studies on acute toxicity of piperonyl butoxide in
    different animal species are summarized in Table 1.

    High oral doses produce haemorrhage into the intestinal tract with
    loss of appetite and prostration (Sarles et al., 1949). It may be
    that these are the effects of local irritation and that the
    hyperexcitability and convulsions produced by large dermal doses
    (Lehman, 1952a) are more indicative of the action of the absorbed
    drug. The compound produces liver injury (Sarles et al., 1949;
    Sarles and Vandergrift, 1952), and at least in dogs, and in rats at
    high dosage levels, liver injury was recognized as the cause of death
    (Sarles and Vandergrift, 1952).

    In rats, large subcutaneous doses produce an increased bleeding
    tendency and "rusty" (bloody) urine (Sarles et al., 1949). Massive
    bleeding was found in some animals at autopsy (Sarles and Vandergrift,
    1952).

    Simultaneous administration of piperonyl butoxide potentiates the
    toxicity of coumaphos and its phosphate by a factor of four to six.
    There is some evidence that piperonyl butoxide interferes with
    detoxification of the organo-phosphorus insecticides (Robbins
    et al., 1959). No additional injury was produced in rats when
    one-sixth the weight of pyrethrin was added to a diet containing
    piperonyl butoxide at a concentration of 1 000 ppm (Sarles and
    Vandergrift, 1952).

    Short-term studies

    Mouse

    Four groups of Swiss mice were given subcutaneous injections of
    tricaprylin solutions containing one of the following compounds:
    trichloromonofluoromethane, tetrachlorodifluoroethane and
    trichlorotrifluoroethane (10% concentration) and piperonyl butoxide
    (5% concentration). Two groups of mice were given the following
    combinations by subcutaneous injection: tetrachlorodifluoroethane
    (10%) plus piperonyl butoxide (5%) and trichlorotrifluoroethane (10%)
    plus piperonyl butoxide (5%). The mice received the injections at the
    ages of 1, 7, 14 and 21 days. In those groups which received piperonyl
    butoxide, either alone or in combination, the total dose of piperonyl
    butoxide was about 5-10 g/kg body-weight. After 50-52 weeks the
    incidence of hepatomas in the groups which received the individual
    compounds was 5 of 126 (about 4%), and the total incidence in the two
    groups which received piperonyl butoxide in combination with a
    "Freon(R)" was 8 of 33 (about 24%). No influence on the incidence of
    malignant lymphomas was seen (Epstein et al., 1967).

    TABLE 1  Acute toxicity of piperonyl butoxide in animals

                                                                             
                             LD50
    Animal         Route     (mg/kg body-weight)      References
                                                                             

    Mouse          oral      4 030                    Negherbon, 1959

    Rat            oral      7 960 -10 600            Sarles et al., 1949

    Rat            oral      13 500                   Lehman, 1948

    Rat            oral      11 500                   Ibid., 1951

    Rat            s.c.      >15 900                  Sarles et al., 1949

    Rabbit         oral      2 650 - 5 300            Ibid.

    Cat            oral      >10 600                  Ibid.

    Dog            oral      >7 950                   Ibid.
                                                                             

    Rat

    In a 17-week study, a dietary level of 5 000 ppm piperonyl butoxide
    caused liver enlargement and periportal hepatic cell hypertrophy with
    slight fatty change and renal tubular pigmentation of a "wear and
    tear" type (Lehman, 1952b,c).

    Single weekly doses of between 530 and 4 240 mg/kg body-weight
    administered six times to rats caused no effects which were evident at
    autopsy three weeks after the final dose (Sarles et al., 1949).

    A 31-day test in rats showed terminal anorexia. Early deaths were
    largely due to damage of ganglionic cells of the brain stem (Sarles
    and Vandergrift, 1952).

    Rabbit

    Single weekly doses of between 1 060 and 4 240 mg/kg body-weight three
    times to rabbits caused no effects which were evident at autopsy 3
    weeks after the final dose (Sarles et al., 1949).

    Dog

    Body-weight gain was reduced compared with controls in dogs dosed with
    32 mg/kg/day for one year; dogs dosed with 106 mg/kg/day or higher
    lost weight. At 3 mg/kg/ day there was a slight increase in liver
    weight without gross or microscopic pathology. The kidneys and
    adrenals were progressively enlarged at dosages of about 100 mg/kg/day
    and above. Microscopic pathology was evident in the liver at dosage
    rates of 32 mg/kg/day and over. Hepatoma and carcinoma were not seen
    (Sarles and Vandergrift, 1952).

    Monkey

    At comparable dosage, symptomatology was somewhat less than in dogs.
    Microscopical pathology of the liver in monkeys at 100 mg/kg/day was
    comparable to that in dogs receiving 30 mg/kg/day, a dosage level that
    produced no observed effect in the monkey. The apparent difference in
    the sensitivity of the two species may be due to the shorter exposure
    of the monkey (1 month) compared with the dogs (1 year) (Sarles and
    Vandergrift, 1952).

    Long-term studies

    Mouse

    Groups of mice (each group consisted of 18 males and 18 females) were
    treated with piperonyl butoxide orally by gavage for 28 days at 100
    mg/kg. One group was not treated further and another was fed 300 ppm
    piperonyl butoxide in the diet until 18 months of age. In a similar
    experiment a group of mice were treated with Butacide(R) (piperonyl

    butoxide (80%) in solvent vehicle) at 464 mg/kg for 28 days and 1 112
    ppm in the diet thereafter (Innes et al., 1969). The authors
    indicated that the piperonyl butoxide treatment required additional
    evaluation, whereas the Butacide(R) treatment did not cause a
    significant increase in tumours after oral administration.

    Rat

    In two-year studies, concentrations of piperonyl butoxide as high as
    1 000 ppm caused no decrease in the growth rate of female rats;
    concentrations as low as 100 ppm produced some reduction in the growth
    rate of males, but the difference was not considered significant. A
    concentration of 10 000 ppm caused a significant reduction in the
    growth rate of both sexes that was accounted for, at least in part, by
    decreased food consumption (78% of control). A concentration of 25 000
    ppm reduced food consumption to 37% of control. However, in subacute
    experiments, anorexia was terminal and therefore not the simple effect
    of unpalatability of the food. A concentration of 10 000 ppm caused a
    distinct increase in mortality rate in both sexes evident at two years
    and a concentration of 25 000 killed about half the animals in half a
    year. Concentrations of 10 000 ppm or higher produced a significant
    increase in the relative weight of the liver and kidney. Histological
    changes in the liver were found at levels of 10 000 ppm and more. Less
    marked changes occurred in the kidney and adrenal. Benign or malignant
    tumours occurred in 30% of the test animals but the authors claimed
    that their occurrence was not related to piperonyl butoxide.
    Reproduction was decreased by a dietary level of 10 000 ppm and
    stopped by a concentration of 25 000 ppm (Sarles and Vandergrift,
    1952).

    OBSERVATIONS IN MAN

    Piperonyl butoxide was acutely administered orally at a dose of 50 mg
    to nine human male volunteers in a double-blind experiment. No effects
    were noted clinically and the metabolism of antipyrine was not
    affected (Brown, 1970). Mean dose was calculated to be 0.71 mg/kg.

    COMMENT

    Additional information requested at the 1966 Joint Meeting has been
    supplied in part. Piperonyl butoxide has been used for over 20 years
    as an insecticide synergist. Long-term studies in rats showed no
    toxicological effect at 100 ppm in the diet. A short-term study in
    dogs showed no toxicological effect at 3 mg/kg/day. Recent
    carcinogenic studies in mice showed no increase in tumours at a level
    of 890 ppm. Administration of extremely high doses of piperonyl
    butoxide together with Freon(R) propellant administered parenterally
    to neonatal mice resulted in an increase in hepatomas. This study was
    considered to be of limited value in assessing the ADI. Reproduction
    studies in a second species and studies on the effects of piperonyl
    butoxide on the liver of dogs as requested by the 1966 Meeting are not
    available. Acute studies in man showed no effects of piperonyl
    butoxide at a level of 0.71 mg/kg.

    Additional data submitted since 1966 now allow the establishment of an
    ADI.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Rat:      100 ppm in the diet, equivalent to 5 mg/kg
                   body-weight/day.

         Dog:      3 mg/kg body-weight/day.

    ESTIMATE FOR ACCEPTABLE DAILY INTAKE FOR MAN

         0 - 0.03 mg/kg body-weight

    METHODS OF RESIDUE ANALYSIS

    (a)  Colorimetric methods

    Secreast and Cail (1971) described a chromatographic-colorimetric
    method for determining low residues of piperonyl butoxide in flour.
    The pentane extract was cleaned up using a Florisil column eluted with
    ethyl acetate/pentane and the final colorimetric determination was
    based on the method of Jones et al. (1952). Satisfactory recoveries
    (95-105%) were obtained, and the sensitivity of the procedure was 0.2
    ppm or 20 g in flour. High-fat content commodities, such as shelled
    nuts, required an initial acetonitrile/pentane cleanup.

    (b)  Thin-layer chromatography

    Gunner (1969) developed a general method for methylenedioxy compounds.
    Separation was achieved on 0.25 mm layers of Adsorbil 1, with ethyl
    acetate:benzene (3:20), benzene:hexane (1:1) or benzene:methanol
    (1:10) as mobile phases. An acidic solution of sodium chromatropate,
    followed by heating, was used for visualization. The resulting purple
    spots were scanned with a densitometer, and residues in the
    microgramme range could be determined.

    (c)  Gas-liquid chromatography

    Moore (1972) proposed a method of analysis of fatty materials using
    the modified electron capture detector of Bruce (1967). The sample was
    extracted with a mixture of ethyl alcohol, ether and hexane. This
    extract was cleaned up by saponification, elution through a silica gel
    column and TLC before the GLC determination using a special design of
    electron capture detector. The minimal detectable quantity was 50 -100
    pg of piperonyl butoxide.

    RESIDUES IN DRIED CODFISH

    No evidence was submitted regarding residues of piperonyl butoxide in
    dried fish, but information regarding usage in Africa was presented.
    Corresponding to the tolerance for residues of pyrethrins of 3 ppm in
    dried fish, the tolerance for piperonyl butoxide would need to be
    increased to 20 ppm. Further data is required on residues in dried
    fish from supervised trials and commercial usage.

    APPRAISAL

    It is considered that suitable methods are now available for
    adaptation for the regulatory determination of residues of piperonyl
    butoxide at the suggested tolerance levels. No further evidence of
    residue levels was presented, but the implications of the data on
    pyrethrin residues on dried fish indicated a need to recommend a
    tolerance for piperonyl butoxide on dried fish of 20 ppm in place of
    the existing temporary tolerance of 1 ppm on dried codfish.

    RECOMMENDATIONS

    TOLERANCE

         Fish (dried)        20 ppm

    FURTHER WORK OR INFORMATION

    REQUIRED (before 30 June 1975)

    Further data on residues in dried fish from supervised trials and from
    commercial usage.

    DESIRABLE

    1.   Studies on the effect of piperonyl butoxide on the liver of dogs.
         (For details see Report of Scientific Group on Procedures for
         Investigating Intentional and Unintentional Food Additives - July
         1966, WHO TRS 348).

    2.   The effect of this compound on reproduction in at least one more
         species.

    REFERENCES

    Albro, P.W. and Fishbein, L. (1970) Short-term effects of piperonyl
    butoxide on the deposition of dietary hydrocarbons in rat tissues.
    Life Sciences, 9(II): 729-739.

    Brown, N.C. (1970) A review of the toxicology of piperonyl butoxide.
    Report Cooper Technical Bureau. (unpublished)

    Bruce, W.N. (1967) Detector cell for measuring picogram quantities of
    organophosphorus insecticides, pyrethrin synergists and other
    compounds by gas chromatography. J. Agr. Fd. Chem., 15: 178-181.

    Casida, J.E., Engel, J.L., Essac, E.G., Kamienski, F.X. and Kuwatsuka,
    S. (1966) Methylene-14C-dioxy-phenyl compounds: metabolism in
    relation to their synergistic action. Science, 153: 1130.

    Chamberlain, R.W. (1950) Am. J. Hyg., 52: 153.

    Epstein, S.S., Joshi, S., Andrea, J., Clapp, P., Falk, H. and Mantel,
    N. (1967) Synergistic toxicity and carcinogenicity of "Freons" and
    piperonyl butoxide. Nature, 214: 526-528.

    Falk, H.L., Thompson, S.J. and Kotin, P. (1965) Arch. Environ. Health,
    10: 847.

    FAO/WHO (1965) Evaluation of the toxicity of pesticide residues in
    food. FAO/PL/1965/10/1; WHO/Food Add./27.65.

    FAO/WHO (1967) Evaluation of some pesticide residues in food.
    FAO/PL:CP/15; WHO/Food Add./67.32.

    FAO/WHO (1968) 1967 evaluations of some pesticide residues in food.
    FAO/PL/1967/M/11/1; WHO/Food Add./68.30.

    FAO/WHO (1970) 1969 evaluation of some pesticide residues in food.
    FAO/PL:1969/M/17/1; WHO/Food Add./70.38.

    Fishbein, L., Falk, H.L., Fawkes, J., Jorden, S. and Corkett, B.
    (1969) The metabolism of piperonyl butoxide in the rat with 14C in
    the methylenedroxy or a-methylene group. J. Chromat., 41: 61-79.

    Fishbein, L. and Gaibel, Z.L.F. (1970) Photolysis of pesticides
    synergists. I. piperonyl butoxide. Bull Environ. Contam. Toxicol., 5:
    546-552.

    Gunner, S.W. (1969) The quantitative determination of methylenedioxy
    compounds by thin-layer chromatography - direct densitometry. J.
    Chromat., 40: 85-89.

    Innes, J.R.M., Ulland, B.M., Valerio, M.G., Petrucelli, L., Fishbein,
    L., Hart, E.R., Pallotta, A.J., Bates, R.R., Falk, H.L., Gart, J.J.,
    Klein, M., Mitchell, I. and Peters, J. (1969) Bioassay of pesticides
    and industrial chemicals for tumourigenicity in mice: a preliminary
    note. J. Nat. Cancer Inst., 42: 1101-1114.

    Jones, H.A., Ackermann, H.J. and Webster, M.E. (1952) Determination of
    piperonyl butoxide colorimetrically. J. Ass. off. analyt. Chem., 35:
    771-780.

    Kamienski, F. and Murphy, S.D. (1971) Biphasic effects of
    methylendroxyphenyl synergists on the action of hexabarbital and
    organophosphates in mice. J. Toxic. appl. Pharmac., 18: 883.

    Lehman, A.J. (1948) Quart. Bull. Assoc. Food and Drug Officials, U.S.,
    12: 82.

    Lehman, A.J. (1951) Quart. Bull. Assoc. Food and Drug Officials, U.S.,
    15: 122.

    Lehman, A.J. (1952a) Quart. Bull. Assoc. Food and Drug Officials,
    U.S., 16: 3.

    Lehman, A.J. (1952b) Quart. Bull Assoc. Food and Drug Officials, U.S.,
    16: 47.

    Lehman, A.J. (1952c) Quart. Bull. Assoc. Food and Drug Officials,
    U.S., 16: 126.

    Lucier, G.W., McDaniel, O.S. and Mathews, H.B. (1971) Microsomal rat
    liver UDP glucuronyltransferase: Effects of piperonyl butoxide and
    other factors on enzyme activity. Arch. Biochem. Biophys., 145:
    520-530.

    Mathews, H.B., Skrinjaric-Spoljar, M. and Casida, J.E. (1970)
    Insecticide synergist interactions with cytochrome P-450 in mouse
    liver microsomes. Life Sciences, 9(I): 1039-1048.

    Moore, J.B. (1972) Paper submitted to the International symposium on
    recent advances in research with pyrethrum, the natural insecticide.
    Minneapolis, U.S.A., 30-31 August.

    Negherbon, W.O. (1959) Handbook of Toxicology, vol.3, Saunders,
    Philadelphia.

    Rai, L. and Roan, C.C. (1956) Effects of piperonyl butoxide on the
    anticholinesterase activities of some organic phosphorous insecticides
    on house fly and purified bovine erythrocyte cholinesterases. J. Econ.
    Entomol., 49: 591-595.

    Robbins, W.E., Hopkins, T.L. and Darrow, D.I. (1959) Synergistic
    action of piperonyl butoxide with Bayer 21/199 and its corresponding
    phosphate in mice. J. Econ. Entomol., 52: 660-663.

    Sarles, M.P., Dove, W.E. and Moore, D.H. (1949) Amer. J. Trop. Med.,
    29: 151.

    Sarles, M.P. and Vandergrift, W.B. (1952) Amer. J. Trop. Med., 1: 862.

    Secreast, M.F. and Cail, R.S. (1971) A chromatographic-colorimetric
    method for determining low residues of piperonyl butoxide in flour. J.
    Agr. Fd. Chem., 19: 192-193.

    Skrinjaric-Spoljar, M., Mathews, H.B., Engel, J.L. and Casida, J.E.
    (1971) Response of hepatic microsomal mixed-function oxidases to
    various types of insecticide chemical synergists administered to mice.
    Biochem. Pharmacol., 20: 1607-1618.
    


    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 (Pesticide residues in food: 1992 evaluations Part II Toxicology)
       Piperonyl butoxide (Pesticide residues in food: 1995 evaluations Part II Toxicological & Environmental)
       Piperonyl Butoxide (IARC Summary & Evaluation, Volume 30, 1983)