PIPERONYL BUTOXIDE JMPR 1972
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
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).
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
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
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,
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
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
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.
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).
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).
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).
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
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.
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,
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.
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
Level causing no toxicological effect
Rat: 100 ppm in the diet, equivalent to 5 mg/kg
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 µg 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.
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.
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
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
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