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 µCi) 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 µCi) 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 µCi) 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 µCi) 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)