The Safety Assessment of Piperonyl Butoxide

Chapter 99

The Safety Assessment of Piperonyl Butoxide Thomas G. Osimitz Science Strategies, LLC

99.1  Chemistry and formulations Piperonyl butoxide (PBO), 2-(2-butoxyethoxy)ethyl 6-propyl-piperonyl ether (IUPAC), is an insecticide synergist produced from the condensation of the sodium salt of 2-(2-butoxyethoxy) ethanol and the chloromethyl derivative of hydrogenated safrole (dihydrosafrole) (Figure 99.1). The dihydrosafrole moiety containing the methylenedioxyphenyl ring constitutes over half of the PBO molecule by weight and is traditionally derived from sassafras oil. Sassafras oil is an essential oil distilled from ­ several species of trees found in China, Laos, and Burma. However, the availability of sassafras oil is limited today due to environmental concerns related to the destructive harvesting of trees, as well as to the nature of safrole as a drug precursor. A novel manufacturing process for PBO where dihydrosafrole is derived no longer from Sassafras oil but from catechol was patented first in 1998 by Endura S.p.A (Endura, 2009). Current world production of PBO averages 94% pure. In the early days, PBO contained small but detectable amounts of safrole and dihydrosafrole (DHS). However, refinements in distillation have resulted in safrole and DHS levels usually below the 40 ppm detection limit by highresolution gas chromatography (Di Blasi, 1998). When the PBO precursor DHS is manufactured from catechol, PBO does not contain safrole as an impurity.

O O

H2 C

C H2

C H2 O

CH3

C H2

H2

H2

C

O

O

Figure 99.1  Chemical structure of PBO. Hayes’ Handbook of Pesticide Toxicology Copyright © 2010 Elsevier Inc. All rights reserved

H2 C H2

O

C H2

C

C H2

CH3

The development of PBO grew out of a need in the late 1930s and early 1940s to extend the usefulness of the naturally derived insecticide pyrethrum, which was considered a strategic insecticide against mosquitoes and other disease-carrying insects. The chemicals that were developed had little intrinsic pesticidal activity of their own; however, they did increase the effectiveness of a given dose of pyrethrins and were thus called synergists. PBO was one of a series of molecules synthesized (Wachs, 1947). PBO is usually formulated with natural pyrethrins or synthetic pyrethroids in ratios (PBO: pyrethrins) ranging from 3:1 to 20:1. Formulations of PBO and carbamates are also available, although their use is minor relative to that of PBO and pyrethrins/pyrethroids.

99.2  Uses As a synergist, PBO inhibits the mixed function oxidase (MFO) system of insects, thereby reducing the oxidative breakdown of other pesticides such as pyrethrum and the synthetic pyrethroids (Casida, 1970). The precise mechanism of inhibition is unknown, but speculation is that a carbene derivative forms and binds to the heme moiety of the cytochrome P450 enzyme, thereby rendering it inactive (Dahl and Brenzinski, 1985; Delaforge et al., 1985; Franklin, 1976; Hodgson et al., 1973; Murray and Reidy, 1989; Philpot and Hodgson, 1971, 1972a,b). More recently, PBO has also been found to inhibit resistance-associated esterases in many insects (Khot et al., 2008) and to inhibit other enzymes such as hydrolases (Kakko et al., 2000) and glutathione S-transferases (Varsano et al., 1992). The result is that higher levels of the insecticide remain in the insect and are thereby available to exercise their lethal effect on the insect. PBO enhances the pesticidal activity of 2127

Hayes’ Handbook of Pesticide Toxicology

2128

Table 99.1  Summary of Acute Toxicity Data and Classifications Route

Species

Result

EEC labeling classification

USEPA toxicity category

Reference

Oral LD50

Rat

4 g/kg (male) 7 g/kg (female)

Unclassified

Category IV

Gabriel (1991b)

Dermal LD50

Rabbit

2 g/kg

Unclassified

Category IV

Gabriel (1991a)

Inhalation LC50

Rat

5.9 mg/l air

Harmful

Category III

Hoffman (1991)

Eye irritation

Rabbit

Minimally irritating

Labeling not indicated

Category III

Romanelli (1991b)

Skin irritation

Rabbit

Minimally irritating

Labeling not indicated

Category IV

Romanelli (1991c)

Skin sensitization (Buehler)

Guinea pig

Negative

Labeling not indicated

Category IV

Romanelli (1991a)

a given level of active ingredient, thus promoting reduced use of the pesticide. Appearing in over 1500 U.S. Environmental Protection Agency (U.S. EPA)-registered products, PBO is one of the most commonly registered pesticides in terms of the number of formulas in which it is present. It is approved for preharvest application to a wide variety of crops including fruits and vegetables. The application rates are low; the highest single rate is 0.5 lbs PBO/acre. It is also used extensively in combination with pyrethrins, various synthetic pyrethroids, and other insecticides to control insect pests in and around the home and in food-handling establishments. A wide variety of water-based PBO-containing products such as crack and crevice sprays, total release foggers, and flying insect sprays are made for use by consumers in the home. Annual use of PBO in the United States is approximately 1.3–1.5 million lbs (PBO Task Force, 2009). About 47% of this total is used for indoor residential purposes, 17% for indoor food uses in warehouses and food handling establishments, and only 13% goes for agricultural crop applications. PBO has also been allowed as a food additive in Japan since 1955, its maximum approved level being 0.024 g/kg (24 ppm) in raw cereals.

99.3  Hazard identification 99.3.1  Acute Toxicity Numerous acute studies have been conducted over the years with PBO in a variety of species and by various exposure routes. This body of data, including the most recent studies, indicates that PBO is generally of low acute toxicity to animals. It is mildly irritating to the eye and skin. It is not a dermal sensitizer. Table 99.1 summarizes this acute toxicity data as well as European Economic Commission (EEC) labeling classifications and U.S. EPA toxicity categories.

99.3.2  Subchronic Toxicity PBO has been tested in dogs, mice, rats, rabbits, and African green monkeys for subchronic toxicity. A summary of the subchronic toxicity studies discussed below is presented in Table 99.2.

99.3.2.1  Dogs Lorber (1972) reported unexpected alterations in blood counts of intact and splenectomized dogs after use of a fogger (containing PBO among other chemicals). The fogger bathed the animals inadvertently in a “dense pesticide mist.” The dogs were part of a research project investigating the relationship of spleen, bone marrow, and blood cells. Further work was undertaken in which 17 intact, 12 splenectomized, and seven partially splenectomized dogs were purposefully exposed to deodorized kerosene containing only PBO (1.5%). Exposure periods consisted of four intervals of 5 min duration, with an 8-min interval between each exposure. The splenectomized dogs showed a reduction in serum platelet count and an occasional increase in reticulocytes. The authors concluded that the demonstrated greater resistance of intact dogs to the hematotoxic potential of the tested chemicals may have been in part due to the larger spleens in these animals, which could perhaps sequester the chemical(s) more effectively. Moreover, they felt that the normal hepatic blood flow in these animals could also enhance the removal of the chemical(s) from the systemic circulation. Goldenthal (1993a) conducted a range-finding study as a prelude to a 1-year chronic study. PBO was administered in the diet to dogs (four animals/sex/dose level) for 8 weeks. The dosage levels were 500, 1000, 2000, and 3000 ppm (approximately equivalent to 12.5, 25, 50, and 75 mg/kg body weight/day, respectively). All dogs survived to study termination. All animals in the 3000 ppm dose group had decreased appetites and reduced defecation during the 1st week. No other abnormal clinical signs were

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

2129

Table 99.2  Summary of Results of Subchronic Toxicity Studies with PBO Species Route Dog

Mouse

Rat

Rabbit

Dose

Inhalation 15,000 ppm

Duration NOAEL

Comments

Reference

Four 5-min intervals

Not applicable

Reduction in serum Lorber (1972) platelet count, increase in reticulocytes (splenectomized animals)

Diet

8 weeks 500–3000 ppm (12.5– 75 mg/kg body weight/day)

500 ppm (12.5 mg/ kg body weight/day)

Decreased body weight, increased liver weight, hepatocyte hypertrophy

Goldenthal (1993a)

Diet

1000–9000 ppm (150– 1350 mg/kg body weight/ day)

20 days

1000 ppm (150 mg/ kg body weight/day)

Increased liver weight, hepatocyte hypertrophy, necrosis, inflammatory cell infiltration

Fujitani et al. (1993)

Oral

10–1000 mg/kg body weight/day

90 days

30 mg/kg body weight/day

Increased liver weight, liver necrosis, centrilobular hypertrophy

Chun and Wagner (1993)

Oral

1500–6000 ppm (236– 880 mg/kg body weight/ day)

7 wks

Not established

Alterations in motor activity

Tanaka (1993)

Gavage

2.5–5 ml/kg body weight/ day

31 days

Not established

Anorexia, loss of weight, death

Sarles and Vandergrift (1952)

Oral

1857 mg/kg body weight/ day

90 days

Not applicable

40% mortality, increased liver Bond et al. (1973) weight

Diet

62.5–2000 mg/kg body weight/day

28 days

125 mg/kg body weight/day

Increased liver weight, microscopic changes in liver

Modeweg-Hausen et al. (1984)

Oral

6000–24,000 ppm (300–1200 mg/kg body weight/day)

13 weeks Not established

Decreased body weight, increased liver and kidney weights, hepatocyte hypertrophy

Fujitani et al. (1992)

Gavage

250–4000 mg/kg body weight/day

10 days

250 mg/kg body weight/day

Ataxia, twitching, dyspnea, gastric ulceration

Chun and Neeper-Bradley (1992)

Inhalation 15–512 mg/m3

90 days

155 mg/m3

Alterations in clinical chemistry Newton (1992) parameters, increased liver and kidney weights

Oral

1 or 4 ml/kg body weight/ wk (5% emulsion)

3 weeks

Not applicable

No signs of toxicity

Dermal

100–1000 mg/kg body weight/day

3 weeks

1000 mg/kg body weight/day (systemic toxicity) 100 mg/kg body weight/day (local effects)

Slight erythema/edema, Goldenthal (1992) fissuring/inflammation of skin

0.03 or 0.1 ml/kg body weight/day

4 weeks

Not applicable

Minor changes in liver

Monkey Oral

present. Three out of four dogs lost weight in the highest dose group. Even at 1000 ppm PBO in the diet, weight gains were lower than the control group. Food intake was similar between treated and control groups, except for a slight reduction in some animals at the 3000 ppm dosage level. There were no treatment-related effects on hematological parameters at any dose level, but slight increases in

Sarles et al. (1949)

Sarles and Vandergrift (1952)

alkaline phosphatase values and slight decreases in cholesterol were noted at doses 2000 ppm. No treatment-related microscopic changes were noted at necropsy in any group, but a compound-related increase in absolute and relative liver and gall bladder weights was recorded in males. Upon histopathologic examination, hypertrophy of hepatocytes was noted in males of all dose levels and in females at

2130

dosages 2000 ppm and above. This finding was consistent with the increases in the liver weights and serum alkaline phosphatase levels described above. No other ­ treatmentrelated microscopic changes were evident. There was a decrease in the absolute and relative weights of the testes and epididymis in the groups treated with 2000 and 3000 ppm. The dose level of 500 ppm was set as a noobservable-adverse-effect level (NOAEL) for this study because the changes recorded in the liver were considered adaptive in nature rather than adverse and were not accompanied by any systemic signs of toxicity.

99.3.2.2  Mice Fujitani et al. (1993) reported the results of dosing CD-1 mice (10 animals/sex/dose level) with 1000, 3000, or 9000 ppm (approximately equivalent to 150, 450, and 1350 mg/kg body weight/day, respectively) PBO in the diet for 20 days. Body weights were depressed in the high-dose animals (about 15%) and in the mid-dose females (about 8%). Kidney and spleen weights were also reduced in the high-dose group. A treatment-related elevation in liver weights was noted with a 79% increase in the high-dose males. Females in the high-dose group showed higher levels of -glutamyl transpeptidase (GGT) activity. The high-dose males and females featured higher levels of cholesterol, phospholipids, and total serum proteins. Hepatocyte hypertrophy, single cell necrosis, and inflammation, most prominently in the centrilobular region, were also seen in the livers of the mid- and high-dose groups. The NOAEL for this study, based on liver toxicity, was 1000 ppm. As a prelude to a 2-year bioassay, Chun and Wagner (1993) reported the conduct of a 90-day oral toxicity study in CD-1 mice (15 animals/sex/dose level) using dose levels of 10, 30, 100, 300, and 1000 mg PBO/kg body weight/ day in the diet. A significant decrease in body weight was noted in the high-dose males (34% versus controls). The target organ for toxicity was the liver as indicated by increased liver weights, hepatocyte necrosis, and centrilobular hypertrophy (NOAEL  30 mg/kg body weight/day).

99.3.2.3  Rats Sarles and Vandergrift (1952) gavaged six male and six female rats with PBO daily, 6 days/week for 31 days. The first seven doses were at 2.5 ml/kg body weight. Two animals died on the 3rd and 4th days. The remaining animals improved after some initial clinical signs of toxicity. They were dosed with a second round of seven doses of 3.5 ml/kg body weight each. Little toxicity was noted at this dose level. Hence, the animals thereafter received doses of 5 ml/kg body weight. Clinical signs of toxicity included anorexia and loss of weight. Additional animal deaths occurred from the 17th to 24th days of testing; the next and last death was at 31 days.

Hayes’ Handbook of Pesticide Toxicology

Bond et al. (1973) administered 1857 mg PBO/kg body weight/day orally to a single group of 20 rats for 90 days. Forty percent of the rats died prior to conclusion of the study. The most significant finding was a dramatic increase in liver weight (i.e., 2.4 times that of untreated controls). The authors also alluded to another study they conducted, which is unpublished, in which rats were fed 500 mg PBO/kg body weight/day. These animals were reported to have liver and kidney damage. A 4-week range-finding study was conducted by Modeweg-Hausen et al. (1984). Rats (10 animals/sex/dose level) were fed 62.5, 125, 250, 500, 1000, or 2000 mg PBO/kg body weight/day. Hepatic eosinophilic infiltration and the increased vacuolization of hepatocytes were seen with increasing severity among the mid- and high-dose groups. These effects were viewed as being degenerative changes representing chronic toxicity. Liver weights were elevated at 250 mg/kg body weight/day and above in the males and at 500 mg/kg body weight/day and above in the females. Except for an increase in alkaline phosphatase at the highest dose level, no treatment-related changes were reported in hematologic and clinical chemical parameters. Based on the observed liver toxicity, the NOAEL for this study was 125 mg/kg body weight/day. A 13-week subacute oral toxicity study was conducted in Fischer F344 rats (10 animals/sex/dose level) at levels of 6000, 12,000, or 24,000 ppm (approximately equivalent to 300, 600, or 1200 mg/kg body weight/day, respectively) PBO in the diet (Fujitani et al., 1992). No mortality occurred. Nasal bleeding and dose-related abdominal distension were reported. A significant decrease in body weight was evident in the high-dose groups (36% decrease in males, 24% decrease in females). Blood hemoglobin levels were reduced in both sexes in the high-dose group and in mid-dose females. Biochemical changes in the high-dose group consisted of increases in albumin, cholesterol, urea, and GGT activity. Liver and kidney weights were increased in a dose-dependent manner. Histopathologic examination revealed hypertrophic hepatocytes (containing a basophilic granular substance) and vacuolation of hepatocytes in periportal areas. Coagulative necrosis and oval cell proliferation were occasionally seen. Atrophy of the epithelial lining of the proximal convoluted tubules in the renal cortex was present in some male rats. A NOAEL for PBO could not be established in this study owing to the presence of liver and kidney effects even at the “low” dose of 6000 ppm. Marked clinical signs of subacute toxicity were seen in the dams of a range-finding study conducted to select doses for a developmental toxicology study (Chun and NeeperBradley, 1992). Pregnant female rats (15 animals/dose level) were gavaged on gestational days 6–15 with PBO at levels of 250, 500, 1000, 2000, or 4000 mg/kg body weight/day. Signs of general stress, such as urogenital wetness and periocular encrustation, were evident in many animals during the first three days of the study at dose levels of

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

at least 500 mg/kg body weight/day. At levels of 2000 mg/kg body weight/day, more severe clinical signs such as ataxia, twitching, prostration, dyspnea, gasping, and lacrimation were noted. Ulceration of the lining of the glandular region of the stomach as well as hemorrhage and sloughing of the lining of the nonglandular region were noted at necropsy. In the only subchronic study conducted by the inhalation route, Newton (1992) exposed CD rats (15 animals/sex/dose level) for 6 h/day, 5 days/week, for 90 days in whole body exposure chambers. PBO was aerosolized to achieve exposure concentrations of 15, 74, 155, and 512 mg PBO/m3 (MMAD of the aerosol was 1.7 m). Neither body weight gain nor food intake was affected by exposure. In the high-dose group, serum alanine transaminase, aspartate transaminase, and glucose levels were decreased, whereas BUN, total protein, and albumin levels were increased. However, not all of these effects were statistically significant, and there was no clear dose–response relationship. Both absolute and relative liver and kidney weights were elevated in the high-dose group. Minimal to slight irritation of the larynx was observed upon necropsy in all treatment groups. Inflammation, congestion, edema, and debris in the lumen were noted as well. Squamous metaplasia of the laryngeal epithelium was noted in all groups but was most marked in both sexes at the highest dose level. Important questions arise from the observation of metaplasia: Does the effect represent true toxicity that is likely to progress to neoplasia upon chronic exposure or does it represent a limited adaptive response to mild irritation? l Does the observation of squamous metaplasia in the rat laryngeal epithelium have relevance for humans? l

A recent comprehensive review of the literature showed that laryngeal metaplasia can be produced by a wide range of chemically dissimilar substances, and even by “nonchemical” means such as irritation by aerosols and particles, and dehydration by alcohols or low humidity air (Osimitz et al., 2009). Other factors that indicate that this response is adaptive and self-limiting include: The well-differentiated character of laryngeal squamous metaplasia l The reversibility of incidence and severity of it during recovery periods demonstrated with several chemicals l The lack of significant clinical observations associated with the effect l The lack of progression of the lesion over time l

Moreover, by virtue of their anatomy, the rat is more sensitive to irritation of the tissue in the larynx than is the human. Gopinath et al. (1987) emphasized that “it would appear that the rodent larynx is particularly sensitive to aerosol damage.” Thus the same dosage of an inhaled chemical delivered to rodent and human larynx would be more likely to cause histopathological alterations in the rat larynx than the human larynx. Taken together, the squamous

2131

metaplasia of the rodent larynx should not be used as a toxicologic endpoint for quantitative risk assessment.

99.3.2.4  Rabbits Sarles et al. (1949) conducted a subacute oral toxicity experiment in rabbits. A 5% PBO emulsion was fed once weekly in the diet to three rabbits over a 3-week period. The dosage used varied between 1.0 and 4.0 ml/kg body weight/ week. There was neither mortality nor clinical signs of toxicity. The rabbit that received the highest dosage was sacrificed 1 week after the last treatment, but no lesions were detected at postmortem examination. A 21-day subchronic dermal toxicity study was conducted in rabbits (five animals/sex/dose level) in which 100, 300, or 1000 mg PBO/kg body weight was applied topically once a day, 5 days a week, for 3 consecutive weeks (Goldenthal, 1992). Treatment-related effects were limited to minor skin changes at the application site. Dermal irritation was present in all treatment groups (although to a lesser extent and incidence at the 100 mg/kg body weight/day dose level). Dermal lesions consisted of very slight erythema and edema. This irritation usually appeared by day 5 and persisted for the remainder of the study. Desquamation and fissuring of the skin appeared in the 300 and 1000 mg/kg body weight/day groups. Moderate acanthosis, hyperkeratosis, and chronic inflammation of the epidermis were present. The severity of these lesions increased with increasing dosage. Body weights were comparable with those in the control group, and food intake was only slightly lower in treated animals. No treatmentrelated changes were seen in hematology and clinical chemistry, and no signs of systemic toxicity were present at any dosage level. The NOAEL is 100 mg/kg body weight/day for local effects, whereas the NOAEL for systemic toxicity is 1000 mg/kg body weight/day.

99.3.2.5  Other Species A 4-week oral toxicity study was conducted by Sarles and Vandergrift (1952) in which two African Green monkeys were fed PBO by capsule, 6 days a week (for 4 weeks), at a dosage level of 0.03 or 0.1 ml/kg body weight/day (one monkey at each dosage level). No gross pathological lesions were evident in the treated monkeys’ livers. Upon histopathologic examination of the liver, the monkey on the higher dose level showed evidence of minimal dystrophy and dysplasia, occasional acidophilic and hyaline-necrosis cells, as well as hydropic swelling.

99.3.3  Reproductive Toxicity A summary of the results of the reproductive and developmental toxicity studies discussed below is presented in Tables 99.3 and 99.4.

Hayes’ Handbook of Pesticide Toxicology

2132

Table 99.3  Summary of Results from Reproductive Studies with PBO Species Route Dose

Study type

Mice

Three-generation Not established

Rat

Dog

NOAEL

Comments

Reference

Pup weights reduced at all dose levels.

Tanaka et al. (1992)

Diet

1000–8000 ppm (268–1583 mg/kg body weight/day)

Diet

1500–6000 ppm Two-generation (400–1250 mg/kg body weight/day)

Not established

Excessive dose levels resulted in decreased pup weights in all treated animals.

Tanaka (1992)

Diet

1500-6000 ppm – males only

Postnatal

3000 ppm

High-dose animals showed increase in motor activity parameters at 11 weeks.

Tanaka (1993)

Diet

100–900 ppm (30–250 mg/kg/ body weight/day)

Two-generation

100 ppm for neurobehavioral effects

Surface righting delayed at PND 7 (300 Tanaka (2003) and 900 ppm). Olfactory orientation reduced at PND 14 (300 and 900 ppm). Total distance in a motor activity monitor increased at 9 weeks (300 and 900 ppm). Average distance and average speed in a motor activity monitor increased at 9 weeks (900 ppm).

Diet

100–25,000 ppm (8–2000 mg/kg body weight/day)

Three-generation 1000 ppm (8 mg/kg body weight/day)

Diet

300–5000 ppm (24–400 mg/kg body weight/day)

Two-generation

Parental toxicity/ Body weights of pups born to dams pup development: treated at the highest dose level were 1000 ppm (80 mg/ reduced in the early postnatal period. kg body weight/day) Reproductive toxicity: 5000 ppm (400 mg/ kg body weight/day)

Diet

30–500 mg/kg body weight/day

2-yr chronic

Not applicable

Increases in ovarian weight observed in Butler et al. some females at highest dose level. (1998)

Diet

500–3000 ppm (12.5–75 mg/kg body weight/day)

Range-finding

Not applicable

Increased absolute and relative weights Goldenthal of testis and epididymis noted. No (1993a) microscopic abnormalities observed in the testis.

99.3.3.1  Dogs In an 8-week dietary range-finding toxicity study in dogs, PBO was fed daily in the diet to beagles at dose rates of 500, 1000, 2000, or 3000 ppm (approximately equivalent to 12.5, 25, 50, or 75 mg/kg body weight/day, respectively) (Goldenthal, 1993a). There was an increase in the absolute and relative weights of the testes and epididymides. No microscopic abnormalities were noted in the testes. Spermatozoa were being produced. Other details of the study are presented in Section 99.3.2.1.

99.3.3.2  Mice A three-generation, one litter per generation, reproductive study was conducted in CD-1 mice by Tanaka et al. (1992).

Very high maternal toxicity at two highest dose levels resulting in marked reductions in the incidence of pregnancies, numbers of litters per dam, general health of the offspring, and average weanling weights of pups.

Sarles and Vandergrift (1952)

Robinson et al. (1986)

Ten animals of either sex were fed diets containing 1000, 2000, 4000, and 8000 ppm PBO (purity not specified). These doses are equivalent to 268, 506, 936, and 1583 mg PBO/kg body weight/day as averaged over F0 and F1 generations from preconception to lactation. Food intake was reduced in the F0 generations at the 8000 ppm dose, indicative of some toxicity, except during the mating period, and was also reduced during the lactation period in the F1 generation, also at 8000 ppm. The 4000 ppm treatment groups of both the F0 and F1 generations also had a reduction in food intake during the lactation period. Mean F1 litter weight was significantly decreased (38%) at 8000 ppm and reduced by 18% at the 4000 ppm treatment level. However, litter size remained unchanged at all levels. Pups born in the 8000-ppm-treated group had a lower survival index at postnatal day 21 (63% vs. 91% for males of control group; 79%

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

2133

Table 99.4  Summary of Results from Developmental Studies with PBO Species Route

Dose

NOAEL

Comments

Mice

Gavage

1065–1800 mg/kg body weight/day

Not established

Total resorption rates significantly Tanaka et al. increased in mid- and high-dose (1994) groups. Significant decrease in body weights of male and female fetuses, appearing to be dose-dependent.

Rat

Gavage

300 or 1000 mg/kg body weight/day

Maternal body weights were Maternal toxicity: Not established Developmental toxicity: 1000 mg/kg reduced at both dose levels tested. body weight/day

Gavage

62.5–500 mg/kg body Maternal and developmental No signs of either maternal or weight/day toxicity: 500 mg/kg body weight/day embryo-fetotoxicity.

Gavage

200–1000 mg/kg body weight/day

Maternal toxicity: 200 mg/kg body weight/day

Reference

Kennedy et al. (1977) Khera et al. (1979)

Gestational body weights and body Chun and weight gains were reduced in the Neeper-Bradley 500- and 1000-mg/kg body weight/ (1991) day groups.

Developmental toxicity: 1000 mg/kg body weight/day Gavage

Rabbit

Gavage

630-1800 mg/kg body Maternal toxicity: 630 mg/kg body weight/day weight/day

50–200 mg/kg body weight/day

Decreased maternal weight gain in 1065- and 1800-mg/kg body weight/ day groups.

Tanaka (1995)

Developmental toxicity: 630 mg/kg body weight/day

Decreased fetal body weight, increased external limb deformities in 1065- and 1800-mg/kg body weight/ day groups.

Maternal toxicity: 50 mg/kg body weight/day

Maternal toxicity was evident at Leng et al. (1986) 100 and 200 mg/kg body weight/day manifested by decreased defecation and a dose-dependent weight loss during the treatment period.

Developmental toxicity: 200 mg/kg body weight/day

vs. 89% for females of the control group). Pup weights in the F1 generation were decreased for all dosage groups, but there was no dose-related response at low- or mid-dose levels. No treatment-related effects were noted in neurobehavioral tests conducted in the F1 animals. The mean F2 litter size was significantly decreased at the 4000 and 8000 ppm treatment levels. Mean F2 treatment weights were decreased in all treatment groups. Pups in the 8000-ppmtreated group of the F2 generation had a lower survival index than controls at day 21 postnatal (59% in males and 79% in females vs. 100% in male and female control groups). Pup weights in the F2 generation were decreased at dosage levels of 2000 ppm and above on postnatal days 4, 7, 14, and 21. Pup weights were reduced on days 4 and 7 at the 1000 ppm dosage level. Changes in neurobehavioral parameters such as surface righting and cliff avoidance at postnatal day 7 were noted in the F2 pups. However, as the author pointed out, no clear dose–response relationship was evident in these neurobehavioral tests. Because pup weights

were reduced at all doses tested, a NOAEL for reproductive effects could not be established from this study.

99.3.3.3  Rats Sarles and Vandergrift (1952) report a study in which groups of 12 male and 12 female rats per dose level were fed diets containing 100, 1000, 10,000, or 25,000 ppm (approximately equivalent to 8, 80, 800, or 2000 mg/kg body weight/ day, respectively) of PBO (technical grade, 80% purity), for three generations. None of the female rats at the highest dose level were fertile and there were marked reductions in the incidence of pregnancies, numbers of litters per dam, general health of the offspring, and average weanling weights of pups born to dams treated at 10,000 ppm. These findings are clearly a result of the high maternal toxicity, especially at 10,000 and 25,000 ppm. No adverse effect on reproduction was observed in three generations of progeny in the 100 and 1000 ppm groups (NOAEL  1000 ppm).

Hayes’ Handbook of Pesticide Toxicology

2134

A two-generation reproduction study was conducted in rats with PBO by Robinson et al. (1986). Groups of 26 male and 26 female Sprague-Dawley rats were utilized and adults of the F0 and F1 generations were treated at dose levels of 300, 1000, or 5000 ppm (approximately equivalent to 24, 80, or 400 mg/kg body weight/day, respectively) in the diet. Animals were treated for 83–85 days prior to placement for mating, and treatment continued throughout mating, pregnancy, and lactation. The only consistent finding throughout the study period was a lower body weight gain at the highest dosage level. This tendency was partially reversed during the lactation period, when females at this dose level showed higher weight gains when compared with control rats. For both F1 and F2 generation pups, the viability, survival, and lactation indices were unaffected by treatment. There were no treatment-related abnormal findings for the pups, and weanlings did not reveal any treatment-related adverse effects. Body weights of pups born to dams treated at the highest dose level were reduced in the early postnatal period. The NOAEL for parental toxicity and pup development was thus 1000 ppm PBO in the diet. The NOAEL for reproductive toxicity was set at 5000 ppm PBO in the diet. In a 2-year chronic oral toxicity study in Sprague-Dawley rats, animals received dietary administration of 15, 30, 100, or 500 mg PBO/kg body weight/day (89% purity) (Butler et al., 1998). Only changes in the reproductive system are discussed here. Full details of the study are presented in Section 99.3.4.3. Increases in ovarian weights were observed among some females receiving 500 mg/kg body weight/day, although no histopathologic changes were noted. Atrophy of the testes was seen histologically in all male groups and when bilateral atrophy was considered alone, there was an increased incidence in the intermediate and high-dose groups with a corresponding reduction in the incidence of unilateral atrophy. However, the finding is unlikely to be related to treatment because the dose–response relationship was unclear and the atrophy was not accompanied by changes in the seminiferous tubules or sperm production. Moreover, there were no statistically significant increases or decreases in testes weight when expressed as either absolute weight or relative-to-brain weight.

99.3.4  Developmental Toxicity 99.3.4.1  Mice Tanaka et al. (1994) reported a study conducted in CD-1 mice in which PBO was administered by gavage on day 9 of gestation to groups of 20 animals at doses of 1065, 1385, or 1800 mg/kg body weight (95% purity; PBO dissolved in olive oil). No abnormal behavior or mortality patterns were observed in dams. Three abortions occurred in the mid- and high-dose groups. Four litters were resorbed in the two higher-dosage groups, but maternal body weights

were comparable between all groups. Total resorption rates were significantly increased in the mid-dose (26%) and the high-dose groups (32%) when compared with the control value (6%). The number of viable fetuses per dam was comparable between all dosage groups. There was a significant decrease in body weights of male and female fetuses derived from treated dams, which did appear to be dose-dependent. Certain external malformations such as exencephaly, cranioschisis, open eyelids, omphalocele, kinky tail, and talipes varus were observed in all groups (including controls) and oligodactyly was recorded in the forelimbs of some fetuses derived from treated dams. The incidence of this latter defect was 6% in those fetuses derived from the highest dosage group. The authors concluded that a single high dose of PBO (1065 mg/kg body weight or above), when given orally to pregnant mice on day 9 of gestation, could cause embryo-fetal toxicity with associated oligodactyly of the forelimbs. The high dose levels of this study make it difficult to interpret the significance of this finding.

99.3.4.2  Rats Kennedy et al. (1977) carried out a developmental toxicity study with PBO in pregnant rats. Twenty female animals per dose level were gavaged with PBO in corn oil at 300 or 1000 mg PBO/kg body weight/day. Other than a decline in body weight gain in both treated groups (especially in the later stages of gestation), no other treatment-related signs of toxicity occurred. The reproductive parameters of the dams were not significantly affected by treatment. One female from each treatment group resorbed most or all of her litter. The fetuses derived from each treatment group exhibited no internal or external skeletal malformations that could be related to treatment. The observation that maternal body weights were reduced at both doses tested meant that a NOAEL was not established for maternal toxicity. Because no developmental effects were seen at any dose including the highest dose tested, the NOAEL for embryo-fetal toxicity for this study was 1000 mg/kg body weight/day. Pregnant female Wistar rats (17–20 per dosage group) were dosed with PBO levels of 62.5, 125, 250, or 500 mg/kg body weight/day from day 6 to day 15 of gestation in a study conducted by Khera et al. (1979). The types and incidences of anomalies in fetuses derived from treated dams were comparable with those of the control group and it was concluded that doses as high as 500 mg/kg body weight/day produced no signs of either maternal or embryo-fetal toxicity. A developmental toxicity study by Chun and Neeper-Bradley (1991) conducted in accord with U.S. EPA Guidelines investigated PBO in Sprague-Dawley rats. Timed pregnant rats were administered PBO (90.78% purity) by gavage on gestation days 6–15. The dosage

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

l­evels were 200, 500, and 1000 mg/kg body weight/day and 25 animals were included in each group. The pregnancy rate was equivalent among groups and ranged from 88% to 96%. No females aborted, delivered early, or were removed from the study. Gestational body weights and body weight gains were reduced in the 500 and 1000 mg/kg body weight/day groups, as was food intake for the first 7 days, indicating that a sufficiently high dose was achieved. Treatment had no effect on gestational parameters including resorption, pre- and postimplantation losses, percentage of live fetuses, and sex ratios, nor did it affect the fetal body weights or the incidence of fetal malformations. However, two common skeletal variations (i.e., nonossification of centrum of vertebrae 5 or 6) had a higher incidence in the two highest dosage groups. These findings were not considered treatment-related, as adjacent vertebrae did not have delayed ossification. The NOAEL for maternal toxicity in the rat was 200 mg/kg body weight/day and the NOAEL for developmental toxicity was at least 1000 mg/kg body weight/day. Tanaka et al. (1995) reported a development toxi­city study in pregnant female Charles River (Crj:CDS) rats dosed with PBO (purity 95%) at levels of 0, 630, 1065, and 1800 mg/kg body weight on days 11–12 of gestation. The most significant treatment-related changes included an increased resorption at the high dose and various limb deformities (oligodactyly, syndactyly, and polydactyly) in the 1065- and 1800-mg/kg body weight dose groups. Two of 10 dams and six of eight dams had anomalous fetuses in the 1065- and 1800-mg/kg body weight dose groups, respectively. No anomalous fetuses were seen at the 630-mg/kg body weight level. These results must be interpreted carefully. Although dosing had no effect on mortality of the treated dams, a large and statistically significant decrease in maternal body weight gain (days 11–20) was seen at 1065 and 1800 mg/kg body weight (23.7% and 36.1%, respectively). Correspondingly, although treatment had no effect on the average litter size, the average fetal body weight of males was decreased at 1065 mg/ kg body weight (4.8%) and for both male and females at 1800 mg/kg body weight (14.5% and 13.5% respectively). It is uncertain what role these significant weight changes have on the development of the limb abnormalities, but the potential significance of those observations is limited given the possible confounding effects of the hightreatment dose levels on the dams.

99.3.4.3  Rabbits New Zealand White female rabbits were gavaged with PBO (purity 100%) in corn oil at levels of 50, 100, or 200 mg/kg body weight between day 7 and day 19 of pregnancy (Leng et al., 1986). Caesarian sections were performed on day 29 of gestation. Maternal toxicity was evident at 100 and 200 mg/kg body weight/day manifested by decreased

2135

defecation and a dose-dependent weight loss during the treatment period (these weight losses were recovered posttreatment). Common developmental defects, including an increase in the number of full ribs and the presence of more than 27 presacral vertebrae, were recorded in all dose groups. However, no dose–response relationship was apparent. The number of litters in the treated groups with these observations was not increased when compared with control values. The NOAEL for maternal toxicity was 50 mg/ kg body weight/day, whereas the NOAEL for developmental toxicity was 200 mg/kg body weight/day.

99.3.4.4  Developmental Neurotoxicity A three-generation reproductive study that included neuro­ behavioral endpoints was carried out in CD-1 mice by Tanaka et al. (1992). Ten animals of either sex were fed diets containing 1000, 2000, 4000, and 8000 ppm PBO (purity not specified). These doses are equivalent to 268, 506, 936, and 1583 mg PBO/kg body weight/day as averaged over F0 and F1 generations from preconception to lactation. No treatment-related effects were noted in neurobehavioral tests conducted on the F1 or F2 animals. Tanaka (1993) further studied the developmental neurotoxicity of PBO (purity not specified) in CD-1 mice in a subsequent two-generation study. Ten mice of either sex per group received diets containing 1500, 3000, or 6000 ppm (approximately equivalent to 400, 700, and 1250 mg/kg/ body weight/day) PBO during a 4-week period prior to mating (F0), during gestation and through the time that the F1 generation was 9 weeks old. Maternal body weights and food consumption were not reported. The open field test demonstrated a statistically significant decrease in ambulating in F0 male mice only at the highest dose (6000 ppm). Because of the excessive dosage levels incorporated in this study, pup body weights were reduced at birth at all dosed animals. By postnatal day (PND) 21, the mean pup body weights of mediumand high-dose pups were decreased by 9.4% and 41%, respectively. The survival index for pups at postnatal day 21 was 79.2% (controls), 92.9% (low-dose group), 80.0% (mid-dose group), and 51.7% (high-dose group). There were no significant differences in the behavioral tests during the lactation period, except for a reduction in olfactory orientation in mid- and high-dose group animals (41.2% and 39.2%, respectively). Other than sporadic nondose-dependent changes, the open field test and multiple water T-maze tests were not significantly altered by PBO. In a postnatal developmental neurotoxicity study, Tanaka (1993) administered PBO to male mice only from 5 to 12 weeks of age at levels of 1500, 3000, and 6000 ppm in the diet. PBO had no effect on food consumption; body weights were not reported. The high-dose animals exhibited motor activity effects at 11 weeks of age, including an increase in number of movements, movement time, total distance, average speed, and number of turnings.

Hayes’ Handbook of Pesticide Toxicology

2136

Tanaka (2003) conducted another two-generation developmental neurotoxicity study in CD-1 mice using dietary dose levels of 100, 300, and 900 ppm from 5 weeks of age in the F0 through 9 weeks of age in F1. No effects were noted in the number of offspring or the survival of offspring through lactation. Treatment had no effect on the body weights of mice after weaning. The following effects were noted in males only:

Test Guidelines OPPTS 870.6300 Developmental Neuro­ tox­icity Study (U.S. EPA, 1998). Moreover, a summary of developmental studies in which neurobehavior was evaluated indicated that out of 191 such studies, only three were conducted in the mouse (Ulbrich and Palmer, 1996).

Surface righting was delayed at PND 7 (300 and 900 ppm). l Olfactory orientation was reduced at PND 14 (300 and 900 ppm). l Total distance in a motor activity monitor was increased at 9 weeks (300 and 900 ppm). l Average distance and average speed in a motor activity monitor was increased at 9 weeks (900 ppm).

Numerous long-term toxicity and oncogenicity studies have been undertaken on PBO over the past 50 years in various species. As evidenced in these studies, the primary target organ is the liver. The results of the studies discussed below are summarized in Table 99.5.

These effects were noted at levels significantly lower than reported in previous studies. Tanaka (1992) previously showed that PBO depressed olfactory orientation in the F1-generation mice in a two-generation toxicity study (1500–6000 ppm in the diet). Tanaka et al. (1992) reported that PBO depressed olfactory orientation in the F1-generation mice and effects on several other parameters in F2generation mice were observed (1000–8000 ppm in the diet). It is not known what accounts for the seemingly high sensitivity of the male mice in this Tanaka (2003) study. Regarding the Tanaka studies, a low incidence of statistically significant measures in a few behavioral measurements should not be taken per se as sufficient evidence of developmental neurotoxicity. Developmental neurotoxicity studies evaluate a wide range of behavioral and histopathological factors. An expert panel has recently evaluated developmental neurotoxicology endpoints (Tyl et al., 2008). This panel has recognized that behavioral endpoints, in particular given the intrinsic variability of age-related development in very young animals, may be highly variable. Rather than drawing conclusions from single “statistical significances,” it is necessary to look for consistent patterns of behavioral effect in “functional domains” and using statistics appropriate to multiple measures over time. Meanwhile, behavioral endpoints must also be taken in the context of systemic toxicity. Test rodents, like their human counterparts, suffer from clinical and behavioral symptoms due to systemic illness (Gerber and O’Shaughnessy, 1986, reference 18). Moreover, developmental neurotoxicity observations in the mice are difficult to evaluate given the paucity of such data in mice. Although rabbits and rats, and, to a lesser extent mice, are the animal species primarily used in routine developmental toxicity, testing the assessment of neurobehavioral toxicity in the offspring is ­evaluated almost exclusively in rats. Rats are the preferred test species in the OECD Test No. 426: Developmental Neurotoxicity Study (OECD, 2001) and the U.S. EPA Health Effects

PBO was administered to dogs in capsule form for a 1-year chronic dietary toxicity study (Sarles and Vandergrift, 1952). Groups of four dogs each were treated at dose levels corresponding to 3, 32, 160, or 320 mg/kg body weight/day. The dosage was adjusted in accordance with any alteration in body weight to maintain the same dose in mg/kg body weight, except for one individual animal per dosage group, which received a constant absolute dose throughout the trial. All dogs belonging to the two highest dosage groups lost weight; however, meaningful comparisons between the lower dose groups and control animals were not possible owing to large variations in body weight gains and the small number of animals involved. All dogs at the highest dosage level died. However, no toxic reaction was seen at 3 mg/kg body weight/day. Red blood cell (RBC) and white blood cell (WBC) counts were unchanged at all dose levels. There was a dose-dependent increase in liver, kidney, and adrenal weights. Microscopic changes were quite similar to those in long-term toxicity studies conducted in rats, with the liver again being the major target organ for toxicity. Hydropic swelling was evident in hepatocytes in the mid-dose group, with hepatic dystrophy and dysplasia becoming more obvious at the two highest dosage levels. The NOAEL for this study was 32 mg/kg body weight/day. A 1-year chronic dietary toxicity study following USPEA Guidelines was conducted in the beagle dog in which groups of four males and four females were fed PBO for 1 year at doses of 100, 600, or 2000 ppm (approximately equivalent to 2.5, 15, or 50 mg/kg body weight/day, respectively) in the diet (Goldenthal, 1993b). All animals survived to study termination. A reduction in body weight gain and food intake was evident in the 2000 ppm group. Physical examinations were otherwise normal throughout the test period. Biochemical analysis showed increases in serum alkaline phosphatase levels at 6 and 12 months in the highest dosage group. Female beagles showed a decrease in serum cholesterol at the 2000-ppm dosage level. Increased liver and gall bladder weights, with mild

l

99.3.5  Chronic Toxicity/Oncogenicity

99.3.5.1  Dogs

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

2137

Table 99.5  Summary of Results of Chronic Toxicity/Oncogenicity Studies with PBO Species Route

Dose

Duration

NOAEL

Comments

Reference

Dog

Oral

3–320 mg/kg body weight/ day

1 year

32 mg/kg body weight/day

Increased liver and kidney weights, hepatic dystrophy and dysplasia

Sarles and Vandergrift (1952)

Diet

100–2000 ppm (2.5–50 mg/ 1 year kg body weight/day)

Diet

300 or 1112 ppm (45 or 167 mg/kg body weight/day)

69 weeks

Diet

45 or 133 mg/kg body weight/day

18 months 45 mg/kg body weight/day

No signs of toxicity

Bond et al. (1973)

Diet

1036–2804 ppm (148– 298 mg/kg body weight/day)

112 weeks Not established

Decreased body weight in both sexes, hepatic nodular hyperplasia in males

U.S. National Cancer Institute (1979)

Diet

6000–12,000 ppm (960– 1 year 1920 mg/kg body weight/day)

Not established

Hepatic adenomas and hepatocarcinomas

Takahashi et al. (1994b)

Diet

30–300 mg/kg body weight/ day

30 mg/kg body weight/day

Increased liver weight, benign hepatic adenomas

Butler et al. (1998)

Diet

2 year 100–25,000 ppm (5– 1250 mg/kg body weight/day)

100 ppm (5 mg/kg body weight/day)

No significant increase in tumor Sarles and incidence; severe liver damage, Vandergrift (1952) increased incidence of liver “hyperdysplastic” nodules

Diet

90 mg/kg body weight/day

2 year

Not applicable

Decreased body weight

Diet

5000–10,000 ppm (~250– 500 mg/kg body weight/day)

107 weeks Not established

Dose-related increase in Cardy et al. (1979) hepatocytomegaly, dose-related increase in lymphomas in female rats, but incidence in controls was also high

Diet

5000–10,000 ppm (250– 500 mg/kg body weight/day)

2 year

Not established

No significant increase in tumor Maekawa et al. incidence; dose-related increase (1985) in ileocecal ulcers

Diet

6000–24,000 ppm (526– 95–96 2187 mg/kg body weight/day) weeks

Not established

Hepatic adenomas and hepatocarcinoma at midand high-dose levels, cecal hemorrhaging, severe general and hepatic toxicity

Takahashi et al. (1994a)

Diet

15–500 mg/kg body weight/ day

2 year

30 mg/kg body weight/day

Increased liver and kidney weights, centrilobular hepatocyte hypertrophy, focal hyperplasia

Butler et al. (1998)

Diet

2.0 ml/day (1000 ppm)

1 year

Not applicable

Slight hepatic dystrophy and dysplasia

Sarles and Vandergrift (1952)

Mouse

Rat

Goat

78 weeks

600 ppm Increased liver and gall bladder (15 mg/kg body weights, hypertrophy of weight/day) hepatocytes Not applicable

hypertrophy of hepatocytes, were also recorded at this highest dosage level. A small increase in thyroid gland and parathyroid gland weights was also noted. However, no microscopic abnormalities were detected in the thyroid gland. No treatment-related histopathologic changes were seen on the study. Based on the changes seen in the liver, the NOAEL for this study was 600 ppm.

Goldenthal (1993b)

No significant increase in tumor Innes et al. (1969) incidence

Hunter et al. (1977)

99.3.5.2  Mice Innes and co-workers (1969) studied the oncogenicity of PBO in mice by administering the maximal tolerated dose. Animals (18/sex/strain) from two hybrid stocks (C57BL/ 6  C3H/Anf or C57BL/6  AKR) were gavaged with 100 mg undiluted PBO/kg body weight or 464 mg PBO/kg

Hayes’ Handbook of Pesticide Toxicology

2138

body weight in solvent vehicle from 7 to 28 days of age. Thereafter, they received 300 ppm undiluted PBO or 1112 ppm PBO in solvent vehicle (approximately equivalent to 45 and 167 mg/kg body weight/day, respectively) in the diet for 69 weeks. These researchers found no significant increase in tumor incidence as a result of PBO treatment. Bond et al. (1973) reported no adverse effects following dosing of mice with 45 or 133 mg/kg body weight/day PBO in the diet for 18 months. Few details are available for this early study, however. The U.S. National Cancer Institute (1979) conducted a mouse carcinogenicity study in which male and female B6C3F1 animals (50/sex/dose level) were initially dosed with 2500 or 5000 ppm of PBO in the diet. Toxicity appearing in both these groups resulted in a reduction in the doses to 500 and 2000 ppm, respectively, after 30 weeks of dosing. The time-weighted average doses in the diets were approximately 1036 and 2804 ppm (approximately equivalent to 148 and 298 mg/kg body weight/day, respectively). Dose-dependent decreases in body weight and body weight gains were observed in both dose groups and both sexes. Nodular hyperplasia of the liver was slightly elevated in males. Although tumors were observed in the liver and lacrimal gland, the incidence was not statistically significant. Thus, the authors concluded that PBO was not oncogenic. Takahashi et al. (1994b) reported the results of a 1-year chronic toxicity study conducted in CD-1 mice using dietary doses of 6000 and 12,000 ppm PBO (equivalent to 960 and 1920 mg/kg body weight/day, respectively). Animals were allocated to three groups consisting of 52, 53, and 100 animals for dose levels of 0, 6000, and 12,000 ppm, respectively. Significant depressions in body weight and body weight gain were noted for low and high doses of PBO. Only 81% of the high-dose animals survived the 12-month study period compared with 98% and 94% of the animals in the low-dose and control groups, respectively. Hepatic adenomas and hepatocarcinomas were observed in both treatment groups as were hemangiosarcomas and hemangioendothelial sarcomas. However, the doses used in this study are clearly in excess of internationally accepted maximum tolerated dose (MTD) criteria and thus they are of questionable relevance in determining the hazard and risk for humans of PBO exposure. Butler et al. (1998) reported a U.S. EPA Guideline study in which groups of 60 male and 60 female CD-1 mice were administered PBO in the diet at doses of 0 (two separate control groups), 30, 100, or 300 mg/kg body weight/day for at least 78 weeks. No treatment-related clinical signs of toxicity or changes in food consumption or clinical chemistry were observed. The mean absolute body weight and mean body weight gains were generally slightly decreased throughout the study at the high dose in both males and females, indicating that the MTD was reached. A dose-related increase in the mean absolute and relative liver weights was seen in the mid- and high-dose groups of both sexes. The mean

absolute and relative liver weights of the low-dose group of male mice were also slightly increased. Both males and females clearly showed an increased incidence of benign hepatic nodules diagnosed as adenomas. The further characterization of the adenomas showed that the increased burden of lesions was due to the increased incidence of eosinophilic adenomas, similar to the lesions induced by a range of enzyme inducers in the mouse (Butler, 1996). There was no increase of either basophilic adenomas or hepatocarcinomas. The NOAEL in this study for nononcogenic effects was 30 mg/kg body weight/day.

99.3.5.3  Rats In an early study, Sarles and Vandergrift (1952) fed Wistar rats with diets containing from 100 to 25,000 ppm (approximately equivalent to 5–1250 mg/kg body weight/day) PBO for 2 years. Twelve males and 12 females were used at each dose level. The entire high-dose group died by week 68 and showed severe liver damage upon necropsy. An increased incidence of “hyperdysplastic” hepatic nodules, characterized by the authors as the appearance of larger cells and increased polyploidy, was seen in the treated groups. Dystrophy and dysplasias were also observed in the livers from animals fed 1000 ppm or greater PBO. The authors concluded that there was no evidence of carcino­ genicity; the 100 ppm dose level was considered nontoxic. Because PBO is most often used as a synergist with pyrethrins, a 2-year dietary study was conducted in SpragueDawley rats using a mixture of pyrethrins (53.1% purity) and PBO (95% purity) (Hunter et al., 1977). Forty-five males and 45 females were fed diets containing 400 ppm pyrethrins plus 2000 ppm PBO. The average daily doses received by the animals over the study period were 16  79 mg/kg body weight/ day (pyrethrins  PBO) for males and 20  101 mg/kg body weight/day (pyrethrins  PBO) for females. Body weights were depressed in the females during the first 78 weeks of treatment and among males during the first 26 weeks of treatment. No other treatment-related effects were noted and no treatment-related change in tumor incidence was seen. The U.S. National Cancer Institute conducted a 2-year cancer bioassay in Fisher 344 rats (Cardy et al., 1979). Fifty male and 50 female animals per dose level were allocated to low- and high-dose groups which received PBO (88.4% purity) in the diet at 5000 or 10,000 ppm (approximately equivalent to 250 or 500 mg/kg body weight/day, respectively) for 107 weeks. A dose-dependent decrease in the mean body weights of treated groups was noted. Other than increased hepatocytomegaly, no dose-related increases in the incidence of tumors or other microscopic findings were observed in the liver. The hepatocytomegaly consisted of foci of enlarged hepatocytes, often associated with large, vesicular nuclei and numerous cytoplasmic vacuoles, giving the cytoplasm a “ground glass” appearance.

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

Distortion of lobular architecture in these foci was minimal, and trabeculae were continuous with adjacent normal hepatocytes. These lesions appear similar to those described by Squire and Levitt (1975) as “eosinophilic foci,” “ground glass foci,” or “clear cell foci.” Although a dose-dependent increase in lymphomas was noted in females, the incidence of lymphomas, leu­kemias, and reticuloses observed was not significantly different from the historical rates from the laboratory. Thus, this study showed that, under the conditions of the bioassay, PBO was not carcinogenic in Fischer 344 rats. The oncogenicity of PBO was also studied in F344/ DuCrj rats by Maekawa et al. (1985). Animals (50 sex/ dose level) were fed a dietary level of 5000 or 10,000 ppm (approximately equivalent to 250 and 500 mg/kg body weight/day, respectively) for 2 years but no significant dose-related increase in the incidence of any tumor was found. A dose-related incidence of ileocecal ulcers, however, was found in animals of both sexes. Takahashi et al. (1994a) conducted a 2-year chronic toxicity study in the rat at dose levels up to 24,000 ppm (1000 times the maximum level approved in raw cereals in Japan). Fischer F344/DuCrj rats (30–33 per group) received a diet containing PBO at 6,000, 12,000, or 24,000 ppm (equivalent to 526, 1052, or 2187 mg/kg body weight/day, respectively) for 95–96 weeks. Beginning at about 40 weeks, 10 rats in the 12,000 ppm male group died due to cecal hemorrhages. By the end of the study, gastrointestinal hemorrhage occurred at all dose levels. Organ weights (with the exception of the liver) were reduced in all animals in the high-dose group. “Probable essential thrombocytopenia” was present in all treated male groups. Body weight gains relative to controls were reduced in all treated groups of both sexes and reached approximately 50% in the highdose group. A dose-dependent increase in hepatocellular hyperplasia (seen as liver nodules) was reported. Although the nomenclature is different, these lesions are much like those described by Sarles and Vandergrift (1952) at toxic doses of PBO. Takahashi also reported hepatocellular adenomas and carcinomas in the mid- and high-dose groups. It is important to note that the study was not intended to be a carcinogenicity study and the procedures for collecting and examining tissues were not performed according to current U.S. EPA/OECD standards. Thus, not all tissues were taken or prepared for histological examination. Because of the high-dose levels used and resulting tox­icity, it is difficult to interpret the carcinogenicity findings and their relevance for hazard assessment. Moreover, several investigators have reported that hepatotoxicity, and the resulting regenerative hyperplasia, can contribute to the formation of liver tumors by nongenotoxic mechanisms (Kociba et al., 1978; McClean et al., 1990; Mutai et al., 1990; Tatematsu et al., 1990; Van Miller et al., 1977). This and other mechanistic aspects of this oncogenicity response are discussed in Section 99.3.5.

2139

Butler et al. (1998) reported the results of a U.S. EPA Guideline dietary study in the Sprague-Dawley rat in which groups of 60 animals of each sex were administered 15, 30, 100, or 500 mg PBO/kg body weight/day. Three control groups were included. Because a 4-week range-finding study did not provide clear evidence of a NOAEL with respect to minor alterations in liver cell morphology, additional animals were used during the early stages of this study. Thus, after completion of 4 weeks of treatment, 10 males and 10 females from each low-dose group and a special control group consisting of 10 males and 10 females were sacrificed for both gross pathological and histopathologic examinations of the liver. Since the results of the histopathologic examination showed no abnormal findings in the livers of these rats, the low-dose level of 30 mg/kg body weight/day was continued on the 2-year study. The 15-mg/kg body weight/day group was discontinued. No adverse treatment-related effects on survival and no treatment-related clinical signs were seen. A reduced growth rate and a minimal reduction in food intake were noted for males and females receiving 500 mg/kg body weight/day. These females were shown to have increased serum cholesterol levels and slightly higher total serum protein levels. The blood urea nitrogen levels were also slightly higher in this group on one occasion. Dose-related increases in liver and kidney weights were noted in both sexes at 100 and 500 mg/kg body weight/day. Histologically, the most common effect in the liver was centrilobular hepatocyte hypertrophy and the presence of eosinophilic and mixed (basophilic and eosinophilic) cell foci. The severity of focal hyperplasia in the liver was also greater in the intermediate- and high-dose groups. The hyperplastic foci contained either basophilic, normal, or enlarged eosinophilic cells that were variable in size. Normal lobular architecture was retained. Portal triads and central veins were present in the lesions. Neither the incidence of adenomas nor incidence of carcinomas was increased. Pituitary adenomas were common in both males and females but showed no treatment-related effect upon incidence of the adenomas. Thyroid changes, including increased pigment in colloid, and follicular hyperplasia (particularly at 500 mg/kg body weight/day) were seen in both sexes at the end of the study as well as in the high-dose group males dying or sacrificed during the study. In the kidney, glomerulonephritis was more common in the male than the female. No significant increase in the incidence of glomerulonephritis was observed, but the severity of the lesions was increased slightly in the intermediate- and high-dose groups. The non-neoplastic changes observed in this rat study are consistent with induction of the hepatic mixed function oxidase system. The liver observations such as increased liver weight, centrilobular hypertrophy, eosinophilic foci, and eosinophilic focal hyperplasia are probably due to

2140

enzyme induction. Likewise, the thyroid follicular hyperplasia is a likely secondary response of the thyroid gland to prolonged TSH stimulation resulting from decreased circulating levels of T3 and T4. Reduced T3 and T4 levels are due to increased conjugation and excretion of the thyroid hormones resulting from liver enzyme induction. This pattern has been observed with other liver enzyme inducers such as phenobarbital (McClain, 1989). Following extensive consideration, rat thyroid tumors formed by this mode of action are not considered to be relevant to humans (Cohen et al., 2004; Hill et al., 1999). Given these considerations, the authors concluded that there was no evidence of carcinogenic activity and that the NOAEL based on liver changes was 30 mg/kg body weight/day.

99.3.5.4  Other Species Sarles and Vandergrift (1952) also reported a chronic oral toxicity experiment where a mature female goat was fed a daily dose of 2.0 ml PBO by capsule, 6 days a week, for 1 year. This dose equated to approximately 1000 ppm PBO in the diet. The dose started 4 days after the goat gave birth to a female kid, with both dam and offspring being observed for 1 year to ascertain any signs of direct or indirect (i.e., PBO in dam’s milk) toxic effects. The general health of both dam and kid was unaffected by treatment. The kid was nursed by the treated dam for approximately 6 months and continued to grow and thrive as expected. Red blood cell (RBC) and white blood cell (WBC) counts were unremarkable. At postmortem examination, the dam’s liver revealed slight dystrophy and dysplasia, with central hydrophilic swelling and slight fatty accumulation. No abnormalities were detected in the organs of the kid goat.

99.3.6  Genotoxicity PBO has shown no evidence of mutagenic activity in a number of bacterial assays involving Salmonella typhimurium, Bacillus subtilis, and Escherichia coli both in the presence or absence of rat liver microsomes (S-9) (Ashwood-Smith et al., 1972; Butler et al., 1996; Ishidate et al., 1984; Kawachi et al., 1980; Moriye et al., 1983; White et al., 1977). Most of the studies in systems using mammalian cells in culture show no evidence of mutation or a chromosomedamaging effect. Galloway et al. (1987) investigated a wide range of compounds including PBO in Chinese hamster ovary cells and failed to find chromosome aberrations and sister chromatid exchange in the presence or absence of rat liver S-9. PBO had no effect on Chinese hamster ovary cells in the report of Butler et al. (1996), and produced a small increase in sister chromatid exchanges only in the absence of S-9 in the recent study by Tayama (1996). Tayama also concluded that the metabolites of PBO are unlikely to be genotoxic. In addition, PBO did not produce chromosomal

Hayes’ Handbook of Pesticide Toxicology

aberrations in Chinese hamster lung cells (Ishidate et al., 1984, 1988; Kawachi et al., 1980) or induce mutations in the CHO/HGPT assay (Butler et al., 1996). However, in L5178Y mouse lymphoma cells, PBO showed evidence of mutagenic activity only in the absence of additional metabolic activation. In this study, the mutagenic activity was observed only where cytotox­icity was evident. The relative total cell growth at the lowest mutagenic concentration of 30 g/ml was around 60% (McGregor et al., 1988). PBO also induced cell transformation in Syrian hamster embryo cells (Amacher and Zelljadt, 1983). In this study, three dose levels (0.5, 1.0, and 3.0 g/ml), were used and only two transformed colonies out of 2761 were observed. No indication was given of the dose level that caused the two transformed colonies. Suzuki and Suzuki (1995) investigated the mutagenicity of PBO in human RSA cells, a cell line of double-transformed human embryonal fibroblasts considered to be hyper­mutable, by determining ouabain resistance. The results show an unusual dose response in that despite having little effect upon survival at dose levels above 0.2 g/ml PBO, the inci­dence of mutation declined. The authors also reported mutation of K-ras codon 12 with an apparent similar dose response. While K-ras mutation has been reported in human tumors at various sites (Almoguera et al., 1988; Bos et al., 1987) no such association has been observed in tumors of the rodent liver, the apparent target site of PBO (Maronpot et al., 1995). Butler et al. (1996) reported that PBO did not induce unscheduled DNA synthesis in rat hepatocytes. Moreover, Beamand et al. (1996) showed a similar negative response to PBO in cultured human hepatocytes. In vivo studies have also failed to demonstrate convin­ cing genotoxic effects of PBO. A dominant lethal assay in ICR/Ha Swiss mice using both single and multiple doses of PBO given either by intraperitoneal injection or by gavage resulted in toxicity and death of the male mice. Although there was some evidence of reduced reproductive efficiency and an increase in early fetal death, the results were not consistent and the authors concluded the study was equivocal (Epstein et al., 1972). Other studies have been reported only briefly as abstracts and have stated that no chromosomal aberrations or sister chromatid exchanges were produced in either rat or mouse bone marrow (Ivett and Tice, 1983; Kawachi et al., 1980).

99.3.7  Mode of Action Considerations   for Oncogenicity The chronic toxicity/oncogenicity studies discussed in Section 99.3.4 indicate that liver is a target for both oncogenic and nononcogenic changes in both the mouse and the rat. In addition, one study showed an apparent hyperplastic response in the thyroid (Butler et al., 1998).

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

Many nongenotoxic compounds have been shown to induce tumors in rodents and consideration of their relevance to humans has been the focus of intense effort over the past two decades (Ames et al., 1993; Butler, 1996; Cohen and Ellwein, 1990; Cohen, et al., 2003, 2004; Grasso and Hinton, 1991; Grasso et al., 1991; Holsapple et al., 2006; Loury et al., 1987; Wilson et al., 1992). Much progress has been made in understanding the mode of action by which liver by nongenotoxic inducers of hepatic xenobiotic metabolism, such as sodium phenobarbital (NaPB) and PBO, may produce liver tumors (Grasso and Hinton, 1991; Grasso et al., 1991; Holsapple et al., 2006). As described above, it appears that two distinct responses are occurring during the lifetime dietary administration of PBO. Clear evidence of carcinogenicity is observed at very high, toxic doses leading to liver tumors in rats and mice (Takahashi et al., 1994a,b, 1997). Studies show that benign liver tumors may be produced in the mouse (but not the rat) at the MTD, likely by a mode of action (MOA) different from what is occurring at the excessively high doses used in the studies by Takahashi et al. (1994a,b, 1997). It is important to note that other studies show the lack of hepatocarcinogenicity of PBO in the mouse and rat at MTD and lower doses. The PBO-induced mouse liver tumors are likely due to a MOA similar to that described for phenobarbital and related compounds (Holsapple et al., 2006). Phenobarbital is known to induce liver tumors in rodents (Whysner et al., 1996). A diagnostic effect of phenobarbital in rodent liver is the induction of CYP forms, particularly of CYP2B forms (Okey, 1990; Nims and Lubet, 1996; Parkinson, 2001). The pleiotropic effects of phenobarbital in rodent liver including increased liver weight, liver hypertrophy, increased replicative DNA synthesis, induction of CYP forms, and liver tumor promotion are mediated through various nuclear receptors, particularly the CAR (Dickins, 2004; Honkakoski et al., 2000; Tien and Negishi, 2006; Ueda et al., 2000). Indeed, in mice lacking CAR, phenobarbital does not induce CYP2B forms, does not increase liver weight, and does not stimulate replicative DNA synthesis (Wei et al., 2000; Yamamoto et al., 2004). Moreover, while phenobarbital promoted liver tumors in normal mice given a single dose of diethylnitrosamine, no hepatocellular adenomas or carcinomas were observed in mice lacking CAR (Yamamoto et al., 2004). Several studies have shown that PBO is an inducer of hepatic xenobiotic metabolism in the mouse and rat (Fennell et al., 1980; Goldstein et al., 1973; Lake et al., 1973; Phillips et al., 1997; Wagstaff and Short, 1971). Phillips et al. (1997) characterized the induction of enzyme activ­ ities in both the mouse and rat and compared it to the classic inducer, phenobarbital. Four groups of 16 male F-344 rats were fed PBO in the diet at 100, 550, 1050, or 1850 mg/kg body weight/day. Animals were treated for either 7 or 42 days and were sacrificed, and liver studies

2141

were performed. Even the low-dose group (100 mg/kg body weight/day PBO) showed increased relative liver weights and microsomal protein content (42 days treatment), increased cytochrome P450 levels (7 days treatment), increased GGT levels (42 days treatment), and increases in certain MFO enzyme activities. PBO appeared to be a mixed-type enzyme inducer in the rat in that it induced hepatic cytochrome P450 isoenzymes in the CYP1A, CYP2B, and CYP3A subfamilies. Recent work by Watanabe et al. (1998) in the rat is in agreement with these findings of Phillips. These workers also noted weak induction of the CYP4A isozyme. The induction pattern was similar to that they observed for NaPB, with the exception that NaPB did not induce CYP1A. A NOEL of 0.05% PBO in the diet (approximately 50 mg/kg body weight) over 4 weeks was reported for this enzyme induction. PBO treatment of the mouse by Phillips and co-workers also resulted in a dose-related induction of cytochrome P450 content and ethylmorphine demethylase activity (CYP3A). Taken with other studies that have shown induction of CYP1A and CYP2B isoenzymes in mouse liver (Adams et al., 1993; Fennell et al., 1980), PBO appears to be able to induce CYP1A, CYP2B, and CYP3A iso­ enzymes in CD-1 mouse liver as well. Aside from the enzyme induction discussed above, certain nongenotoxic rodent liver carcinogens produce either a transient or a sustained stimulation of cell replication (Goldsworthy et al., 1991). Both PBO and NaPB produced a stimulation of cell replication after 7 but not 42 days of treatment in the mouse (Phillips et al., 1997). Like NaPB, PBO also increased relative liver weight in CD-1 mice and produced liver hypertrophy, although a difference in the lobular distribution of this effect was noted. Generally, the effects of PBO on relative liver weight, liver morphology, replicative DNA synthesis, and xenobiotic metabolism occurred at the 100 and 300 mg/kg body weight/day dose levels, the same doses where eosinophilic nodules were observed in a 2-year study by Butler et al. (1998). In addition, the eosinophilic nodules produced by PBO in mouse liver (Butler, 1996) appear similar to those formed by NaPB (Evans et al., 1992). Many studies have demonstrated that PBO can induce CYP forms and CYP-dependent enzyme activities in mice and rats (Adams et al., 1993a,b; Goldstein et al., 1973; Hodgson and Philpot, 1974; Hodgson et al., 1995; Fennell et al., 1980; Lake et al., 1973; Mugumura et al., 2006; Phillips et al., 1997; Ryu et al., 1996, 1997; Wagstaff and Short, 1971; Watanabe et al., 1998). The induction of CYP forms has been evaluated by the measurement of enzyme activities, mRNA levels, and CYP apoprotein levels. Studies in the mouse have demonstrated that PBO can induce hepatic CYP2B forms and also other CYP subfamily forms including CYP1A forms. These studies have been performed in various mouse strains including strains both responsive (e.g., C57BL/6) and nonresponsive

2142

(e.g., DBA/2) to CYP1A induction by aromatic hydrocarbons. Male C57BL/6 N mice were given a single intraperitoneal injection of 338 mg/kg (1 mmol/kg) PBO and killed 24 h later (Adams et al., 1993a). Treatment with PBO significantly induced hepatic microsomal 7-ethoxyresorufin Odeethylase, acetanilide hydroxylase and 7-pentoxyresorufin O-depentylase activities, which were employed as enzymatic markers of CYP1A1, CYP1A2, and CYP2B10, respectively. Western immunoblotting of liver microsomes revealed an induction of CYP1A1, CYP1A2, and CYP2B10 apoproteins, whereas while PBO induced CYP1A2 and CYP2B10 mRNA levels, only a weak induction of CYP1A1 mRNA was observed. In another study, male C57BL/6 mice were given single intraperitoneal doses of 52, 104, 156, 208, and 416 mg/kg PBO and killed 24 h later (Adams et al., 1993b). Treatment with PBO induced hepatic microsomal 7-ethoxyresorufin O-deethylase, acetanilide hydroxylase and 7-pentoxyresorufin O-depentylase activities, CYP1A1, CYP1A2, and CYP2B10 mRNA levels, and CYP1A1, CYP1A2, and CYP2B10 apoprotein levels by Western immunoblotting. The effect of PBO on enzyme activities and mRNA and apoprotein levels was generally dose-dependent. Male C57BL/6 and DBA/2 mice were given single intraperitoneal 254 and 508 mg/kg (0.75 and 1.5 mmol/kg) doses of PBO and killed 24 h later. While CYP1A2 and CYP2B10 apoprotein levels were induced by PBO in both mouse strains, CYP1A1 apoprotein levels were only induced in liver microsomes from C57BL/6 mice (Adams et al., 1993b). Male C57BL/6 and DBA/2 mice were given a single intraperitoneal injection of 400 mg/kg PBO and killed 24 h later (Ryu et al., 1997). PBO induced CYP1A1 mRNA in both mouse strains, the effect being more marked in C57BL/6 mice. Treatment with PBO also induced CYP1A2 mRNA levels and immunocytochemical studies demonstrated a centrilobular induction of CYP1A1/2 in liver sections from both mouse strains. Additional studies in aryl hydrocarbon receptor (AhR) knockout mice have confirmed that AhR-independent pathway(s) are involved in induction of CYP1A2 by PBO (Ryu et al., 1996). The treatment of male ICR mice with 6000 ppm PBO in the diet for 1, 4, and 8 weeks significantly elevated CYP1A1, CYP2A5, CYP2B9, and CYP2B10 mRNA levels (Muguruma et al., 2006). The treatment of male C57BL/10 and DBA/2 mice with 500 mg/kg/day PBO by intraperitoneal injection induced some hepatic microsomal CYP-dependent enzyme activities (Fennell et al., 1980), whereas in another study the treatment of male CD-1 mice with 100 and 300 mg/kg/day PBO by dietary administration for 42 days resulted in increased microsomal total CYP content (Phillips et al., 1997). Male F344 rats were given 0 (control), 100, 550, 1050, and 1850 mg/kg/day PBO by dietary administration for periods of 7 and 42 days (Phillips et al., 1997). With the exception of the lowest PBO dose level (where

Hayes’ Handbook of Pesticide Toxicology

the changes were not always statistically significant), significant increases in microsomal total CYP content and 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin Odepentylase and ethylmorphine N-demethylase activities were observed at both time points. The three enzyme activities are markers for induction of CYP1A, CYP2B, and CYP3A/other CYP forms, respectively. In another study, male F344 rats were treated with diets containing 0.05, 0.2, and 2% PBO for 1 and 4 weeks (Watanabe et al., 1998). Significant increases in microsomal total CYP content, some CYP-dependent enzyme activities, glutathione S-transferase activities, and one UDP glucuronosyltransferase activity were observed at the highest PBO dietary level after 1 and 4 weeks, with some increases being observed in rats given 0.2% PBO, whereas no significant effects were observed in rats given 0.05% PBO. Western immunoblotting studies were conducted with liver microsomes from control rats and rats given 2% PBO for 4 weeks. Treatment with PBO resulted in an induction of CYP1A1, CYP2B1/2, and CYP3A apoprotein lev­els, with a weak induction of CYP4A apoprotein levels also being reported (Watanabe et al., 1998). Finally, immunocytochemical procedures were employed to demonstrate a centrilobular induction of CYP1A1/2 and CYP2B1/2 in liver sections from rats given 2% PBO for 4 weeks. The treatment of male Wistar rats with 1200 mg/kg/day PBO by oral administration for up to 7 days was reported to upregulate CYP genes in the 1, 2, 3, and 4 families (EllingerZiegelbauer et al., 2005). With respect to the rat, Watanabe et al. (1998) demonstrated the production of centrilobular hypertrophy in the rat liver following 4 weeks of dosing with 2% PBO in the diet. The degree of response was similar to that observed after 4 weeks dosing with 0.1% NaPB. Further evidence of the similarity between the action of PBO and NaPB comes from a report by Okamiya et al. (1998) showing an increase in proliferating cell nuclear antigen in rats fed 0.2% PBO in the diet for 4 weeks. They also demonstrated a decrease in the gap junction protein connexin 32 (Cx32) at the highest dose tested (2.0%) after dosing for 1, 2, but not 4 weeks. Phillips et al. (1997) reported that high doses of PBO (1850 mg/kg body weight/day) given to rats caused a significant reduction of body weight gain and of food consumption throughout the 42 days of dosing. Morphological examination of liver showed individual cell necrosis in rats dosed with 1050 and 1850 mg PBO/kg body weight/ day. While the severity of the individual cell necrosis was similar in rats given 1050 and 1850 mg PBO/kg body weight/day, the incidence was greater (seven of eight animals examined) in rats given 1850 mg/kg body weight/day. Like NaPB, the increase in relative liver weight in PBOtreated animals was associated with hypertrophy, although a difference in the lobular distribution of this effect was noted. Replicative DNA synthesis was stimulated by 550

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

and 1050 mg PBO/kg body weight/day and 0.05% NaPB after 7 days of administration, most likely due to transient mitogenesis typical of enzyme inducers. In contrast, the stimulation of cell replication observed after 42 days treatment by 1050 mg/kg body weight/day is more likely to be associated with the onset of a regenerative hyperplasia. The mitogenic and hypertrophic effects of PBO were observed at doses (e.g., 550 mg/kg body weight/day) lower than those required to produce individual cell necrosis, whereas a high incidence of necrosis was only observed in rats given 1850 mg/kg body weight/day for 42 days. Chronic treatment (i.e., 42 days) with PBO at high-dose levels such as that in an oncogenesis study could result in a sustained stimulation of replicative DNA synthesis and an increased likelihood of oncogenesis. It is important to note that Takahashi et al. (1994b) reported no increase in liver tumors following 2 years of dosing at the lowest study dose of 547 mg/kg body weight/day, a dose calculated by the authors to be about 18,000 times the allowable daily intake (ADI) for humans. In contrast, higher doses of PBO were both toxic to the rat liver and produced tumors. Both of these mechanisms are threshold phenomena and suggest that at doses likely to be encountered by humans, PBO poses essentially no oncogenic risk.

99.3.8  Human Studies Wintersteiger and Juan (1991) investigated the absorption of combination pyrethrins and PBO sprays across the skin of six healthy subjects in Austria. The spray was applied over a wide area of the back with a total dose of approximately 3.3 mg pyrethrum extract and 13.2 mg PBO being administered. No untoward clinical signs were noted. Cutaneous absorption of PBO was shown to be extremely low, with plasma samples containing no more than 10 ng PBO/ml. Wester et al. (1994) investigated the percutaneous absorption of both PBO and pyrethrin compounds across the skin of the ventral forearm in six volunteers. Based on the recovery of radioactivity in the urine, it was calculated that 2.1%  0.6% of the dose of PBO was absorbed through the skin. Not surprisingly, higher levels of absorption of PBO were achieved when the compound was applied to the skin of the scalp (8.3%). There was no evidence of any local or systemic toxicity of PBO when used as a topical agent in humans. The most definitive study on the absorption and excretion of PBO was a mass balance study reported by Selim (1995) using 14C PBO from two different formulations following dermal application to healthy volunteers. The first preparation applied was a 4% (w/w) solution of PBO in an aqueous formulation. This product was applied to four healthy human volunteers. The second preparation tested was a 3% (w/w) solution of PBO in isopropyl alcohol. In the former case, the mean amount of PBO applied was 3.8 mg per volunteer (approximately 39.9 Ci of radioactivity per volunteer), while the average exposure was 3.0 mg

2143

PBO per volunteer (approximately 40 Ci of radioactivity per volunteer) in the case of the isopropyl alcohol solution. Results from this study show that there was a similar dermal absorption pattern for both formulations. The principal route of excretion of absorbed radioactivity was via urine. Fecal samples from volunteers contained negligible levels of radioactivity. Dermally applied PBO was rapidly excreted from the volunteers. The majority of the applied radioactivity remained at the application site, with less than 3% of the applied dose being absorbed during the 8-h test period. Radioactivity did not accumulate in the skin. The only study to examine the ability of PBO to inhibit xenobiotic metabolism in humans was reported by Conney et al. (1972). Using the rate of antipyrine metabolism as a gauge of P450 activity, two healthy men (weighing 87.4 kg and 82.6 kg, respectively) were given capsules containing increasing amounts (5, 10, 20, and 50 mg) of PBO at consecutive intervals of approximately 1 week. No signs of toxicity were seen. Clinical chemical analysis of blood and urine samples taken at 4, 8, and 24 h after ingestion of the capsules did not reveal any adverse effects. In a subsequent study, eight men were given PBO as a single dose of 0.71 mg/kg body weight. A control group received a placebo. Two hours later, both the treated and control groups received a 250-mg oral dose of antipyrine. Antipyrine was analyzed in blood samples taken at intervals over the next 31 h. PBO had no effect on the rate of clearance of antipyrine. Although no systematic epidemiology studies have been conducted on PBO-exposed individuals, no evidence sug­gests that PBO has resulted in any significant adverse effects to human health. Occupational health data from a PBO manufacturing site in Italy, consisting of routine health checks, were collected on potentially exposed workers from 1974 to 1994. Sixty workers were examined, 11 of whom were employed in the manufacture of PBO for periods of 15–26 years. Workers received x-rays and spirometric evaluations every 3 years and annual clinical chemistry analysis. No adverse clinical signs or symptoms related to PBO were found (Endura, 1996). Similarly, at a manufacturing site in Scotland, where PBO was made from 1962 to 1990, “no cases of toxic symptoms or adverse effects attributable to PBO manufacture” were noted in production workers at the plant. Moreover, no adverse effects were reported in operations involved in the handling and use of PBO at a site in England (JMPR, 1993; Pitman Moore, 1990; Wellcome, 1991). The only clinical report referring to PBO exposure is the case of two sisters who gave birth, within 2 weeks of each other, to children who each had coarction of the aorta (Hall et al., 1975). Both mothers had been on a camping trip at 2 months gestation where they used “large amounts” of insect repellents and insecticides containing, among other chemicals, PBO, pyrethrins, DEET, and the organophosphorus insecticide DDVP. No cause-and-effect relationship was established.

2144

Hayes’ Handbook of Pesticide Toxicology

There have been no reported suicide attempts by humans with PBO. Based on its acute toxicity to experimental animals, a probable oral lethal dose for humans is estimated to be 5–15 g/kg body weight (i.e., approximately 300–900 g PBO for a 60-kg human).

The authors conclude that in view of their widespread use, the data indicate that PY/PBO products can be used with a relatively low risk of adverse effects. Moreover, the data suggest that they are not likely to cause reactions in people with asthma or allergies.

99.3.9  Human Experience

99.4  Pharmacodynamics

As mentioned earlier, although PBO is not used alone, it has been used extensively in combination with the pyrethrins. Insight into the human experience with pyrethrins and PBO-containing products comes from an investigation of human incidents reported through the American Association of Poison Control Centers (AAPCC) Toxic Exposure Surveillance System (TESS) associated with regulated insecticides containing pyrethrins and PBO (PY/PBO) from 2001 to 2003 (Osimitz et al., 2009). Sales of household insecticides containing PY/PBO during the period of this review (2001–2003) were estimated to be over 40,000,000 individual product units. Assuming that a unit is used on four occasions, this suggests that over 160,000,000 uses of PY/PBO products occurred during the review period. Given that many households have more than one person in them, it is reasonable to assume that perhaps over 300,000,000 people may have had some exposure to PY/PBO products over the 3 years of the study period. Although tabulation of incidents included all pyrethrins/ PBO exposures regardless of effect, the focus was placed on those incidents reporting potential dermal and respiratory effects. Because the TESS system does not contain a data field designated to capture the caller’s allergy or preexisting medical condition information, such as allergy status of hypersensitivities, a surrogate measure of association was selected. The treatment options documented in each case record were used to identify treatments typically associated with more intense respiratory or dermal effects. Records were searched for indication of bronchodilator use as part of the treatment, suggesting that simple fresh air or ventilation was insufficient to alleviate signs and symptoms likely associated with shortness of breath in patients with preexisting airway disease. With respect to dermal effects, steroid treatment as suggestive of a need for treatment involving more than simple irrigation or washing of the affected area was reviewed. The limitations notwithstanding, the analysis showed that: Despite extensive use, incidents with reports of moderate or major adverse effects were relatively rare (717 moderate and 23 major outcomes out of 17,873 calls). l Following label-directed use of the products, adverse dermal or respiratory reactions were very rare; (dermal: 17 moderate, 1 major; respiratory: 18 moderate, 0 major). l The data suggest that asthmatics and people sensitive to ragweed (Ambrosia artisifolia) are not unusually sensitive to PY/PBO. l

Yamamoto (1973) suggests that the primary function of synergists such as PBO when formulated with pyrethrins or pyrethroids is to provide an alternative substrate for the MFO enzyme system, which would normally metabolize such insecticides. Inhibition of MFO-mediated oxidation of the transmethyl groups and the alcohol moiety on the pyrethrin molecule appear to be the most important functions of PBO. Inhibition of ester hydrolysis may also contribute to the effectiveness of PBO as a synergist. As a known alternative substrate for the liver microsomal enzyme system, PBO will inhibit the metabolism of many xenobiotics including drugs and pesticides. Brown (1970) reported that the detoxification of certain drugs such as pentobarbital, zoxazolamine, antipyrine, and benzopyrene were inhibited by PBO, presumably due to the inhibition of their microsomal oxidation. Conney et al. (1972) investigated the inhibition of antipyrine metabolism in rats and mice. Both species were treated intraperitoneally (i.p.) with a single dose of PBO, followed by a further i.p. injection of antipyrine (200 mg/kg body weight) 1 h later. A marked species difference was noted in the response; the no-observable-effect level (NOEL) for inhib­ ition of antipyrine metabolism in the mouse was 0.5–1.0 mg PBO/kg body weight, whereas the NOEL for the rat was 100 mg PBO/kg body weight. The effects of PBO on the metabolism of benzopyrene in Sprague-Dawley rats were studied by Falk et al. (1965). PBO was administered by the oral, intraperitoneal (i.p.), or intravenous (i.v.) routes at various times before the i.v. injection of labeled benzopyrene. The level of radioactivity was then measured in bile at frequent intervals up to 4 h. This author demonstrated marked inhibition of benzopyrene metabolism when PBO was administered i.v. at 262 mg/kg body weight, some 5 min to 16 h before the administration of benzopyrene. However, this effect is much reduced at 121 mg/kg body weight. Virtually no effect was seen at 25 h post-dosing. This implies that single large doses of PBO are quickly metabolized by rats. Administration of PBO by the oral and i.p. routes resulted in a greatly reduced effect when compared with the i.v. route. A second similar study carried out by Conney et al. (1972), where the effects of i.p. administration of PBO on the metabolism of benzopyrene were investigated, showed less sensitive results when rats of lighter weight were used (approximately 180 g versus 400 g in Falk’s study). It was postulated by Brown (1970) that the extra fat in the

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

animals in Falk’s study could possibly act as a reservoir of PBO and lead to a longer duration of action. It would appear from these studies in rats that 250 mg of PBO per kg body weight is the minimum oral dose required to give any significant effect on benzopyrene metabolism. Whereas a single dose of PBO will generally inhibit the metabolism of pentobarbital, repeated PBO doses will generally induce the metabolism of phenobarbital and other xenobiotics. Brown (1970) reports an experiment in rats where a single i.p. dose of PBO (333–1000 mg/kg body weight) increased the sleeping time of the animals following administration of pentobarbital. However, the i.p. administration of eight injections of 50 mg PBO/kg body weight, each at 12-h intervals, followed by the injection of pentobarbital some 18 h later, caused a reduction in sleeping time in rats. The administration of 50 mg PBO per kg body weight i.p. to rats (Anders, 1968) and mice (Graham et al., 1970) prior to treatment with hexobarbital approximately doubled the sleeping time of both species. CD-1 mice given a single i.p. dose of 600 mg PBO per kg body weight were found to have suffered less hepatotoxicity when treated with acetaminophen (600 mg/kg body weight, p.o.) at either 2 h prior to or 1 h following PBO administration. This reduced hepatotoxicity was measured via GSH and sorbitol dehydrogenase levels, as well as subsequent histo­ pathology of the liver. Since the hepatic MFO system metabolizes acetaminophen to a toxic metabolite, the decreased toxicity seen in this experiment is likely due to inhibition of such oxidase enzymes by PBO (Brady et al., 1988). Many other studies have been undertaken relevant to the pharmacodynamics of PBO and are reported elsewhere (Skrinijaric-Spoljar et al., 1971; Conney et al., 1972; Goldstein et al., 1973). More recent work is discussed in Section 99.3.5 of this chapter.

99.5  Exposure assessment 99.5.1  Dietary Exposure The first formal discussion of human exposure to PBO took place at a joint FAO/WHO Codex Alimentarius in 1987. FAO/WHO estimated that the average daily human diet (1.4 kg) might contain as much as 1 ppm of PBO, corresponding to a daily dose of 1.4 mg. This is likely an over­estimation as cooking may destroy up to 90% of PBO present. In a refinement of this exposure estimate, Crampton (1994) calculated that the daily exposure of adults to PBO residues in food was 0.0037 mg/kg body weight/day. During the 1990s, the PBO Task Force (PBTF), a consortium of PBO producers and marketers, developed extensive exposure data for PBO for use in dietary exposure assessments. In addition to a series of crop residue studies on various crops representing 12 crop groups, studies were conducted where residue levels and transfer factors were obtained following application of PBO to livestock.

2145

The USPEA used these data, when appropriate, along with two models, the Lifeline model (Version 2.0) and the Dietary Exposure Evaluation Model software with the Food Commodity Intake Database (DEEM-FCID, Version 2.03) to estimate acute and chronic dietary exposure to PBO in the United States. Both models incorporated food consumption data from the USDA’s Continuing Surveys of Food Intakes by Individuals (CSFII) from 1994 to 1996 and 1998. USDA Pesticide Data Program (PDP) data were used for commodities that have pre-harvest registered uses and for cereal grain crops that have a stored grain use. All other commodities were assigned residues from either a simulated warehouse space spray experiment or a simulated restaurant experiment. Residue data from studies conducted following dermal treatment of livestock were used as input values for meat, milk, poultry, and eggs. The residues of concern for plants include the parent PBO and a twofold factor to account for metabolites, unless field trial data for metabolites on related crops indicated a lower factor was appropriate. Percent crop treated data were used for all commodities for which percent crop treated data are available. Where no percent crop treated data were available, the dietary analyses assumed 100% crop-treated. Based on the methodology discussed above, the acute dietary exposure at the 99.9th percentile was estimated as 0.2865 mg/kg/day and 0.3467 mg/kg/day for adults (50  years) and females (13–49 years), respectively. Chronic dietary exposures were estimated at 0.0066 mg/kg/day and 0.0070 mg/kg/day for adults (50  years) and females (13–49 years), respectively. The dietary exposure estimates likely significantly overestimate real exposure. Important refinements include obtaining additional residue data and additional percent crop-treated information

99.5.2  Non-Dietary Exposure FAO/WHO estimated the exposure from aerosol consumer products (from ingestion and inhalation) to be approximately 0.63 mg/day (FAO/WHO, 1987). Given the extensive non-dietary use of PBO, the NonDietary Exposure Task Force (NDETF) was established in 1996 to develop a long-term program to conduct a series of transferability studies to better understand the phenomenon of human exposure to pesticides used in the home. Most of the studies were conducted with formulations of pyrethrins/PBO and permethrin/PBO and focused on the use of fogger and aerosol products indoors. Carpet and vinyl were selected as the flooring surfaces of interest because of their different physical and chemical properties and because they represent a significant amount of the floor coverings used in homes in North America. While the focus of the NDETF efforts was on total release foggers, a study was also conducted to determine both dispersion (air levels) and deposition (on flooring) of pyrethrin/PBO resulting from the use of a hand held aerosol spray can.

2146

Potential direct exposure of the user was also measured. Air sampling from the breathing zone of the applicator and analysis of residues on cotton gloves was performed. The Food Quality Protection Act (FQPA) requires that the U.S. EPA consider available information concerning the cumulative effects of a particular pesticide and other substances that have a common mechanism of toxicity. The U.S. EPA has confirmed that PBO does not appear to produce a toxic metabolite produced by other chemicals nor does it share a common mechanism of action with others. Using the data developed by the NDETF as well as other sources, they estimated exposures for the following scenarios (U.S. EPA, 2005a): Handler Liquid spray formulation by low-pressure handwand for indoor surface spray application Liquid spray formulation by low-pressure handwand for indoor crack and crevice treatment Liquid spray formulation by hose-end sprayer for lawn and garden application l Postapplication Inhalation exposure from airborne application of mosquito adulticide Inhalation exposure from application of mosquito adulticide from truck mounted sprayer Toddler incidental ingestion of residue from treated turf grass via hand-to-mouth activities Toddler incidental ingestion of residue via object-tomouth activity while on treated grass Toddler incidental ingestion of soil from treated area Toddler incidental ingestion of residues deposited on carpet via hand-to-mouth activities after use of total release foggers Toddler incidental ingestion of residues deposited on vinyl flooring via hand-to-mouth activities after use of total release foggers Toddler incidental ingestion of residues on pets via hand-to-mouth activities after pet treatment Inhalation exposure to aerosol spray during and after space spray application The details of these assessments are beyond the scope of this chapter, but estimated exposures ranged from 0.000079 mg/kg/day (handler exposure while using hoseend sprayer, short- and intermediate-term exposure) to 0.28 mg/kg/day (short-term child ingestion of residues on pets via hand-to-mouth activities after pet treatment). l

99.6  Risk characterization 99.6.1  Cancer The Joint FAO/WHO Meeting on Pesticide Residues (JMPR) evaluated the toxicology of PBO in 1965, 1966, 1972, 1992,

Hayes’ Handbook of Pesticide Toxicology

and, most recently in 1995. They concluded that, at doses up to internationally accepted standards for a Maximum Tolerated Dose, PBO is not oncogenic in the mouse or rat. Based on this assessment, no risk assessment is warranted (JMPR, 1995). The U.S. EPA evaluated the weight of evidence relating to the potential oncogenicity of PBO and classified it as a Group C-Possible Human Carcinogen (U.S. EPA, 1995a). This was based on the increases in hepatocellular tumors in both male and female mice (adenomas, carcinomas, and combined adenomas and carcinomas in the males and adenomas only in the females). However, because of the generally low concern for mutagenicity, and the minor significance of other tumors observed in the rat studies, rather than recommend the Q*1 linearized multistage model for risk characterization based on oncogenicity, the U.S. EPA endorsed the use of a Reference Dose (RfD) and Margin of Exposure (MOE) approaches using nononcogenic endpoints, such as body weight changes. Although, as discussed previously, the data suggest that the MOA resulting in PBO-induced liver tumors in mice is not plausible in humans, quantitative differences exist between the doses of PBO that produce liver tumors in mice and human exposure to PBO. In the PBO mouse bioassay showing liver tumors male mice were given 100 and 300 mg/kg/day PBO and female mice were given 300 mg/ kg/day PBO (Butler et al., 1996). The lowest dose level at which tumors were seen was thus 100 mg/kg/day. The proposed Acceptable Daily Intake (ADI) of PBO established by the Joint FAO/WHO Meeting on Pesticide Residues in 1995 is 0.2 mg/kg/day (JMPR, 1995). The U.S. EPA has estimated the Population Adjusted Dose (PAD) for acute human PBO exposure to be 6.3 mg/kg/day and chronic human exposure to be 0.155 mg/kg/day (U.S. EPA, 2005b) (The PAD is the Acute reference dose (RfD) or the Chronic RfD modified by the FQPA Safety Factor. The safety factor for both the acute and chronic dietary assessments of PBO is 1X.) The lowest dose level for liver tumors in mice (100 mg/kg/day) is 645 times greater than the “permitted” level for human chronic exposures to PBO. Actual expos­ ure is much less: the U.S. EPA estimates chronic dietary exposure to PBO for the general population to be approximately 0.008 mg/kg/day. Thus, the lowest dose level that was associated with liver tumors in mice (100 mg/kg/day) is approximately 12,500 times greater than the estimated level for human chronic exposures to PBO (U.S. EPA, 2005b).

99.6.2  Non-Cancer Effects As mentioned above, the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) evaluated the toxicology of PBO in 1965, 1966, 1972, 1992, and, most recently in 1995. Based on the NOAEL of 600 ppm (16 mg/kg body weight/day) in the most sensitive toxicology study (1 year feeding study in dogs, Goldenthal, 1993b), they established

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

an Allowable Daily Intake (ADI) for humans of 0.2 mg PBO/kg body weight (JMPR, 1995). Following a careful evaluation of the hazard data, the U.S. EPA, as part of the reregistration of PBO, identified a series of toxicological endpoints used to estimate risk using the Margin of Exposure (MOE) approach. They concluded that no quantitative dermal assessment was required because no systemic effects were observed at the limit dose (1000 mg/kg/day) in the 21-day dermal absorption study in rabbits. The EPA selected benchmark studies for acute inhalation exposure, short-, intermediate- and long-term inhalation exposure, and short- and intermediate-term incidental oral exposure. MOE levels of concern ranged from 100 to 1000, meaning that MOEs below these values may trigger risk management measures. The results of the nondietary risk assessments indicate that all residential ex­posure scenarios result in MOEs greater than the applicable MOE levels of concern and thus are of no safety concern.

99.6.2.1  Dietary Risk Dietary risk assessment incorporates both exposure to and toxicity of a given pesticide. Both the FAO/WHO estimate of dietary exposure of 1.4 mg/person/day (0.02 mg/ kg/day) and the Crampton (1994) estimate of daily expos­ ure of adults to PBO residues in food of 0.0037 mg/kg body weight/day are much less than the ADI of 0.2 mg/kg/day. The U.S. EPA expresses dietary risk as a percentage of a level of concern (referred to as the population adjusted dose, or PAD). The PAD is the dose predicted to result in no unreasonable adverse health effects to humans, including sensitive subgroups. Thus, it is a function of the reference dose (RfD) and the appropriate FQPA safety factor. Exposures less than 100% of the PAD are below EPA’s level of concern. For acute dietary exposure, the EPA used the no-observed-adverse-effect-level (NOAEL) of 630 mg/ kg/day from a rat oral developmental toxicology study (Tanaka et al.,1995). The aPAD was calculated as 630 mg/ kg/day ÷ 100 safety factor  6.3 mg/kg/day. Exposure estimates for the U.S. population and the highest exposure group (children 1–2 years old) show exposures at 6% and 20% of the aPAD, respectively. The chronic dietary endpoint came from a dog study with a NOAEL of 15.5 mg/kg/day (Goldenthal, 1993b). The exposure estimate for the U.S. population is 5% of the cPAD and 12% for the highest exposed subpopulation, children (1–2 years of age).

99.6.2.2  Non-dietary Risk The FAO/WHO estimate of 0.63 mg/day exposure from aerosol consumer products corresponding to a daily dose of 0.009 mg/kg body weight for a 70-kg adult and 0.042 mg/ kg body weight for a 15-kg child is well below the ADI

2147

for humans of 0.2 mg/kg body weight for PBO set by the JMPR (1995). The more recent and more highly refined assessment by the U.S. EPA considered three product user (handler) and nine postapplication residential exposure scenarios. The results of the residential exposure assessment indicated that all residential exposure scenarios assessed showed MOEs greater than the applicable target MOEs (ranging from 600 for a child playing with a pet treated with spray to more than 1,000,000 for incidental ingestion risks to toddlers reentering treated lawns). All residential scenarios result in exposures below the level of concern.

References Adams, N. H., Levi, P. E., and Hodgson, E. (1993a). Differences in induction of three P450 isozymes by piperonyl butoxide, sesamex. Safrole and Isosafrole. Pesticide Biochem. Physiol. 46, 15–26. Adams, N. H., Levi, P. E., and Hodgson, E. (1993b). Regulation of cytochrome P-450 isozymes by methylenedioxyphenyl compounds. Chem.-Biol. Interact. 86, 255–274. Almoguera, C., Shibatan, D., Forrester, K., Martin, J., Arnheim, N., and Peruduo, M. (1988). Most human carcinogenesis of the exocrine pancreas contain mutant c-K-ras genes. Cell 53, 549–554. Amacher, D. E., and Zelljadt, I. (1983). The morphological transformation of Syrian hamster embryo cells by chemicals repeatedly nonmutagenic to Salmonella typhimurium. Carcinogenesis 4, 291–295. Ames, B. N., Shigenaga, M. K., and Gold, L. S. (1993). DNA lesions, inducible DNA repair, and cell division: Three key factors in mutagenesis and carcinogenesis. Environ. Health Perspect. 101(Suppl. 5), 35–44. Anders, M. W. (1968). Inhibition of microsomal drug metabolism by methylenedioxybenzenes. Biochem. Pharmacol. 17, 2367–2370. Ashwood-Smith, M. J., Trevino, J., and Ring, R. (1972). Mutagenicity of dichlorvos. Nature (London) 240, 418. Beamand, J. A., Price, R. J., Phillips, J. C., Butler, W. H., Glynne Jones, G. D., Osimitz, T. G., Gabriel, K. L., Preiss, F. J., and Lake, B. G. (1996). Lack of effect of PBO on unscheduled DNA synthesis in precision-cut human liver slices. Mutation Res. 371, 273–282. Bond, H., Mauger, K., and DeFeo, J. J. (1973). The oral toxicity of pyrethrum, alone and combined with synergists. Pyreth. Post 12, 59. Bos, J. L., Fearson, E. R., Hamilton, S. R., Verlaan-de Uries, M., Van Boom, J. H., van der Eb, A. J., and Vogelstein, B. (1987). Prevalence of ras gene mutations in human colorectal cancers. Nature 327, 293–297. Brady, J. T., Montelius, D. A., Beierschmitt, W. P., Wyand, D. S., Khairalla, E. A., and Cohen, S. D. (1988). Effect of PBO post-treatment on acetaminophen hepatotoxicity. Biochem. Pharmacol. 37, 2097–2099. Brown, N. C. (1970). Report A28/52, Research and Development, The Wellcome Foundation, UK. Butler, W. H. (1996). A review of the hepatic tumours related to mixedfunction oxidase induction in the mouse. Toxicol. Pathol. 24, 484–492. Butler, W. H., Gabriel, K. L., Preiss, F. J., and Osimitz, T. G. (1996). Lack of genotoxicity of PBO. Mutation Res. 371, 249–258. Butler, W. H., Gabriel, K. L., Osimitz, T. G., and Preiss, F. J. (1998). Oncogenicity studies of PBO in rats and mice. Hum. Exper. Toxicol 17, 323–330. Cardy, R. H., Renne, R. A., Warner, J. W., and Cypher, R. L. (1979). Carcinogenesis bioassay of technical-grade PBO in F344 rats. J. Nat. Canc. Inst. 62, 569–578.

2148

Casida, J. E. (1970). MFO involvement in the biochemistry of insecticide synergists. J. Agric. Food. Chem. 18, 753–772. Chun, J. S., and Neeper-Bradley, T. L. (1991). Developmental Toxicity Evaluation of PBO Administered by Gavage to CD® (SpragueDawley) Rats. Unpublished Rep. 54–586 from Bushy Run Research Center, undertaken for the PBO Task Force, Washington, DC. Chun, J. S., and Neeper-Bradley, T. L. (1992). Developmental Toxicity Dose Range-Finding Study of PBO Administered by Gavage to CD® (Sprague-Dawley) Rats. Unpublished Rep. 54-578 from Bushy Run Research Center, undertaken for the PBO Task Force, Washington, DC. Chun, J. S., and Wagner, C. L. (1993). 90 Day Dose Range-Finding Study with PBO in Mice. Study No. 91 N0052, Bushy Run Research Centre, Union Carbide Chemicals and Plastics Company Inc., Export, PA, undertaken for the PBO Task Force. Cohen, S. M., and Ellwein, L. B. (1990). Cell proliferation in carcinogenesis. Science 249, 1007–1011. Cohen, S. M., Meek, M. E., Klaunig, J. E., Patton, D. E., and FennerCrisp, P. (2003). The human relevance of information on carcinogenic mode of action: Overview. Crit. Rev. Toxicol. 33, 581–589. Cohen, S. M., Klaunig, J., Meek, M. E., Hill, R. N., Pastoor, T., LehmanMcKeeman, L., Bucher, J., Longfellow, D. G., Seed, J., Dellarco, V., Fenner-Crisp, P., and Patton, D. (2004). Evaluating the human relevance of chemically induced animal tumors. Toxicol. Sci. 78, 181–186. Conney, A. H., Chang, R., Levin, W. M., Garbut, A., Munro-Faure, A. D., Peck, A. W., and Bye, A. (1972). Effects of PBO on drug metabolism in rodents and man. Arch. Environ. Health 24, 97–106. Crampton, P. L. (1994). PBO human intake estimates. (Europe) G. R. 94–0009. Dahl, A. R., and Brenzinski, D. A. (1985). Inhibition of rabbit nasal and hepatic cytochrome P-450-dependent hexamethylphosphoramide (HMPA)-N-demethylase by methylenedioxyphenyl compounds. Biochem. Pharmacol. 34, 631–636. Delaforge, M., Ioannides, C., and Parke, D. V. (1985). Ligand-complex formation between cytochrome P-450 and P-448 and methylenedioxyphenyl compounds. Xenobiotica 15, 333–342. Di Blasi, G. (1998). A review of the chemistry of PBO. In “PBO—The Insecticide Synergist” (D. Glynne Jones, ed.). Academic Press, San Diego. Dickins, M. (2004). Induction of cytochromes P450. Curr. Top. Med. Chem. 4, 1745–1766. Ellinger-Ziegelbauer, H., Stuart, B., Wahle, B., Bomann, W., and Ahr, H. J. (2005). Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver. Mutat. Res. 575, 61–84. Endura (1996). “Personal correspondence,” Endura, SpA, Bologna, Italy. Endura (2009). “Personal correspondence,” Endura, SpA, Bologna, Italy. Epstein, S. S., Arnold, E., Andrea, J., Bass, W., and Bishop, Y. (1972). Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol. 233, 288–325. Evans, J. G., Collins, M. A., Lake, B. G., and Butler, W. H. (1992). The histology and development of hepatic nodules and carcinoma in C3H/ He and C57BL/6 mice following chronic phenobarbitone administration. Toxicol. Pathol. 20, 585–594. Falk, H. L., Thompson, S. J., and Kotin, P. (1965). Carcinogenic potential of pesticides. Arch. Environ. Health 10, 847–858. Fennell, T. R., Sweatman, B. C., and Bridges, J. W. (1980). The induction of hepatic cytochrome P-450 in C57BL/10 and DBA/2 mice by isosafrole and PBO. A comparative study with other inducing agents. Chem.-Biol. Interact 31, 189–201. Franklin, M. R. (1976). Methylenedioxyphenyl insecticide synergists as potential human health hazards. Environ. Health Perspec. 14, 29–37.

Hayes’ Handbook of Pesticide Toxicology

Fujitani, T., Ando, H., Fujitani, K., Ikeda, T., Kojima, A., Kubo, Y., Ogata, A., Oishi, S., Takahashi, H., Takahashi, O., and Yoneyama, M. (1992). Subacute toxicity of PBO in F344 rats. Toxicology 72(3), 291–293. Fujitani, T., Tanaka, T., Hashimoto, Y., and Yoneyama, M. (1993). Subacute toxicity of PBO in ICR mice. Toxicology 83, 93–100. Gabriel, D. (1991a). Acute Dermal Toxicity, Single Level—Rabbits. Unpublished Rep. 91-7317A from Biosearch Inc., Philadelphia, PA, undertaken for the PBO Task Force, Washington, DC. Gabriel, D. (1991b). Acute Oral Toxicity, LD50—Rats. Unpublished Rep. 91-7317A from Biosearch Inc., Philadelphia, PA, undertaken for the PBO Task Force, Washington, DC. Galloway, S. M., Armstrong, M. J., Reubec, C., Coleman, S., Brown, B., Cannen, C., Bloom, A. D., Nakamura, F., Alimed, F. M., Duk, S., Rimpo, J., Margolin, B. H., Resnick, M. A., Anderson, B., and Zeiger, E. (1987). Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Environ. Mol. Mutagen. 10(Suppl. 10), 1–175. Gerber, G. J., and O’Shaughnessy, D. (1986). Comparison Of The Behavioral Effects Of Neurotoxic And Systemically-Toxic Agents: How Discriminatory Are Behavioral Tests Of Neurotoxicity?. Neurobehav. Toxicol. Teratol. 8, 703–710. Goldenthal, E. I. (1992). 21-day Repeated Dose Dermal Toxicity Study with PBO in Rabbits. Unpublished Rep. 542-007 from International Research and Development Corp., Mattawan, Michigan, undertaken of the PBO Task Force, Washington, DC. Goldenthal, E. I. (1993a). Evaluation of PBO in an Eight-Week Toxicity Study in Dogs. Unpublished Rep. 542-004 from International Research and Development Corp., Mattawan, Michigan, undertaken for the PBO Task Force, Washington, DC. Goldenthal, E. I. (1993b). Evaluation of PBO in a One Year Chronic Dietary Toxicity Study in Dogs. Unpublished Rep. 542–005 from International Research and Development Corp., Mattawan, Michigan, undertaken for the PBO Task Force, Washington, DC. Goldstein, J. A., Hickman, P., and Kimbrough, R. D. (1973). Effects of purified and technical PBO on drug-metabolizing enzymes and ultrastructure of rat liver. Toxicol. Appl. Pharmacol. 26, 444–458. Goldsworthy, T. L., Morgan, K. T., Popp, J. A., and Butterworth, B. E. (1991). Guidelines for measuring chemically-induced cell proliferation in specific rodent target organs. In “Chemically Induced Cell Proliferation: Implications for Risk Assessment” (B. E. Butterworth, T. D. Slaga, W. Farland, and M. McClain, eds.), pp. 253–284. WileyLiss, New York. Gopinath, C., Prentice, D. E., and Lewis, D. J. (1987). “Atlas of Experimental Toxicological Pathology,” MTP Press, Lancaster, England. Graham, P. W., Hellyer, R. O., and Ryan, A. J. (1970). The kinetics of inhibition of drug metabolism in vitro by some naturally occurring compounds. Biochem. Pharmacol. 19, 769–775. Grasso, P., and Hinton, R. H. (1991). Evidence for and possible mech­ anisms of nongenotoxic carcinogenesis in rodent liver. Chem.-Biol. Interact 248, 271–290. Grasso, P., Sharratt, M., and Cohen, A. J. (1991). Role of persistent, nongenotoxic tissue damage in rodent cancer and relevance to humans. Annu. Rev. Pharmacol. Toxicol. 31, 253–287. Hall, J. G., McLaughlin, J. F., and Stamm, S. (1975). Coarctation of the aorta in male cousins with similar maternal environmental exposure to insect repellent and insecticides. Pediatrics 55, 425–427. Hodgson, E., and Philpot, R. M. (1974). Interaction of methylenedioxyphenyl [1,3-benzodioxole] compounds with enzymes and their effects on mammals. Drug Metab. Rev. 3, 231–301. Hodgson, E., Philpot, R. M., Backer, R. C., and Mailman, R. B. (1973). Effect of synergists on drug metabolism. Drug Metab. Dispos. 1, 391–401.

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

Hodgson, E., Ryu, D-Y., Adams, N., and Levi, P. E. (1995). Biphasic responses in synergistic interactions. Toxicol. 105, 211–216. Hoffman, G. M. (1991). An Acute Inhalation Toxicity Study of PBO in the Rat. Unpublished Rep. 91-8330 from Bio/dynamics, East Millstone, NJ, undertaken for the PBO Task Force, Washington, DC. Holsapple, M. P., Pitot, H. C., Cohen, S. H., Boobis, A. R., Klaunig, J. E., Pastoor, T., Dellarco, V. L., and Dragan, Y. P. (2006). Mode of action in relevance of rodent liver tumors to human cancer risk. Toxicol. Sci. 89, 51–56. Honkakoski, P., and Negishi, M. (2000). Regulation of cytochrome P450 (CYP) genes by nuclear receptors. Biochem. J. 347, 321–337. Hunter, B., Bridges, J. L., and Prentice, D. G. (1977). “Long Term Feeding of Pyrethrins and PBO to Rats,” Conducted by Huntingdon Research Center for the Pyrethrum Board of Kenya. Innes, J., Ulland, B., Valerio, M., Petrucelli, L., Fishbein, L., Hart, E., and Pallotta, A. (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. Nat. Canc. Inst 42, 1101–1114. Ishidate, M., Sofuri, T., Yoshikawa, K., Hayashi, M., Nohmi, T., Sawada, M., and Matsnoka, A. (1984). Primary mutagenicity screening of food additives currently used in Japan. Food. Chem. Toxicol. 22, 623–636. Ishidate, M., Harnois, M. C., and Sofuni, T. (1988). A comparative analysis of data on the clastogenicity of 951 substances tested in mammalian cell cultures. Mutation. Res. 195, 151–213. Ivett, J. L., and Tice, R. R. (1983). Response of C57BL/6 and DBA/2 mouse strains to benzene-induced genotoxicity after inhibition of cytochrome P-450. Environ. Mutagen. 5, 450–451. JMPR (1993). PBO. In “Pesticide Residues in Food—1992,” Toxicology Evaluations, pp. 317–334. World Health Organization, Geneva. JMPR (1995). “PBO.” A monograph prepared by the Joint FAO/WHO Meeting on Pesticide Residues, Geneva. Kakko, I., Toimela, T., and Tahti, H. (2000). Piperonyl butoxide potentiates the synaptosome ATPase inhibiting effect of pyrethrin. Chemosphere 40, 301–305. Kawachi, T., Komatsu, T., Kada, T., Ishidate, M., Sasaki, M., Sugiyama, T., and Tazima, Y. (1980). Results of recent studies on the relevance of various short-term screening tests in Japan.. Appl. Methods Oncol 3, 253–276. Kennedy, G. L., Smith, S. H., Kinoshita, F. K., Keplinger, M. L., and Calandra, J. C. (1977). Teratogenic evaluation of PBO in the rat. Food Cosmet. Toxicol. 15, 337–339. Khera, K. S., Whalen, C., Angers, G., and Trivett, G. (1979). Assessment of the teratogenic potential of PBO, biphenyl and phosalone in the rat. Toxicol. Appl. Pharmacol. 47, 353–358. Khot, A. C., Bingham, G., Field, L. M., and Moores, G. D. (2008). A novel assay reveals the blockade of esterases by piperonyl butoxide. Pest Manag Sci 64, 1139–1142. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., Dittenber, D. A., Kalnins, R. P., Frauson, L. E., Park, C. N., Barnard, S. D., Hummel, R. A., and Humiston, C. G. (1978). Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-­tetrachlorodibenzo-pdioxin in rats. Toxicol. Appl. Pharmacol. 46(2), 279–303. Lake, B. G., Hopkins, R., Chakraborty, J., Bridges, J. W., and Parke, D. V. (1973). The influence of some hepatic enzyme inducers and inhibitors on extrahepatic drug metabolism. Drug Metab. Dispos. 1, 342–349. Leng, J. M., Schwartz, C. A., and Schardein, J. L. (1986). Teratology Study in Rabbits. Unpublished Rep. 542-002 from International Research and Development Corp., Mattawan, Michigan, submitted to WHO by Endura SA, Bologna, Italy, for the PBO Task Force, Washington, DC.

2149

Lorber, M. (1972). Hematotoxicity of synergized pyrethrum insecticides and related chemicals in intact and totally and subtotally splenectomized dogs. Acta Hepato-Gastroenterol 19, 66–78. Loury, D. J., Goldsworthy, T. L., and Butterworth, B. E. (1987). The value of measuring cell replication as a predictive index of tissuespecific tumorigenic potential (Banbury Report 25). In “Nongenotoxic Mechanisms in Carcinogenesis” (B. E. Butterworth and T. J. Slaga, eds.), pp. 119–136. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY. Maekawa, A., Onodera, H., Furuta, K., Tanigawa, H., Ogiu, T., and Hayashi, T. (1985). Lack of evidence of carcinogenicity of technicalgrade PBO in F344 rats: Selective induction of ileocaecal ulcers. Fd. Chem. Toxic. 23(7), 675–682. Muguruma, M., Nishimura, J., Jin, M., Kashida, Y., Moto, M., Takahashi, M., Yokouchi, Y., and Mitsumori, K. (2006). Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatcarcinogenesis in mice. Toxicology 228, 178–187. Maronpot, R. R., Fox, T., Malarkey, D. E., and Goldsworthy, T. L. (1995). Mutations in the ras proto-oncogene: clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101, 125–156. McClain, R. M. (1989). The significance of hepatic microsomal enzyme induction and altered thyroid function in rats: Implications for thyroid gland neoplasia. Toxicol. Path 17, 294–303. McClean, E. M., Driver, H., and McDanell, R. (1990). Nutrition and enzyme inducers in liver tumor promotion in human and rat. Bull. Cancer 77(5), 505–508. McGregor, D. B., Brown, A., Cattanach, P., Edwards, I., McBride, D., Riach, C., and Caspary, W. J. (1988). Response of the L5178Y TK  /TK mouse lymphoma cell forward mutation assay III, 72 coded chemicals. Environ. Mol. Mutagenesis 12, 85–154. Modeweg-Hausen, L., Lalande, M., Bier, C., Lossos, G., Osborne, B. E. (1984). A Dietary Dose Range-Finding Study of PBO in the Albino Rat. Unpublished Rep. 81820B from Bio-Research Laboratories, Edgewater, MD, undertaken for the PBO Task Force, Washington, DC. Moore, P. (1990). Manufacturers of PBO in Scotland, 1962–1990. Moriye, M., Ohta, T., Watanabe, K., Miyazawa, T., Kato, K., and Sturaso, Y. (1983). Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutation Res. 116, 185–216. Murray, M., and Reidy, G. F. (1989). In vitro formation of an inhibitory complex between an isosafrole metabolite and rat hepatic cytochrome P-450. Drug Metab. Dispos. 17, 449–454. Mutai, M., Tatematsu, M., Aoki, T., Wada, S., and Ito, N. (1990). Modulatory interaction between initial clofibrate treatment and subsequent administration of 2-acetylaminofluorene or sodium phenobarbital on glutathione S-transferase positive lesion development. Cancer Lett. 49(2), 127–132. Nims, R. W., and Lubet, R. A. (1996). The CYP2B subfamily. In “Cytochromes P450 : Metabolic and Toxicological Aspects” (C. Ioannides ed.), pp. 135–160. CRC Press, Boca Raton. Newton, P. E. (1992). A subchronic (3-month) inhalation toxicity study of PBO in the rat via whole-body exposures. Unpublished Rep. 91-8333 from Bio/Dynamics, East Millstone, NJ, undertaken for the PBO Task Force, Washington, DC. Okamiya, H., Onodera, H., Ito, S., Imazawa, T., Yasuhara, K., and Takahashi, M. (1998). Mechanistic study on liver tumor promoting effects of PBO in rats. Arch. Toxicol. 72(11), 744–750. Okey, A. B. (1990). Enzyme induction in the cytochrome P-450 system. Pharmacol. Ther. 45, 241–298.

2150

Organization for Economic Cooperation and Development. (2001). OECD Test No, 426: Developmental Neurotoxicity Study. Osimitz, T. G., Sommers, N., and Kingston, R. (2009). Human exposure to insecticide products containing pyrethrins and piperonyl butoxide (2001–2003). Food Chem. Toxicol. E-publication 2009 Mar 21, ahead of print. Parkinson, A. (2001). Biotransformation of xenobiotics. In “Casarett and Doull’s Toxicology: The Basic Science of Poisons” (C. D. Klaassen, ed.) 6th ed, pp. 133–224. McGraw Hill, New York. Phillips, J. C., Cunningham, M. E., Price, R. J., Osimitz, T. G., Gabriel, K. L., Preiss, F. J., Butler, W. H., and Lake, B. G. (1997). Effect of PBO on cell replication and xenobiotic metabolism in rat liver. Fund. Appl. Toxicol. 38, 64–74. Philpot, R. M., and Hodgson, E. (1971). A cytochrome P-450-PBO spectrum similar to that produced by ethyl isocyanide. Life Sci. II 10, 503–512. Philpot, R. M., and Hodgson, E. (1972a). The effect of PBO concentration on the formation of cytochrome P-450 difference spectra in hepatic microsomes for mice. Mol. Pharmacol. 8, 204–214. Philpot, R. M., and Hodgson, E. (1972b). The production and modification of cytochrome P-450 difference spectra by in vivo administration of methylenedioxyphenyl compounds. Chem. Biol. Interact. 4, 185–194. PBO Task Force (2009). “Estimate of Use of PBO in the US for 1997,” Personal communications. Consumer Specialty Products Association, Washington, DC. Robinson, K., Pinsonneault, L., and Procter, B. G. (1986). A Two-Generation (Two-Litter) Reproduction Study of PBO Administered in the Diet to the Rat. Unpublished Rep. 81689 from Bio-Research Laboratories Ltd., Montreal, Canada, undertaken for the PBO Task Force, Washington, DC. Romanelli, P. (1991a). Guinea Pig Dermal Sensitization—Modified Buehler Method. Unpublished Rep. 91-7317A from Biosearch Inc., Philadelphia, undertaken for the PBO Task Force, Washington, DC. Romanelli, P. (1991b). Primary Eye Irritation—Rabbits. Unpublished Rep. 91–7317A from Biosearch Inc., Philadelphia, undertaken for the PBO Task Force, Washington, DC. Romanelli, P. (1991c). Primary Skin Irritation—Rabbits. Unpublished Rep. 91-7317A from Biosearch Inc., Philadelphia, undertaken for the PBO Task Force, Washington, DC. Ryu, D-Y., Levi, P. E., Fernandez-Salguero, F. J., and Hodgson, E. (1996). Piperonyl butoxide and acenaphthylene induce cytochrome P450 1A2 and 1B1 mRNA in aromatichydrocarbon-responsive receptor knockout mouse liver. Mol. Pharmacol. 50, 443–446. Ryu, D-Y., Levi, P. E., and Hodgson, E. (1997). Regulation of hepatic CYP1A isozymes by piperonyl butoxide and acenaphthylene in the mouse. Chem.-Biol. Interact 105, 53–63. Sarles, M. P., Dove, W. E., and Moore, D. H. (1949). Acute toxicity and irritation tests on animals with the new insecticide, PBO. Am. J. Trop. Med 29, 151–166. Sarles, M. P., and Vandergrift, W. B. (1952). Chronic toxicity and related studies on animals with the insecticide and pyrethrum synergist PBO. Am. J. Trop. Med. 1, 862–883. Selim, S. (1995). Absorption, Excretion, and Mass Balance of 14C PBO from Two Different Formulations after Dermal Application to Healthy Volunteers. Unpublished Rep. PO594006 from Biological Test Center, Irvine, CA, undertaken for the PBO Task Force, Washington, DC. Skrinijaric-Spoljar, M., Matthew, 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.

Hayes’ Handbook of Pesticide Toxicology

Squire, R. A., and Levitt, M. H. (1975). Report of a workshop on classification of specific hepatocellular lesions in rats. Cancer Res. 35, 3214–3223. Suzuki, H., and Suzuki, N. (1995). PBO mutagenicity in human RSa cells. Mutation. Res. 344, 27–30. Takahashi, O., Oishi, T., Fujitani, T., Tanaka, T., and Yoneyama, M. (1994a). Chronic toxicity studies of PBO in F344 rats: Induction of hepatocellular carcinoma. Fundam. Appl. Toxicol. 22, 293–303. Takahashi, O., Oishi, T., Fujitani, T., Tanaka, T., and Yoneyama, M. (1994b). PBO induces hepatocellular carcinoma in CD1 mice. Arch. Toxicol. 68, 467–469. Takahashi, O., Oishi, S., Fujitani, T., Tanaka, T., and Yoneyama, M. (1997). Chronic toxicity studies of piperonyl butoxide in CD-1 mice: induction of hepatocellular carcinoma. Toxicology 124, 95–103. Tanaka, T. (1992). Effects of PBO on F1 generation mice. Toxicol. Lett. 60, 83–90. Tanaka, T. (1993). Behavioral effects of PBO in male mice. Toxicol. Lett. 69, 155–161. Tanaka, T. (2003). Reproductive and neurobehavioral effects of PBO administered to mice in the diet. Food Additives and Contam. 20, 207–214. Tanaka, T., Takahashi, O., and Oishi, S. (1992). Reproductive and neuro­ behavioral effects in three-generation toxicity study of PBO administered to mice. Food Chem. Toxicol. 30, 1015–1019. Tanaka, T., Fujitani, T., Takahashi, O., and Oishi, S. (1994). Developmental toxicity evaluation of PBO in CD-1 mice. Toxicol. Lett. 71, 123–129. Tanaka, T., Fujitani, T., Takahashi, O., Oishi, S., and Yoneyama, M. (1995). Developmental toxicity evaluation of PBO in CD rats. Toxicol. Ind. Hlth. 11, 175–184. Tatematsu, M., Ozaki, K., Mutai, M., Shichino, Y., Furihata, C., and Ito, N. (1990). Enhancing effects of various gastric carcinogens on development of pepsinogen-altered pyloric glands in rats. Carcinogenesis 11(11), 1975–1978. Tayama, S. (1996). Cytogenic effects of PBO and safrole in CHO-K1 cells. Mutation Res. 368, 249–260. Tien, E. S., and Negishi, M. (2006). Nuclear receptors CAR and PXR in the regulation of hepatic metabolism. Xenobiotica 36, 1152–1163. Tyl, R. W., Crofton, K., Moretto, A., Moser, V., Sheets, L. P., and Sobotka, T. J. (2008). Identification and Interpretation of Developmental Neurotoxicity Effects: A Report from the ILSI Research Foundation/ Risk Science Institute Working Group on Neurodevelopmental Endpoints. Neurotoxicol. Teratol. 30, 39–381. Ueda, A., Hamadeh, H. K., Webb, H. K., Yamamoto, Y., Sueyoshi, T., Afshari, C. A., Lehmann, J. M., and Negishi, M. (2000). Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to Phenobarbital. Mol. Pharmacol. 61, 1–6. Ulbrich, B., and Palmer, A. K. (1996). Neurobehavioral aspects of developmental toxicity testing. Environ. Health Perspect. 104(Suppl. 2), 407–412. U.S. Environmental Protection Agency (U.S. EPA) (1995a). “List of Chemicals Evaluated for Carcinogenic Potential,” Office of Pesticide Programs, Washington, DC. U.S. Environmental Protection Agency (U.S. EPA) (1995b). “Pesticide Handlers Exposure Database (PHED) Evaluation Guidance,” PHED VI. 1. Occupational and Residential Exposure Branch, Office of Pesticide Programs, Washington, DC. U.S. Environmental Protection Agency (U.S. EPA) (1998). “Health Effects Test Guidelines-OPPTS 870.6300 Developmental Neurotoxicity Study,” Office of Pesticide Programs, Washington, DC.

Chapter | 99  The Safety Assessment of Piperonyl Butoxide

U.S. Environmental Protection Agency (1998). “Assessment of Thyroid Follicular Cell Tumors,” EPA/630/R-97/002. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2005a). “Occupational and Resi­dential Exposure Assessment and Recommendations for the Reregis­ tration Eligibility Decision (RED) For Piperonyl Butoxide,” Office Of Prevention, Pesticides And Toxic Substances, Washington, DC. U.S. Environmental Protection Agency (2005b). “Piperonyl Butoxide HED Risk Assessment for Reregistration Eligibility Document (RED),” Office Of Prevention, Pesticides And Toxic Substances, Washington, DC. U.S. National Cancer Institute (1979). “Bioassay of PBO for Possible Carcinogenicity,” DHEW Publ. 79–1375. U.S. Department of Health, Education and Welfare, Bethesda, MD. Van Miller, J. P., Lalich, J. J., and Allen, J. R. (1977). Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8-tetrachlorodibenzo-rhodioxin. Chemosphere 6(9), 537–544. Varsano, R., Rabinowitch, H. D., and Rubin, B. (1992). Mode of action of piperonyl butoxide as herbicide synergist of atrazine and terbutryn in maize. Pestic. Biochem. Physiol. 44, 174–182. Wachs, H. (1947). Synergistic insecticides. Science 105, 397–401. Wagstaff, D. J., and Short, C. R. (1971). Induction of hepatic microsomal hydroxylating enzymes by technical PBO and some of its analogues. Toxicol. Appl. Pharmacol. 19, 54–61. Watanabe, T., Manabe, S., Ohashi, Y., Okamiya, H., Onodera, H., and Mitsumori, K. (1998). Comparison of the induction profile of hepatic drug-metabolizing enzymes between PBO and phenobarbital in rats. J. Toxicol. Pathol. 11, 1–10.

2151

Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., and Moore, D. D. (2000). The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407, 920–923. Wellcome Environmental Health Unit (1991). Users of PBO. Wester, R. C., Bucks, D. A. W., and Maibach, H. I. (1994). Human in vivo percutaneous absorption of pyrethrin and PBO. Food Chem. Toxicol. 32, 51–53. White, T. J., Goodman, D., Shulgin, A. T., Castagnoli, N. Jr., Lee, R., and Petrakis, N. L. (1977). Mutagenic activity of some centrally active aromatic amines in Salmonella typhimurium. Mutat. Res. 56, 199–202. Whysner, J., Ross, P. M., and Williams, G. M. (1996). Phenobarbital mechanistic data and risk assessment: Enzyme induction, enhanced cell proliferation, and tumor promotion. Pharmacol. Ther. 71, 153–191. Wilson, D. M., Goldsworthy, T. L., Popp, J. A., and Butterworth, B. E. (1992). Evaluation of genotoxicity, pathological lesions, and cell proliferation in livers of rats and mice treated with furan. Environ. Mol. Mutagen. 19, 209–222. Wintersteiger, R., and Juan, H. (1991). “Resorption Study of Tyrason after Dermal Application (Study Performed on Healthy Subjects),” 01/91, p. 1. J.S.W.—Experimental Research, Studie Analytik. Yamamoto, I. (1973). Mode of action of synergists in enhancing the insecticidal activity of pyrethrum and pyrethroids. In Pyrethrum—Natural Insecticide, pp. 195–210. Academic Press, London/New York. Yamamoto, Y., Moore, R., Goldsworthy, T. L., Negishi, M., and Maronpot, R. R. (2004). The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by Phenobarbital in mice. Cancer Res. 64, 7197–7200.