Mammalian toxicology overview and human risk assessment for sulfosulfuron

Regulatory Toxicology and Pharmacology Regulatory Toxicology and Pharmacology 39 (2004) 310–324 www.elsevier.com/locate/yrtph

Mammalian toxicology overview and human risk assessment for sulfosulfuron Charles E. Healy,* William F. Heydens, and Mark W. Naylor1 Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63141, USA Received 18 August 2003 Available online 1 April 2004

Abstract Sulfosulfuron is a low-use rate sulfonylurea herbicide. A review of the toxicity database for sulfosulfuron indicates that the molecule has a low order of acute toxicity. It is not genotoxic and is not a reproductive, developmental, or nervous system toxicant. There were no indications of endocrine disruption in any study performed with the molecule. The only findings considered to be an adverse effect in mammalian laboratory animals following prolonged subchronic or chronic exposure to sulfosulfuron were isolated to the urinary tract. These findings occurred in conjunction with findings of urolith formation following high-level chemical dosing, resulting in epithelial hyperplasia that, in a few cases, progressed to tumor formation. Mode-of-action information supports the conclusion that these tumors result from a non-genotoxic, threshold-based process that is well established and widely considered to be not relevant to humans. Based on its short-term, infrequent application pattern and very low use rate and crop residues, aggregate and cumulative risk assessments indicate that sulfosulfuron has substantial margins of exposure and does not represent a significant risk to human health. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Sulfosulfuron; Toxicity; Urolithiasis; Human risk assessment

1. Introduction Sulfosulfuron (N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-2-ethylsulfonyl-imidazo[1,2a]pyridine-sulfonamide) (a.k.a. MON 37500) (Fig. 1) is a unique sulfonylurea herbicide that helps farmers protect wheat from yield loss by selectively controlling brome, quack/ couch, wild oats, gallium, and apera. The product is safe to wheat and provides excellent application flexibility since it is applied pre-emergence or early post-emergence to the target weed. Significantly, sulfosulfuron is active at very low use rates that result in reduced crop chemical use and improved container disposal considerations (registered maximum use rates in wheat are 40 g/ha). Sulfosulfuron is compatible with current agronomic systems and has a

*

Corresponding author. Fax: 1-314-694-4028. E-mail address: [email protected] (C.E. Healy). 1 Retired. 0273-2300/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2004.02.005

technical fit in all major wheat production areas including Australia, Europe, North America, India, parts of Africa, the Confederation of Independent States (CIS), and (pending) Saudi Arabia. A comprehensive toxicology data package on sulfosulfuron was developed to evaluate the safety of the material and to meet worldwide crop registration requirements. With one exception (Arnold et al., 2001), the information from the studies that comprise this database has not been previously published in the open literature. All of the details and findings of the studies described in this paper have been peer reviewed and extensively evaluated by scientists and others from international regulatory agencies, however, and, in addition, were conducted according to U.S., European, and Japanese regulatory testing guidelines and Good Laboratory Practices. This document provides an overview of the existing toxicology database and also contains an assessment of the potential risks to human health that might result from the use of sulfosulfuron in wheat.

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2.2. Genotoxicity

Fig. 1. The chemical structure of sulfosulfuron.

2. Toxicity studies 2.1. Acute toxicity studies 2.1.1. Active ingredient The potential of sulfosulfuron to produce acute mammalian toxicity or irritation is low. The acute rat oral LD50 and rabbit dermal LD50 are both greater than 5000 mg/kg (Bonnette, 1993a,b). No mortality was observed in rats following a 4 h inhalation exposure at 3.0 mg/L, the highest attainable concentration (Bechtel, 1994a). Slight-to-moderate conjunctival redness and slight-to-moderate swelling were observed in the eyes of each of six rabbits at 1 h after dosing, with discharge in five of six rabbits (Bonnette, 1993c). Iritis was also observed in four of six rabbits at 1 h post-installation. No corneal effects were observed throughout the study. Irritation of the iris cleared within 24 h and conjunctival findings were resolved by 72 h post-installation in five rabbits and by day 7 in the sixth. Essentially no dermal irritation was noted in rabbits following a 4 h dermal exposure (Bonnette, 1993d). No evidence of dermal irritation or sensitization was noted in guinea pigs using a guinea pig maximization test (Blaszcak, 1995a). 2.1.2. Formulation The registered formulation of sulfosulfuron is a water dispersible granule containing approximately 75% sulfosulfuron (percent active ingredient, PAI). In the United States the product is called Maverick while in other world areas it is known as Monitor, Leader, Apyros, Athos, Monza, and Sundance. The acute oral LD50 (Blaszcak, 1995b) and dermal LD50 (Blaszcak, 1995c) for Maverick in rats were greater than 5000 mg/kg. The 4 h rat inhalation LC50 was greater than 2.6 mg/L (Bechtel, 1994b). Slight conjunctival irritation, including redness, discharge, and swelling, was observed when Maverick was placed into the eyes of six New Zealand rabbits (Blaszcak, 1995d). The eyes of all rabbits were clear by 72 h post-exposure (Blaszcak, 1995e). Very slight dermal erythema was observed after 1 h when Maverick was applied to the skin of rabbits. All animals were essentially free of irritation by 72 h post-exposure. Maverick did not induce dermal sensitization in a guinea pig maximization test (Blaszcak, 1995f).

Study results show that sulfosulfuron is not genotoxic in standard bacterial or mammalian assays. In vitro studies conducted include an Ames Salmonella point mutation assay in five test strains (Stegeman et al., 1995a), a CHO/HGPRT mammalian point mutation assay (Stegeman et al., 1995b), and a chromosomal aberration test in human lymphocytes (Murli, 1996). An in vivo mouse micronucleus assay (Stegeman et al., 1995c), with a confirmatory radiolabel assay (Stegeman et al., 1996) that demonstrated that the test material, or a metabolite, reached the bone marrow, was also conducted. All of the above studies were negative for genotoxic effects. A cytogenetics assay (Yoshida, 1996) using Chinese hamster lung cells was conducted to fulfill Japanese manufacturing requirements that require exposure at elevated concentrations up to and including 5000 lg/ml. This concentration of sulfosulfuron in the test media exceeded its solubility limit. In this study, dose-dependent toxicity was observed in the presence of S9 at dose levels exceeding solubility (>1000 lg/ml), with precipitating test material present. Concurrently, increases in chromosome aberrations were observed only in the absence of S9 and at dose levels exceeding solubility (>2000 lg/ml), again with precipitating test material present. Further, historical results at this testing facility indicate that of the 147 substances tested from 1994 to 1997, 68 (46%) induced chromosomal aberrations in this assay. Of those 68, 44 (65%) had a high incidence of chromatid exchanges at both low- and high-dose levels. The induction of toxicity and chromosomal aberrations in vitro only after extended treatment at precipitating dose levels is of doubtful biological relevance. The observation that an in vitro chromosomal aberration test in human lymphocytes and an in vivo mouse micronucleus assay were negative indicate, in this case, that this in vitro CHL assay does not represent any real genotoxic potential for sulfosulfuron. 2.3. Subchronic toxicity studies 2.3.1. Dog oral study In a 90-day study (Naylor and Thake, 1996a), male and female beagle dogs were given sulfosulfuron by gelatin capsule at dose levels of 0, 30, 100, 300, and 1000 mg/kg/day for 5 days/week. There were no adverse effects on body weights, food consumption, or hematological or clinical chemistry parameters. Urinary bladder/urethral stones (calculi) were found at the 1000 mg/ kg/day level in one male dog that was sacrificed in extremis on day 75. Urinary tract gross and microscopic effects were present in females at 300 mg/kg/day or greater, and in males at 1000 mg/kg/day. Thus, 300 mg/ kg/day in male dogs and 100 mg/kg/day in female dogs

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were considered to be the No Observable Adverse Effect Levels (NOAELs) for this study. 2.3.2. Mouse oral study CD-1 mice received sulfosulfuron (99.1% pure) in the diet at concentrations of 0, 100, 1000, 3000, or 7000 ppm (0, 17.9, 162.8, 549.5, and 1143.9 mg/kg/day in males and 0, 32.8, 313.1, 887.3, and 2123.3 mg/kg/day in females) for approximately 90 days (Naylor and Thake, 1995a). There were 10 mice/sex/dose level. There were no differences between treated and control groups relative to body weights, body weight gains, or food consumption. There were no treatment-related effects in hematology evaluations, gross pathology, and microscopic pathology. Clinical chemistry evaluations were also unremarkable with the exception of an equivocal decrease in alkaline phosphatase levels in high-dose females. Based on these observations, the NOAEL for male mice was at least 7000 ppm and at least 3000 ppm in females. 2.3.3. Rat oral study In this study (Naylor and Thake, 1995b), eight groups of 10 male and 20 female Sprague–Dawley rats were administered sulfosulfuron at dietary concentrations of 0, 20, 200, 2000, 6000, or 20,000 ppm. This study was designed to meet all of the requirements for a 90day feeding study in rodents, with an additional reproduction component that served as a rangefinder for a subsequent reproduction study. The additional 10 females/dose level were mated to the males after 10 weeks of exposure and allowed to deliver their litters. The males and the 10 females not used for breeding were sacrificed after 13 weeks; the dams and their pups were sacrificed at lactation day 4. Organ weights and standard hematological and blood chemistry parameters were evaluated in all males and in those females sacrificed at 13 weeks. Reproductive evaluations were made for mating rate, fertility rate, and length of gestation. Dietary exposure to sulfosulfuron resulted in mild but statistically significant body weight/weight gain reductions at the 20,000-ppm level in adult males and in pregnant females during gestation. There were no adverse effects on food consumption, hematological or clinical chemistry parameters, organ weights, or gross or microscopic pathology observations. The No Observed Adverse Effect Level (NOAEL) for subchronic toxicity was considered to be 6000 ppm in males and females (370 and 448 mg/kg/day, respectively). There were no effects observed on reproductive indices or offspring growth/development at any dose level. 2.3.4. Rat dermal study Sulfosulfuron was applied to the unabraded skin of Sprague–Dawley rats for 6 h/day, 5 days/week for four weeks at dose levels of 0, 100, 300, and 1000 mg/kg/day

(Naylor and Thake, 1997a). No treatment-related effects were observed at any dose level. Therefore, the NOAEL for this study was considered to be at least 1000 mg/kg/ day. 2.4. Developmental and reproductive toxicity studies 2.4.1. Rat developmental study In this study (Holson, 1994a), sulfosulfuron was administered to four groups of 25 mated female Sprague–Dawley rats at dose levels of 0, 100, 300, and 1000 mg/kg/day on days 6–15 of gestation. The test animals were observed for mortality and clinical signs of toxicity and were evaluated for body weights, food consumption, gross pathology, and histopathology. Following determination of the gravid uterine weight, the uterus and ovaries were examined and the number of viable and nonviable fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Fetuses were sexed, weighed, and examined for external, visceral, and skeletal malformations or developmental variations. No signs of maternal toxicity or developmental effects were observed at any dose level. Therefore, 1000 mg/kg/day or greater was considered to be the NOAEL for developmental toxicity in rats. 2.4.2. Rabbit developmental study In another developmental toxicity study (Holson, 1994b), sulfosulfuron was administered to four groups of 20 artificially inseminated New Zealand white rabbits at dose levels of 0, 50, 250, and 1000 mg/kg/day on days 7–19 of gestation. The animals were observed for mortality and clinical signs of toxicity and were evaluated for changes in body weights, food consumption, gross pathology, and histopathology. Following determination of the gravid uterine weight, the uterus and ovaries were examined and the number of viable and nonviable fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Fetuses were sexed, weighed, and examined for external, visceral, and skeletal malformations or developmental variations. No signs of maternal toxicity or developmental effects were observed at any dose level. Therefore, 1000 mg/kg/day or greater was considered to be the NOAEL for developmental toxicity in rabbits. 2.4.3. Rat reproduction study In a two-generation reproduction toxicity study (Naylor and Thake, 1996b), sulfosulfuron was administered to 30 male and 30 female Sprague–Dawley rats per dose group at dietary concentrations of 0, 50, 500, 5000, and 20,000 ppm. The animals were observed twice daily for mortality and moribundity and weekly for clinical signs of toxicity. Cohabitation of males with females was performed until there was copulatory evidence, which was designated as gestation day 1.

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The number of live and dead pups was counted on postnatal days 0, 4, 7, 14, and 21. On day 4 of lactation, all litters were culled randomly to eight pups, maintaining equal numbers of each sex where possible. On day 21 of lactation, at least one but no more than two pups/sex/ litter were selected at random from F1a litters to provide 30 pups/sex/group for the next parental generation. Standard gross pathology and histopathology evaluations were performed on both parental and offspring generations. Dietary exposure to sulfosulfuron resulted in mild body weight/weight gain reductions at 20,000 ppm in adult animals. Urinary tract microscopic changes (related to urolith formation) in adults and some offspring were also observed at this dose level. There were no treatment-related adverse effects on any reproductive indices or on pup survival/development at any dose level. The NOAEL was considered to be 5000 ppm (312 mg/kg/day in males and 378 mg/kg/day in females) for subchronic toxicity and at least 20,000 ppm (1313 mg/kg/day in males and 1598 mg/kg/day in females) for reproductive toxicity. 2.5. Neurotoxicity studies 2.5.1. Rat acute oral study In this study (Branch et al., 1997), four groups of 10 rats/sex were given sulfosulfuron at a single oral gavage dose of 0, 125, 500, or 2000 mg/kg body weight in corn oil. Behavioral tests, consisting of the Functional Observational Battery (FOB) and motor activity test, were conducted prior to dosing, at the time of peak effect (7 h), and at 7 and 14 days post-dosing. Animals were sacrificed 15 days after dosing. Five animals/sex were whole-body perfused intracardially with sodium nitrite followed by 4% formaldehyde/1.5% glutaraldehyde while under general anesthesia. Selected central and peripheral nervous tissues and a sample of skeletal muscle from these animals were obtained for histopathological examination. The remaining animals from each group were given a gross necropsy examination approximately three weeks after test material administration. Nervous system tissues and skeletal muscle from perfused high-dose and control animals were examined microscopically. There were differences between treated and control rats in a few parameters, but none occurred in a doserelated pattern and all were considered unrelated to treatment. For instance, diarrhea was observed in several rats on the day of dosing (mostly likely due to the corn oil vehicle), rearing, when calculated as a percentage of pretest values, was statistically significantly increased in 125 mg/kg females at 7 h and 1-week post-dosing (but was not statistically significant when expressed as raw counts), and hindlimb grip strength was statistically significantly reduced in 500 mg/kg

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females at 7-h and 14-day post-dosing but not when calculated as a percentage of pretest values. No behavioral, functional, or neuropathological changes were associated with sulfosulfuron administration at any acute oral dose level tested up to 2000 mg/kg. Under the conditions of this study, sulfosulfuron was not considered to be a neurotoxicant. The NOAEL was considered to be in excess of 2000 mg/kg for a single, acute gavage dose for rats of both sexes. 2.5.2. Rat subchronic oral study In this study (Kaempfe et al., 1997), sulfosulfuron was administered in the feed to four groups of Sprague– Dawley (CD) rats, each group consisting of 10 rats/sex. The groups were exposed to target dietary levels of 0, 200, 2000, or 20,000 ppm of sulfosulfuron for approximately 14 weeks. Clinical observations, body weights, and food consumption were recorded weekly. Neurobehavioral tests were performed on each animal before initiation of exposure and during the 4th, 8th, and 13th weeks of exposure. During the 14th week of exposure, five animals/sex/group were whole-body perfused intracardially with sodium nitrite followed by 4% formaldehyde/1.5% glutaraldehyde, and selected central and peripheral nervous tissues and a sample of skeletal muscle from these animals were collected. The remaining animals from all groups were sacrificed and given a complete necropsy examination at the start of the 15th exposure week. All retained nervous tissues and skeletal muscle from the control and high-dose animals were examined microscopically. No treatment-related signs of neurotoxicity were noted following administration of sulfosulfuron in the diet at concentrations up to and including 20,000 ppm. There were differences between treated and control rats in a few parameters, but none occurred in a dose-related pattern and all were considered unrelated to treatment. A small number of additional clinical abnormalities were noted during the FOB examinations, but none of these were attributed to treatment since they did not occur in a dose-related fashion. Sulfosulfuron produced no signs of neurotoxicity or general toxicity in male and female rats at daily doses up to and including 20,000 ppm (1211 mg/kg/day for males and 1467 mg/kg/ day for females). Under the conditions of this study, sulfosulfuron was not considered to be a neurotoxicant. The NOAEL was considered to be in excess of 20,000 ppm. 2.6. Chronic toxicity and oncogenicity studies 2.6.1. Dog one-year study Sulfosulfuron was administered by gelatin capsule to five male and five female beagle dogs/dose level at doses of 0, 5, 20, 100, and 500 mg/kg/day, 5 days/week (Naylor and Ruecker, 1997a). Urinary crystals were present

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in both sexes at the 500 mg/kg/day dose level at the midstudy sampling period, and a calculus, thickened/irregular mucosa, and red foci were observed in the urinary bladder of one male dog at the same dose level at the conclusion of the study. The mucosal lesions were considered related to the presence of the calculus. Sulfosulfuron was thus considered to have demonstrated chronic toxicity secondary to urinary crystal and calculus formation in male dogs at a dose level of 500 mg/ kg/day. Urinary crystal formation was also observed at 500 mg/kg/day in female dogs but without histopathological findings. The NOAEL for chronic toxicity in this study was 100 mg/kg/day. 2.6.2. Mouse 18-month oncogenicity study Sixty male and 60 female CD-1 mice/dose level were administered sulfosulfuron at dietary concentrations of 0, 30, 700, 3000, and 7000 ppm (Naylor and Thake, 1997b). The survival of animals given test diets was not significantly affected in this study. Treatment-related effects included clinical signs of urine-stained hair, intraabdominal swellings, and gross pathology findings of urinary calculi and related dilation/distension/enlargement of urinary tract organs in males at the 3000 and/or

7000-ppm dose levels. Mean serum levels of blood urea nitrogen (BUN) were statistically significantly increased in males at the 7000-ppm dose level at the end of the study. This effect was considered related to treatment and was probably the result of the gross and microscopic renal effects. Hyperplasia of the transitional epithelium, chronic/ active inflammation, dilatation, and/or calculi was present in the urinary bladder of male mice from the 3000 to 7000 ppm dose groups (Table 1). In addition, at the highest dose level, squamous metaplasia of the urinary bladder mucosal was observed. In the kidneys of male mice from the 7000 ppm level, there was an increase in the incidence of atrophy of the cortex and dilatation of the renal pelvis (Table 1). All these effects were associated with the presence of and/or a direct result of urinary calculi/microcalculi. The chronic toxicity NOAEL was 700 ppm (93.4 mg/kg) for males. There were no effects attributed to the test material at any dose level in female mice. Therefore, the NOAEL was 7000 ppm (1388.2 mg/kg) in females. There was a small increase in benign mesenchymal tumors of the urinary bladder submucosa (a tumor reported to be unique to Swiss-derived mice) in males at the 7000 ppm dose level

Table 1 Incidence of selected lesions in the urinary bladder and kidney of mice exposed to sulfosulfuron for 18 months (Naylor and Thake, 1997b) Group

0 ppm

30 ppm

700 ppm

3000 ppm

7000 ppm

Males Urinary bladder Epithelial hyperplasia Squamous metaplasia Ulceration Chronic active inflammation Dilatation Calculus/microcalculus Mesenchymal tumors

4 0 0 2 5 1 0

1 0 1 3 4 0 0

2 0 0 2 3 0 0

25

1 3 23

8 4 1

41

8
4 41

20

10
5

Kidney Cortex atrophy Pelvis dilatation Tubular adenoma

2 5 0

0 0 0

0 2 0

3 4 0

11
27

1

Femalesa Urinary bladder Epithelial hyperplasia Squamous metaplasia Ulceration Chronic active inflammation Dilatation Calculus/microcalculus Mesenchymal tumors

0 0 0 0 0 0 1

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

1 0 0 1 0 0 1

Kidney Cortex atrophy Pelvis dilatation Tubular adenoma

5 0 0

1 0 0

2 1 0

2 1 0

2 1 1

a

a The number of males was 59/dosing group except 7000 ppm, where the number was 60. For females, the number of animals was 59 at 0 ppm, 10 at 30, 56, and 700 ppm, and 58 at 3000 and 7000 ppm. * p < 0:05. ** p < 0:01.

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(Table 1). The NOAEL for oncogenic effects was 3000 ppm (393.6 mg/kg/day) in males and P7000 ppm (1388.2 mg/kg/day) in females. A renal pathology expert performed further evaluation of the single renal tumors in each of the genders in the high-dose group (Hard, 2000). The reviewer stated that careful examination of the renal tubules failed to show evidence of treatment-related cellular injury or death, increased mitotic activity, or increased nuclear size. Also, there was no incidence of compound-induced hyperplasia. Chronic progressive nephropathy (CPN) was observed with almost 100% occurrence in the four treated groups and controls. The severity of the lesions was comparable among groups. Hard (2000) concluded that there was no pathological effect, including enhancement of CPN, in the mouse kidney that was sulfosulfuron induced except for an increased incidence in renal pelvis dilation with its secondary effects in the parenchyma in males. There was no direct injurious effect on renal tubule epithelium and no induction of atypical tubule hyperplasia, a precursor lesion for renal tubule adenomas. It was concluded that despite the uncommon nature of spontaneously arising renal tubule tumors in laboratory mice, one such occurrence in 60 high-dose male mice and 58 high-dose female mice at 18 months does not constitute a viable hypothesis of cause and effect. Furthermore, even though such tumors are uncommon, they have nonetheless been observed historically in control CD-1 mice (Giknis and Clifford, 2000). Because of this, and in particular, the absence of any precursor tumor stage or intermediate end-point associated with renal tubule carcinogenesis, the single adenomas occurring in isolation in the high-dose male and female groups are considered incidental findings unrelated to treatment with sulfosulfuron. The significance of this finding is discussed in a later section of this paper. 2.6.3. Rat two-year combined chronic toxicity/oncogenicity study Five groups of 60 male and 60 female Sprague– Dawley rats were administered sulfosulfuron at dietary concentrations of 0, 50, 500, 5000, and 20,000 ppm for up to 22 months (Naylor and Ruecker, 1997b). Surviving males at the 20,000 ppm level were sacrificed on day 259 due to excessive mortality in the group up to that point in time. All survivors in the other dose groups were sacrificed at month 22 due to poor long-term survival across all sulfosulfuron-dosing groups and controls. (Prior approvals to terminate the 20,000 ppm level males and to conclude the study at 22 months were obtained from U.S. EPA and the study was considered acceptable for regulatory purposes.) There were no treatment-related clinical signs of toxicity (i.e., diarrhea, behavioral symptoms of toxicity, etc.). From day 51 to day 485, there were statistically significant decreases in

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mean body weight and/or cumulative body weight gain at the 20,000 ppm dietary level in females that were attributed to treatment. Decreased albumin and albumin/ globulin ratio in 5000 ppm level males at the end of testing may have been related to treatment, although no explanatory correlative histological findings were noted. Abnormal crystals detected in the urine of 5000 ppm level males and 5000 and 20,000 ppm level females and increased numbers of urinary red blood cells and/or occult blood at the highest dose level of both sexes were considered related to test material administration. Urinary tract calculi were observed in some females at these same dietary levels and three of these calculi were chemically analyzed (Dubelman, 2000). The calculi were completely dissolved in an acetonitrile/water solvent system with the aid of sonication and then analyzed for sulfosulfuron content by HPLC with UV detection. The results of the analyses showed that the calculi consisted almost entirely of sulfosulfuron (i.e., 94.1, 101.6, and 70.9% of the stones consisted of parent compound), indicating that sulfosulfuron present in rat urine precipitates in essentially an unmetabolized state. Gross necropsy examination findings considered to be treatment related occurred in females at the two highest dose levels include: renal calculi, renal pelvic dilatation, and granular/pitted/irregular kidney surface; urinary bladder calculi; ureteral calculi, ureter dilatation, and abnormal contents; and thyroid/parathyroid enlargement. Microscopic effects occurred at 5000 and 20,000 ppm in kidney, urinary bladder, and ureter of females and included: renal pelvic calculus/microcalculus, pelvic dilatation, pelvic epithelial hyperplasia, squamous metaplasia, pyelonephritis, and increased mean severity of nephropathy; urinary bladder mucosal hyperplasia, acute inflammation and polymorph exudate; and ureteral calculus/microcalculus, dilatation, inflammation, mucosal hyperplasia, squamous metaplasia, and erosion. Microscopic calculi/microcalculi in the urethra or kidney of two 5000 ppm level males were also considered to be direct treatment effects. Increased incidences of mineralization in several tissues, diffuse parathyroid hyperplasia, femoral and sternal fibrous osteodystrophy, and pyloric stomach erosions in females at the two highest dose levels were considered to be secondary to nephropathy. Urinary bladder transitional cell carcinomas and urinary bladder transitional cell papillomas in each of two 5000 ppm level females were probably treatment related, most likely as secondary reactive effects to urinary system calculi. This response has been shown to occur in the presence of calculi and with chronic exposure, although the reason(s) for the lack of tumors at the higher doses (20,000 ppm) and lack of effect in males was not apparent. In summary, sulfosulfuron produced chronic toxicity at dietary concentrations of 5000 and 20,000 ppm in

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both sexes in rats. The NOAEL for chronic toxicity in this study was 500 ppm (24.4 and 30.4 mg/kg/day in males and females, respectively). A urinary bladder transitional cell carcinoma and a urinary bladder transitional cell papilloma in two females at the 5000 ppm level were probably secondary reactive effects to urinary system calculi irritation. The NOAEL for oncogenic effects in this study was 5000 ppm (244.2 mg/kg/day) for males and 500 ppm (30.4 mg/kg/day) for females. 2.6.4. Mode of action study—bladder oncogenicity In a study designed to determine if the rat urinary bladder carcinogenesis was indeed the result of calculus formation, groups of male Sprague–Dawley rats were fed sulfosulfuron at doses of 0, 50, 500, 5000, and 20,000 ppm for 10 weeks (Arnold et al., 2001). Ten animals were co-administered 5000 ppm sulfosulfuron with 12,300 ppm NH4 Cl to determine if inhibition of the formation of calculi would prevent any urothelial effects of treatment with sulfosulfuron. Ten animals in the control group and in the high-dose sulfosulfuron group were fed only basal diet for an additional 10 weeks to determine if the effects of sulfosulfuron on the bladder epithelium were reversible. There was an increased incidence of microcrystalluria observed at 5000 and 20,000 ppm. There was no increase in microcrystalluria observed in the urine of rats coadministered sulfosulfuron and NH4 Cl. Urinary bladder calculi were found in the bladder of one animal fed 20,000 ppm. Examination by light microscopy showed diffuse papillary/nodular hyperplasia of the bladder epithelium in this animal. No increased microcrystalluria was observed after withdrawal of the chemical from the diet, and the bladder epithelium was normal by light microscopy. It was concluded that the hyperplastic effects, and therefore the carcinogenic effects, associated with the feeding of high doses of sulfosulfuron occur only with the appearance of urinary tract calculi. Thus, the hyperplastic and carcinogenic effects of sulfosulfuron in rats are high-dose, threshold phenomena that are secondary to calculus formation in the urinary bladder. 2.7. Rat absorption, distribution, metabolism, and excretion study Pilot groups of Sprague–Dawley rats were dosed with one of two ring-specific-labeled [14 C]sulfosulfuron ([14 C]Pd-sulfosulfuron or [14 C]Im-sulfosulfuron) (Lau et al., 1996). (Pd means that sulfosulfuron was labeled at the C-5 position of the pyrimidine ring. Im means that sulfosulfuron was labeled at the C-3 position of the imidazopyridine ring. Fig. 2 shows the structure of [14 C]sulfosulfuron with both radiolabels shown by asterisk on the same molecule.) Two different dose levels were tested; groups P1 (dosed with [14 C]Pd-sulfosulfuron) and P3 (dosed with [14 C]Im-sulfosulfuron) received

Fig. 2. The chemical structure of

14

C-labeled sulfosulfuron.

10 mg/kg and groups P2 (dosed with [14 C]Pd-sulfosulfuron) and P4 (dosed with [14 C]Im-sulfosulfuron) received 1000 mg/kg. Fig. 3 shows the proposed metabolic pathway for sulfosulfuron in rats. Results from the pilot groups showed that, for all treatment groups, less than 0.04% of the radioactivity was recovered as 14 CO2 after 24 h. For this reason, expired gases were not measured in the main study. In the definitive portion of the study, the animals were dosed with a 1:1 mixture of [14 C]Pd-sulfosulfuron and [14 C]Im-sulfosulfuron. The study consisted of four groups: a single low oral dose (Group 1—target dose level of 10 mg/kg), a single low intravenous dose (Group 2—target dose level of 10 mg/kg), a single high oral dose (Group 3—target dose level of 1000 mg/kg), and a repeat low oral dose consisting of 14 daily doses of unlabeled sulfosulfuron followed by a single oral dose of 14 C-labeled sulfosulfuron (Group 4—target dose level of 10 mg/kg). Five days after dosing the animals were sacrificed and major tissues (liver, lung, kidney, etc.) were removed as well as blood and gut contents. These tissues and the residual carcass were then analyzed for the amount of radioactivity present. [14 C]Sulfosulfuron was shown to be well absorbed at the low oral dose and repeated low oral dose (approximately 95% for males and 91% for females). Absorption at the high-dose level was reduced to approximately 36 and 39% for males and females, respectively. The liver revealed the highest traces of radioactivity (<0.13% of the administered dose). Radioactivity in all other individual tissues was less than 0.01% of the administered dose. The radioactivity remaining associated with the residual carcass ranged from 0.02 to 0.36% of the administered dose. Four distinct sulfosulfuron metabolites (desmethyl sulfosulfuron, 5-hydroxy sulfosulfuron, sulfonamide, and pyrimidine sulfate, from trace levels to 3.59% of the administered dose) and unmetabolized sulfosulfuron were identified in excreta from rats dosed with sulfosulfuron. Metabolism of sulfosulfuron occurred by ring hydroxylation at the 5-position of the pyrimidine ring, and demethylation of the methoxy group at either the 4or 6-position of the pyrimidine ring. Cleavage of the sulfonylurea bond between the imidazopyridine and pyrimidine rings was limited. Similar percentages of urinary and fecal metabolites were found at both the

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317

Fig. 3. Proposed pathway for metabolism of sulfosulfuron in rats.

10 mg/kg dose level and the 1000 mg/kg dose level from oral administrations, indicating that the formation of sulfosulfuron metabolites in rats was not dose dependent. No significant differences in metabolism were observed due to route of administration or single versus repeated dosing. Only minor gender differences in the distribution of metabolites were noted. Greater than 90% of the dose was excreted three days after administration. The mean total recovery of the administered dose for all groups of animals ranged from 97 to 101% of the dose. The rate of excretion of radioactivity in the urine revealed a biexponential elimination process with mean half-life for the initial phase of approximately 2.2–5.8 h and for the terminal elimination phase of 21.4–56.7 h for all groups. In summary, in the low-dose groups, absorption was greater than 90%, while at the high dose absorption averaged about 40%. Metabolism of sulfosulfuron in the rat occurred to only a limited extent (<4%), with demethylation and pyrimidine ring hydroxylation as the metabolic routes. Expiration as carbon dioxide or volatiles was not a significant route of elimination. The cleavage of the sulfonylurea bond of sulfosulfuron to form separate imidazopyridine and pyrimidine metabolites is a minor metabolic pathway. The rat readily excreted sulfosulfuron and its metabolites, with urinary excretion as the major route of elimination at the low dose and fecal excretion being the predominant route of elimination after a high dose. There was no evidence of bio-retention of sulfosulfuron or its metabolites in rats and tissue and blood levels were negligible, with no individual tissue showing levels exceeding 0.2% of the dose.

3. Oncogenicity—weight-of-evidence evaluation 3.1. Relevance of bladder tumors in rodents The only evidence of oncogenicity with sulfosulfuron was a low incidence of urinary bladder tumors in female rats and in male mice at relatively high dose levels. Single incidences of urinary bladder transitional cell carcinoma and papilloma were observed in female rats at a dietary concentration of 5000 ppm (314 mg/kg/day). Benign mesenchymal tumors of the urinay bladder submucosa occurred in five male mice at the highest dose tested (7000 ppm or 943 mg/kg/day) and in one male mouse at the next lower dose level (3000 ppm or 394 mg/kg/day). A cooperative effort between the Health Effects Division of EPAÕs Office of Pesticide Programs (OPP) and the ILSI Risk Science Institute organized a working group to examine the relevance of rodent bioassay data for human carcinogenic risk assessment (Rodent Bladder Carcinogenesis Working Group, 1995). This group concluded that non-genotoxic chemicals can induce bladder tumors in rats and mice as a result of mechanical irritation produced by calculi formation. The chronic irritation and damage caused by the calculi eventually lead to urothelial proliferation, regenerative hyperplasia and ultimately, tumor formation (Clayson et al., 1995). The group noted important differences between rodents and humans including: anatomical differences that can enhance the formation of stones in the urinary bladder of rodents (DeSesso, 1995); body orientation and gravitational effects that encourage the

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passage or removal of calculi in humans; urine composition, protein content, and pH, as well as metabolic differences that can differ substantially between species; and the ability of the rodent to maintain a relatively normal life despite the continued presence of urinary bladder stones and their irritation effect on the urinary bladder mucosa. Because of these species differences, the rodent is not a satisfactory model for human urinary bladder carcinogenesis, and the finding of rodent bladder tumors in the presence of calculi formation cannot be directly extrapolated to humans. The group further noted that this mode of tumor formation is a high-dose and, in some cases, a species-specific phenomenon. The subject of rodent bladder carcinogenesis was also reviewed by an expert scientific advisory group of the International Agency for Research on Cancer (IARC, 1999). This group also concluded that the production of chemically induced urinary tract calculi in rats and mice is a high-dose phenomenon (studies cited in the IARC reference indicate levels in excess of 1% of the diet for various chemicals as constituting a ‘‘high dose’’). It was further noted that rodents are much more susceptible to both the formation of calculi and resulting oncogenic response. Even more recently, Cohen et al. (2002) examined the occurrence of urinary tract calculi and thresholds in carcinogenesis. It was concluded that tumor formation resulting from urinary tract calculi is a high-dose, threshold phenomenon. Important anatomic and physiologic differences make rodents more susceptible than humans to the precursor events and lesions responsible for eventual tumor formation. For chemicals producing tumors by this mode of action and for which human exposure is very low, the authors concluded that there is no real carcinogenic hazard to humans. For sulfosulfuron, it was shown that there was an increased incidence of crystalluria at 5000 and 20,000 ppm levels compared with minimal to no crystal formation at lower exposure levels (Cohen, 1996) In addition, the gradation of crystal formation increased at the higher dose levels (levels tested were 0, 50, 500, 5000, and 20,000 ppm, fed to the animals over a 10-week period). Calculi were found in the urinary bladder of one 20,000-ppm rat. In this animal, light microscopic examination revealed extensive urothelial toxicity and regenerative hyperplasia, including diffuse papillary nodular hyperplasia in the urinary bladder. No such effects were seen in any other animals. 3.2. Mesenchymal bladder tumors in mice Mesenchymal bladder tumors in mice were the predominant tumor type observed in the chronic bioassays with sulfosulfuron. While these tumors may occur spontaneously at a low level, they occur predominantly in male mice over 10 months of age where there is a

concurrent incidence of obstructive uropathy (Halliwell, 1998). This indicates that the changes leading to the formation of submucosal mesenchymal tumors (SMT) are reactive to the obstructive process (International Life Sciences Institute, 1997). The smooth muscle of the mouse urinary bladder arises embryologically from mesenchymal cells that give rise to three layers of muscle cells. When the nascent muscle is sufficiently stretched to separate these layers, cells arising from the mesenchymal ridge repopulate the space between the layers. It seems likely that the bladder distention associated with calculi formation and subsequent irritation, inflammation, blockage, etc. could lead to proliferation of undifferentiated or partially differentiated mesenchymal cells and ultimately an oncogenic response. In the study with sulfosulfuron, all treated animals that developed mesenchymal tumors had urinary bladder calculi, further strengthening the link between calculus formation and the cycle of irritation, cell proliferation, and tumor development. The relevance of mouse mesenchymal bladder tumors for human risk assessment has been questioned on additional grounds. The species and strain susceptibility for submucosal mesenchymal tumors is extremely narrow. These tumors occur only in mice and their occurrence is primarily limited to mice of the closely related Swiss Webster and CD-1 strains (Halliwell, 1998). There have been no tumors reported in other animals that mimic the histomorphologic appearance of submucosal mesenchymal tumors. Even more important, tumors have not been reported in humans that originated from the submucosal lamina propria that morphologically resemble the submucosal mesenchymal tumors of the mouse urinary bladder. 3.3. Summary and conclusions for oncogenicity Chronic administration of sulfosulfuron produced a low incidence of bladder tumors in female rats and in male mice. These tumors occurred only at relatively high dose levels (314–943 mg/kg/day) and resulted from calculi formation and chronic irritation. Sulfosulfuron did not produce any evidence of genotoxicity in a battery of in vitro and in vivo assays. Based on these findings and anatomical differences between rodents and humans, it is concluded that the oncogenic effect of sulfosulfuron in animals is a high-dose, threshold-sensitive phenomenon that is not operative in humans under actual conditions of exposure. Therefore, a margin-of-exposure (MOE) approach to carcinogenic risk assessment is appropriate. (An MOE, sometimes used interchangeably with the term ‘‘margin of safety,’’ is the comparison of an estimated daily exposure to a chemical with an experimentally derived NOAEL. If the MOE is in excess of 100, it is generally assumed that the exposure does not pose a significant health risk for most humans.

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The greater the MOE value, the higher the level of comfort exists that the chemical does not pose a significant health risk.)

4. Dietary exposure assessments In a study to determine the total residues of sulfosulfuron and its metabolites in wheat forage, hay, straw, and grain (Kunda and Mueth, 1997), sulfosulfuron applications were made to both winter and spring wheat pre-plant incorporated (PPI), preemergent (PRE), or postemergent (POST), at a maximum use rate of 40 g/ha (0.035 lb/acre). Analyses were conducted on the forage sampled on the day of POST application, and approximately 14 and 30 days after POST application. The residues in the forage samples ranged from a high of 3.036 ppm in winter wheat and 2.081 ppm in spring wheat on the day of POST application to 0.044 ppm and <0.008, the analytical limit of quantitation (LOQ), respectively, 30 days after POST application. Residues in the wheat grain were always at or below the LOQ, regardless of application timing or crop season. Table 2 illustrates the residues in grain and the proposed maximum residue levels (MRLs). Potential dietary exposure from meat, milk, poultry, and eggs is negligible. There were only extremely low residues (<0.1 ppm) in a hen metabolism study (Nadeau et al., 1996). Results from a 28-day feeding study with Holstein dairy cows showed that sulfosulfuron residues in raw milk had attained plateau levels by day 4 of the study, indicating a lack of accumulation (Sidhu, 1997). At the highest dietary-exposure level (8 ppm), maximum residues were 0.004 ppm (LOQ) in raw milk, skim milk, fat, and muscle, 0.005 ppm in cream, and about 0.1 ppm in liver and kidney. The 8-ppm level is the highest theoretical dose that could occur in the unlikely scenario that dairy cows begin grazing on a wheat field immediately following application of sulfosulfuron for a period of 28 days. (This assumes that there is no degradation of sulfosulfuron in the plant over the 28-day period.)

Table 2 The total grain residues (ppm) of sulfosulfuron and its metabolites in winter and spring wheat pre-plant incorporated (PPI), preemergent (PRE), or postemergent (POST), and proposed maximum residue levels (MRLs (equivalent to U.S. tolerance levels)) at a use rate of 40 g/ha (0.035 lb/acre) (Kunda and Mueth, 1997) Source of grain

Range

Proposed MRL

POST Winter wheat Spring wheat

<0.008 <0.008

0.02 0.02

PRE or PPI Winter wheat Spring wheat

<0.008 Not detected

0.02 0.02

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Actual residues would undoubtedly be much lower for both the cattle feed and for consumers. Therefore, human dietary exposure to sulfosulfuron will be derived almost exclusively from wheat flour used for baking purposes. Dietary exposures for various populations within the United States were estimated with the Dietary Exposure Estimating Model (DEEM) software (Novigen Sciences) using the 1994–1996 USDA food consumption surveys. The conservative exposure scenario assumed that 100% of the wheat grain in commerce contains residues at the tolerance level. Thus, for this estimate, residue in wheat was assumed to be 0.02 ppm. Tolerances for milk, fat, and meat are 0.006, 0.005, and 0.005 ppm, respectively, and for meat by-products 0.05 ppm (U.S. Environmental Protection Agency, 1999). The exposure calculations assumed that residues did not decrease with time or during processing or cooking/baking. For the reasons discussed above, potential exposures from poultry and eggs were considered to be insignificant and were not included. The predicted human dietary exposures to sulfosulfuron using this set of assumptions and the USDA surveys are presented in Table 3. Children aged 1–6 were considered to receive the highest potential dietary exposures to sulfosulfuron. Using the conservative estimate of crop and animal residues scenario described above, the predicted dietary exposures were estimated to be 255 ng/kg bw/day for children aged 1–6 and 80–87 ng/ kg bw/day for the general U.S. population. As expected, essentially all of the exposure is derived from wheat, with the contribution from animal commodities being negligible. 4.1. Risk characterization 4.1.1. Chronic effects Based on the lowest NOAEL of 24 mg/kg/day (from the rat chronic study), the 1994–1996 USDA food survey, and assumed residues of 100% of the maximum allowable residue (tolerance), the estimated MOE for children aged 1–6 and for the overall population would be at least approximately 94,118 and 289,157, respectively. The true MOEs would undoubtedly be much higher for the following reasons: (1) At best, market penetration for sulfosulfuron is expected to be no more than 50%, instead of 100% as assumed in this assessment. (2) The vast majority of wheat farmers are expected to use sulfosulfuron at rates ranging from 0.015 to 0.025 lb/acre rather than the 0.035 lb/acre used in the residue studies. (3) Residues are likely to be reduced by normal grain processing procedures. (4) Intervals between herbicide application and harvesting or cattle feeding would probably be longer than was used in these studies. (5) Actual residues were lower than the LOQ value on which the proposed tolerances are based

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Table 3 Estimated dietary exposure to sulfosulfuron for selected U.S. populationsa Population subgroup

mg/kg bw/day

Percent of RfD (%)

0.000083 0.000083 0.000080 0.000087 0.000083

0.0 0.0 0.0 0.0 0.0

Northeast region Midwest region Southern region Western region

0.000086 0.000088 0.000077 0.000086

0.0 0.0 0.0 0.0

Hispanics Non-Hispanic whites Non-Hispanic blacks Non-Hispanic other than black or white

0.000090 0.000083 0.000079 0.000083

0.0 0.0 0.0 0.0

All infants (<1 year old) Nursing infants Non-nursing infants Children (1–6 years) Children (7–12 years)

0.000090 0.000027 0.000109 0.000255 0.000142

0.0 0.0 0.0 0.1 0.0

Females Females Females Females Females

0.000067 0.000049 0.000054 0.000073 0.000077

0.0 0.0 0.0 0.0 0.0

0.000088 0.000057 0.000049

0.0 0.0 0.0

U.S. U.S. U.S. U.S. U.S.

population—all seasons population—spring season population—summer season population—autumn season population—winter season

(13–19 years/not pregnant or nursing) (20+ years/not pregnant or nursing) (13–50 years) (13+/pregnant/not nursing) (13+/nursing)

Males (13–19 years) Males (20+ years) Seniors (55+)

Wheat grain residues were assigned a value of 0.02 ppm (tolerance), with 100% market penetration. Milk commodities were assigned a residue of 0.006 ppm. Cow, veal, sheep, goat, and horse were assigned residues of 0.005 ppm for lean meat and fat, and 0.05 ppm for meat by-products. Reference dose (RfD, chronic) ¼ 0.24 mg/kg bw/day (based on rat chronic NOAEL of 24 mg/kg/day and a 100-fold safety factor). a Generated using Dietary Exposure Estimating Model (DEEM) software, Novigen Sciences, version 7.81. Results are based on the combined data from the National Food Consumption Surveys from 1994 to 1996.

(Naylor, 1997; U.S. Environmental Protection Agency, 1999). Thus, it is concluded that there would be no significant risk of toxicity from ingesting products containing wheat derived from crops that had been treated with sulfosulfuron. 4.1.2. Conclusions Based on the toxicology and residue data discussed above, there should not be any toxicological concerns from animal or human consumption of wheat grain or animal products with regard to sulfosulfuron. The RfD for sulfosulfuron is 0.24 mg/kg/day, which is 1% of the NOAEL for males in the chronic rat toxicity study. No more than 0.1% of the RfD would be used by consumption of grain and animal products by the highest exposure group, even in the unlikely scenario where wheat containing sulfosulfuron residues at tolerance levels or maximum allowable residue was used as the sole source of grain for an extended period of time. The lifetime risk for a 70-year exposure for adults is calculated along with those for infants, children of

varying ages, and others. Shown in Table 4 are the maximum possible levels of exposure to sulfosulfuron and the coincident MOE. Using a standard 100-fold safety or uncertainty factor (10 for intraspecies variability and 10 for interspecies variability) for determining a lack of risk for exposure to sulfosulfuron, all of the MOEs are several orders of magnitude in excess of this safety factor. In addition, these already substantial MOEs are probably very conservative based on the opinion of Cohen et al. (2002) that only a 10-fold safety factor for intraspecies variability is sufficient for chemicals, like sulfosulfuron, that act through the mechanism of urinary bladder calculus formation. 4.2. Food quality protection act and related considerations Under the auspices of the Food Quality Protection Act (FQPA), aggregate assessments are performed in order to account for risk based on exposure to a single chemical from varied sources (diet, drinking water, and non-occupational) and/or different routes of exposure. These are described next.

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Table 4 Maximum expected sulfosulfuron exposure, percent of the RfD used by that exposure, and coincident margins of exposure for various population groups Subgroups

Exposure (mg/kg/day)

Percent of RfD
(%)

MOE

U.S. population (all seasons) U.S. population (Midwest) All infants (<1 year old) Nursing infants Non-nursing infants Children (1–6 years old) Children (7–12 years old) Females (13+/pregnant/non-nursing)

0.000083 0.000088 0.000090 0.000027 0.000109 0.000255 0.000142 0.000073

<1 <1 <1 <1 <1 <1 <1 <1

289,157 272,727 266,667 888,889 220,183 94,118 169,014 328,767

*

Calculated by U.S. Environmental Protection Agency.

4.2.1. Aggregate exposure 1. Diet exposure The estimates of dietary exposure incorporated grain (flour) and milk, fat, meat, and meat by-product residues, assumed no loss due to processing or cooking, and that 100% of the wheat grain used in commerce would contain sulfosulfuron residues at the level of the tolerance level. In this very conservative scenario, less than 0.1% of the RfD would be used by consumption of grain and animal products by the highest exposure group. 4.2.2. Aggregate exposure 2. Drinking water In a lysimeter study (q.v.) using sandy soil, levels of sulfosulfuron in the leachate ranged from <0.01 to 0.03 lg/L after 18 months (McMullan, 1996). These results, together with soil half-life study results, indicate that sulfosulfuron is fairly tightly bound to the soil. Considering the low application rates, soil-binding characteristics, and low soil mobility, the risk of significant ground and surface water contamination and exposure by drinking water is negligible. Therefore, aggregate risk of exposure to sulfosulfuron does not include drinking water. Assuming that 10% of the RfD is allocated to drinking water exposure, and the average 70 kg person consumes two liters of water per day, a Maximum Allowable Concentration value for drinking water of 0.84 mg/L is proposed: MAC ¼

RfD  body weight  P ; C

where body weight ¼ body weight (70 kg) for adults, P is the fraction of the RfD allocated to drinking water (i.e., 10%), and C is the daily consumption of drinking water (2 L). For sulfosulfuron, this gives the following calculation: 0:24 mg=kg  70  0:1 ¼ 0:84 mg=L: 2 4.2.3. Aggregate exposure 3. Non-occupational Sulfosulfuron has some non-crop uses including roadsides, fence rows, industrial sites, parks, apartment

complexes, schools, and other public areas. Exposure assessments have been made for mixer/loaders and applicators in these situations (occupational exposure), and the cumulative (amortized) daily exposure from both these activities has been estimated to be less than 0.5 lg/kg/day or approximately 0.2% of the RfD (Naylor, 1997; U.S. Environmental Protection Agency, 1999). The non-occupational exposure in these locations to the casual passer-by would be expected to be orders of magnitude less. The exposure in either instance does not present a significant exposure risk, and exposure to the general public will be almost exclusively through dietary consumption of wheat products. In other words, people would not be exposed to sulfosulfuron in sufficient quantities for adequate periods of time to allow for the formation of urinary tract crystals and calculi and a subsequent hyperplastic response in the urinary tract. Therefore, MOEs for sulfosulfuron in residential and other non-occupational settings would be orders of magnitude greater than the already high MOEs presented above for dietary exposures. 4.3. Cumulative risk As opposed to aggregate risk assessment, cumulative risk is assessed by consideration of the potential effects of combined exposures to two or more chemicals sharing a common mechanism of action. Toxic effects due to oral exposure to sulfosulfuron are almost exclusively due to urinary precipitates at relatively high exposure levels for lengthy periods. This process would not occur under any foreseeable conditions of human exposure given the low use rate, low residues in consumer commodities, and large MOEs for applicators. In concurrence with this thought, and in the absence of data with which they could include sulfosulfuron in a cumulative risk assessment, the U.S. Environmental Protection Agency (1999) stated that the chemical does not produce a toxic metabolite and therefore does not share a mechanism of toxicity in common with by other sulfonylurea chemicals or by other pesticides commonly used on wheat. Urinary precipitates and/or resulting calculi are not common in humans and, because neither crystalluria

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nor calculi are generally associated with tumor formation in humans, it is not considered necessary to combine the effects of this chemical with those of any other that may produce crystalluria in humans. Substances that have been shown to produce urinary crystals and/or calculi at relatively high dose levels in animal studies, such as sodium saccharin, uracil, diethylene glycol, terephthalic acid, and melamine, have not proved to be general health concerns for humans. Even in the case of sulfonamide drugs, which are well known as crystalforming agents in the urine of humans, the ordinary precaution of increased hydration proves effective at preventing stone formation or adverse health consequences from urinary crystals. Further, sulfonamide drug use has been in decline for many years and is seldom the treatment of choice in combating infections. It is felt, therefore, that the toxic effects of sulfosulfuron, principally crystalluria and/or urolithiasis, should not be considered along with other crystal-producing chemicals. 4.4. Estrogenic effects While no studies that are specifically designed to assess estrogenic effects have been performed with sulfosulfuron, there was no evidence that exposure to sulfosulfuron had any effect on reproduction, fertility, or mating indices, development or maturation of embryos, or development, growth, or survival of offspring in the range of short-term, chronic, or reproductive mammalian, avian, and aquatic studies that were conducted. There were no gross or microscopic pathologic effects in endocrine organs or endocrine-sensitive tissues, or in any reproductive organs, tissues, or endpoints that were considered related to exposure to sulfosulfuron. With no evidence of bioaccumulation and low environmental concentrations, there is no risk of endocrine disruption in humans or wildlife. The studies considered included a 90-day pilot study in rats with a reproduction component, a two-generation reproduction study in rats, pilot and full developmental toxicity studies in rats and rabbits, pilot and full reproduction studies in bobwhite quail (Frey, 1995, 1996a) and mallard ducks (Frey, 1996b,c), a life-cycle toxicity test with daphnia (Graves et al., 1996a), and an early life-stage toxicity test with the rainbow trout (Graves et al., 1996b). 4.5. Evaluation of exposure of infants and children In assessing the potential for additional sensitivity of infants and children to residues of sulfosulfuron, data were considered from developmental toxicity studies in the rat and rabbit and a two-generation reproduction study in rats. No developmental or reproductive effects were observed up to the highest dose tested in each of the three studies. The observed NOAELs were

1000 mg/kg/day, 1000 mg/kg/day, and 20,000 ppm, respectively. Using the same conservative assumptions that were made previously for the dietary exposure analysis for the U.S. general population, the percent of the RfD used by pre-adult subpopulations are: all infants— 0.038%; nursing infants—0.011%; non-nursing infants— 0.045%; children, 1–6 years—0.106%; and children, 7–12 years—0.059%. It may be concluded that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to sulfosulfuron residues.

5. Overall conclusions The toxicity data for sulfosulfuron indicate that it has a low order of acute toxicity. Therefore, U.S. EPA has decided that an acute risk assessment is not needed (U.S. Environmental Protection Agency, 1999). Sulfosulfuron is not genotoxic. Furthermore, in subchronic studies it has been tested at very high dose levels, i.e., 1000 mg/kg/day, the limit dose, with only very minor effects in rats and mice at the highest dose tested. The only effect in dogs was calculus formation and associated pathology indicative of irritation in the urinary tract at high-dose levels. There are no indications that sulfosulfuron is neurotoxic based on the lack of adverse effects in both acute and subchronic neurotoxicity studies performed at very high dose levels. Additionally, no effects that may be attributed to a neurotoxic effect have been observed in clinical observations and histopathological evaluations in standard subchronic and chronic studies. Sulfosulfuron is clearly not a neurotoxic agent. The chemical is also not a developmental or a reproductive toxin and there are no indications of endocrine disruption in any of the studies performed. Consequently, U.S. EPA decided that it did not need to apply an additional Food Quality Protection Act (FQPA) safety factor above the standard 100-fold-safety factor for inter- and intra-species variability (U.S. Environmental Protection Agency, 1999). In addition, while specific developmental neurotoxicity and immunotoxicity studies have not been performed with sulfosulfuron, there are no indications that the chemical is a developmental neurotoxin or an immunotoxicological agent based on the data observed in the standard acute, subchronic, chronic, developmental, and reproductive toxicity studies that were performed. The primary finding in mammalian laboratory animals following chronic exposure to sulfosulfuron appears to be isolated to the urinary tract and was the result of urolith formation following high-level chemical dosing. In rats and mice, this progressed to tumor formation in a few animals. However, the finding of the tumors in laboratory animals is irrelevant to humans

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because of (a) anatomical differences, (b) the lack of genotoxicity, (c) the high-dose threshold required for manifestation of the effect, and (d) the findings presented in the ‘‘mode of action’’ study that also showed reversibility of the effect. Therefore, based on its shortterm, infrequent application pattern and low use rate and crop residue analyses, sulfosulfuron has substantial MOEs and does not represent a risk to human health. References Arnold, L.L., Cano, M., St. John, M.K., Healy, C.E., Cohen, S.M., 2001. Effect of sulfosulfuron on the urine and urothelium of male rats. Toxicol. Pathol. 29, 344–352. Bechtel, C.L., 1994a. Acute inhalation study of MON 37532 herbicide. Monsanto Company, unpublished report. Bechtel, C.L., 1994b. Acute inhalation study of MON 37500 herbicide. Monsanto Company, unpublished report. Blaszcak, D.L., 1995a. Guinea pig maximization test with MON 37500. Monsanto Company, unpublished report. Blaszcak, D.L., 1995b. Acute oral toxicity study in rats with MON 37532. Monsanto Company, unpublished report. Blaszcak, D.L., 1995c. Acute dermal toxicity study in rats with MON 37532. Monsanto Company, unpublished report. Blaszcak, D.L., 1995d. Primary eye irritation study in rabbits with MON 37532. Monsanto Company, unpublished report. Blaszcak, D.L., 1995e. Primary dermal irritation study in rabbits with MON 37532. Monsanto Company, unpublished report. Blaszcak, D.L., 1995f. Guinea pig maximization test with MON 37532 (Method of Magnusson and Kligman). Monsanto Company, unpublished report. Bonnette, K.L., 1993a. Acute oral toxicity study in rats with MON 37500. Monsanto Company, unpublished report. Bonnette, K.L., 1993b. Acute dermal toxicity study in rats with MON 37500. Monsanto Company, unpublished report. Bonnette, K.L., 1993c. Primary eye irritation study in rabbits with MON 37500. Monsanto Company, unpublished report. Bonnette, K.L., 1993d. Primary dermal irritation study in rabbits with MON 37500. Monsanto Company, unpublished report. Branch, D.K., Thake, D.C., Kaempfe, T.A., Li, A.A., 1997. Acute neurotoxicity study of MON 37500 in Sprague–Dawley rats. Monsanto Company, unpublished report. Cohen, S.M., 14 November, 1996. Personal communication. Cohen, S.M., Johansson, S.L., Arnold, L.L., Lawson, T.A., 2002. Urinary tract calculi and thresholds in carcinogenesis. Food Chem. Toxicol. 40, 793–799. Clayson, D.B., Fishbein, L., Cohen, S.M., 1995. Effects of stones and other physical factors in the induction of rodent bladder cancer. Food Chem. Toxicol. 33, 71–784. DeSesso, J.M., 1995. Anatomical relationships of urinary bladders compared: their potential role in the development of bladder tumours in humans and rats. Food Chem. Toxicol. 33, 705– 714. Dubelman, S., 2000. Determination of sulfosulfuron concentration in rat bladder calculi. Addendum to: Combined chronic toxicity/ oncogenicity study of MON 37500 administered in the diet to Sprague–Dawley rats. Monsanto Company, unpublished report. Frey, L.T., 1995. MON 37500: A pilot reproduction study with the Northern Bobwhite (Colinus virginianus). Monsanto Company, unpublished report. Frey, L.T., 1996a. MON 37500: A reproduction study with the Northern Bobwhite (Colinus virginianus). Monsanto Company, unpublished report.

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Naylor, M.W., Ruecker, F.A., 1997a. One year dog oral toxicity study. Monsanto Company, unpublished report. Naylor, M.W., Ruecker, F.R., 1997b. Combined chronic toxicity/ oncogenicity study of MON 37500 administered in the diet to albino rats. Monsanto Company, unpublished report. Naylor, M.W., Thake, D.C., 1997a. Rangefinding and one month dermal study of MON 37500 in CD rats. Monsanto Company, unpublished report. Naylor, M.W., Thake, D.C., 1997b. Oncogenicity study of MON 37500 administered in feed to CD-1 mice for 18 months. Monsanto Company, unpublished report. Rodent Bladder Carcinogenesis Working Group, 1995. Urinary bladder carcinogenesis: Implications for risk assessment. Food Chem. Toxicol. 33, 797–802. Sidhu, R.S., 1997. Stability of MON 37500 residues in dairy cow milk and meat tissues during frozen storage. Monsanto Company, unpublished report.

Stegeman, S.D., Warren, J., Kier, L.D., 1995a. Ames/Salmonella mutagenicity assay of MON 37500. Monsanto Company, unpublished report. Stegeman, S.D., Kier, L.D., Asbury, K.J., Garrett, S.L., McAdams, J.G., 1995b. CHO/HGPRT gene mutation assay of MON 37500. Monsanto Company, unpublished report. Stegeman, S.D., Kier, L.D., Asbury, K.J., Garrett, S.L., McAdams, J.G., Warren, J., 1995c. Mouse bone marrow micronucleus assay of MON 37500. Monsanto Company, unpublished report. Stegeman, S.D., Kier, L.D., Albin, L.A., Lau, H., 1996. Mouse bone marrow micronucleus assay of C14-radiolabeled MON 37500. Monsanto Company, unpublished report. U.S. Environmental Protection Agency, 1999. Sulfosulfuron, Pesticide Tolerance, Federal Register 64, 27186-27192. Yoshida, N., 1996. Chromosomal aberration test of TKM-19 using cultured mammalian cells. Monsanto Company, unpublished report.