Toxicology of Triazolopyrimidine Herbicides

Chapter 87

Toxicology of Triazolopyrimidine Herbicides Richard Billington, Sean C. Gehen Dow AgroSciences LLC, Indianapolis, Indiana

Thomas R. Hanley Jr. Syngenta Crop Protection, Inc., Greensboro, North Carolina

87.1  Introduction The triazolopyrimidines are herbicides used for the preemergent and postemergent control of broadleaf weeds in a variety of crops. The general structure of this class of chemistry is a substituted triazolopyrimidine connected to a substituted phenyl (cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam) or pyridine (pyroxsulam) ring through a sulfonamide bridge, as presented in Figure 87.1. The substituents of the various members of this class are presented in Table 87.1. The herbi­cidal mode of action is through inhibition of acetolactate synthase (ALS) in plants, though the mechanism appears to be different from that of sulfonylureas. Acetolactate synthase (EC 4.13.18), also known as acetohydroxyacid synthase, is a key enzyme in the synthesis of

the ­ branched-chain aliphatic amino acids leucine, isoleucine, and valine. Inhibition of this enzyme in plants results in cessation of cell growth and division, leading to the death of suscep­tible plants. However, this enzyme is lacking in humans and other animals, which accounts for the low mammalian toxicity of these chemicals. R1

R4

R2 N 4

N

1

5

N

3

R3

2

R5

R6

Figure 87.1  Generic structure of the triazolopyrimidine herbicides.

Table 87.1  Substituents of Triazolopyrimidine Sulfonamide Herbicides 1

2

3

4

5

R1

R2

R3

R4

R5

R6

(1,5c) Clorasulam-methyl

N

C

C

NH

SO2

H

CO2CH3

Cl

OCH2CH3

F

H

Diclosulam

N

C

C

NH

SO2

H

Cl

Cl

OCH2CH3

F

H

Florasulam

N

C

C

NH

SO2

H

F

F

OCH3

H

F

Penoxsulama

N

C

C

SO2

NH

H

OCH2CHF2

CF3

OCH3

H

OCH3

(1,5a) Flumetsulam

C

N

C

NH

SO2

H

F

F

H

CH3

—

Metosulam

C

N

C

NH

SO2

CH3

Cl

Cl

OCH3

OCH3

—

C

N

N

SO2

NH

H

CF3

OCH3

OCH3

OCH3

—

a

Pyroxsulam a

Sulfonamide linkage reversed

Hayes’ Handbook of Pesticide Toxicology Copyright © 2010 Elsevier Inc. All rights reserved

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Comprehensive toxicological testing has been conducted with all of these substances according to national and international regulatory frameworks for the approval and registration of pesticides and their products. The methods used for the studies summarized in this review typically comply with the regulatory test guidelines – as published by the Organization for Economic Cooperation and Development (OECD), the U.S. Environmental Protection Agency (U.S. EPA), the European Union (EU), and Japan Ministry of Agriculture, Forestry, and Fisheries (JMAFF) to determine potential health effects – and Good Laboratory Practice (GLP) regulations. Studies typ­ ically involve oral administration of the test substance by incorporation into the animal feed, which can be achieved either using a fixed dietary concentration throughout the course of the study or by regularly varying the concentration of the substance in the feed to achieve a constant dosage in terms of milligrams per kilogram of body weight per day. The notable exceptions to this typical approach are acute oral studies, including neurotoxicity, developmental toxicity studies, and in vivo genotoxicity studies, which typically involve oral administration of the substance suspended in an aqueous vehicle by gavage. These studies are used to characterize any potential hazards resulting from acute, subchronic, or chronic exposure and to derive reference doses for humans (e.g., an Acceptable Daily Intake, ADI). These are required to enable the acceptability of predicted exposures to the triazolopyrimidines under all anticipated use scenarios to be determined as part of the comprehensive human health-based risk assessments. In general, triazolopyrimidine herbicides have very low mammalian toxicity as determined by acute, shortterm, long-term (chronic), genotoxicity, reproduction, developmental, and neurotoxicity tests. The kidneys and/or liver are the primary organs affected by repeated exposure, and, even then, in most cases the effects represent adaptive responses to very high levels of exposure. From a pharmacokinetic perspective, oral absorption is rapid, as is excretion, with no evidence of accumulation. In all cases, the sulphonamide bridge remains intact and the parent molecule is the principle component in excreta, mainly in urine. Although metabolism is limited, in some cases many metabolites are produced, which typically comprise ring demethylation and hydroxylation with or without formation of various types of conjugations. The mammalian toxicity of these materials is reviewed in this chapter.

87.2  Cloransulam-methyl 87.2.1  Identity, Properties, and Uses 87.2.1.1  Chemical Name The IUPAC name for cloransulam-methyl is methyl 3-chloro-2-(5-ethoxy-7-fluoro[1,2,4]triazolo[1,5-c]pyrimidin2-ylsulfonamido)benzoate; the CAS name is methyl

3-chloro-2-[[(5-ethoxy-7-fluoro[1,2,4]triazolo[1,5-c] pyrimidin-2-yl)sulfonyl]amino]benzoate.

87.2.1.2  Structure See Figure 87.1 and Table 87.1.

87.2.1.3  Synonyms Cloransulam-methyl is also known as XR-565, or XDE565, and is sold as FirstRate herbicide in the United States, and as Pacto and Supra herbicides in South America. (All trade names used in this chapter are registered trademarks of Dow AgroSciences LLC.) The Chemical Abstract Service (CAS) registry number is 147150-35-4.

87.2.1.4  Physical and Chemical Properties The empirical formula for cloransulam-methyl is C15H13 ClFN5O5S, with a molecular weight of 429.8. It is a solid at room temperature, with a low vapor pressure (3   1016 mmHg at 25°C). The water solubility is pH-dependent, with values of 2.96 mg/l at pH 5, 184 mg/l at pH 7, and 3430 mg/l at pH 9 (20°C). The log Kow is estimated at 3.7; the pKa is 4.81.

87.2.1.5  Uses Cloransulam-methyl is used as a soil-applied or incorporated preemergence or postemergence broadleaf herbicide in soybeans at maximum label rates of 44 g per hectare soil applied and 18 g per hectare postemergence.

87.2.2  Toxicity to Laboratory Animals 87.2.2.1  Acute Exposure The acute toxicity of cloransulam-methyl is low. The acute oral LD50 in the rat was greater than 5000 mg/kg body weight (bw) in both males and females and the dermal LD50 in the rabbit was greater than 2000 mg/kg bw. The 4-h inhalation LC50 in the rat was greater than 3.77 mg/l of air, which was the highest attainable respirable aerosol concentration. Cloransulam-methyl produced no indications of dermal irritation in rabbits or delayed contact skin sensitization in the guinea pig, and only slight transient eye irritation in the rabbit following acute exposure (Bradley et al., 1992; Cosse and Berdasco, 1992a,b,c,d; U.S. EPA, 1997a,b).

87.2.2.2  Repeated Exposure Cloransulam-methyl was evaluated in subacute and subchronic dietary studies in rats, mice, and dogs. The primary target organs identified in these studies were the kidney (rat and mouse), the liver (rat, mouse, and dog), and the thyroid (rat).

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

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In the Fischer 344 (F344) rat, dosages of 100–1000 mg/ kg/day for 2 weeks produced slight decreases in red blood cell parameters and urine-specific gravity in males, and slightly increased cecal and liver weights in females at 1000 mg/kg/day. The no-observed-effect level (NOEL) was 500 mg/kg/day. Dosages of 100–1000 mg/kg/day for 13 weeks produced treatment-related kidney changes comprising very slight to moderate hypertrophy of collecting tubule epithelial cells and/or slight vacuolation of the renal proximal tubular epithelium consistent with fatty changes in all treated groups. Decreased body weight gain and feed consumption, very slight hepatocellular vacuolation, and slight thyroid follicular hypertrophy were also seen at 500 and 1000 mg/kg/day (Haut et al., 1991, 1992a; Stebbins and Haut, 1994). Dosages of 100, 500, or 1000 mg/kg/day fed to B6C3F1 mice for 2 weeks produced slight hepatocellular hyper­trophy at 500 mg/kg/day and above in males, and at 1000 mg/kg/day in females. The NOEL was 100 mg/kg/day. Dosages ranging from 50 to 1000 mg/kg/day given for 13 weeks produced slight centrilobular and midzonal hepatocellular hypertrophy at 100 mg/kg/day and above in males, and at 500 mg/kg/day and above in females. Electron microscopy characterized the hypertrophy as an increase in rough endoplasmic reticulum (RER) with a decrease in cytoplasmic glycogen content. Kidney effects in mice consisted of decreased vacuolation of the renal tubules, consistent with decreased cytoplasmic lipid, accompanied by lower kidney weights at 500 mg/kg/day and higher. The subchronic lowest observed effect level (LOEL) and NOEL values in mice were 100 and 50 mg/kg/day, respectively (Haut et al., 1992b; Stebbins and Haut, 1993). Cloransulam-methyl, when fed to dogs for 2 weeks at dosages of 500 mg/kg/day or higher, produced hepatic inflammation, degeneration, and necrosis. No effects were seen at dosages of 200 mg/kg/day or lower. In a subchronic study, dogs exhibited a taste aversion to this material at dosages of 200 mg/kg/day and above, which resulted in a combination of impaired nutritional status and toxicity of the material. A dosage of 40 mg/kg/day resulted in lower body weight gains. Histologic examination did not identify a target organ, although a subsequent chronic study in dogs identified the liver as the primary target organ. Based on decreased body weights, a subchronic NOEL was not established in dogs (Stebbins et al., 1996; Szabo et al., 1992). In a 21-day repeated dermal application study in rabbits, cloransulam-methyl at dosages of 100, 500, or 1000 mg/kg/day produced slight anemia in female rabbits at the highest dosage. Male rabbits were unaffected at 1000 mg/kg/day and the NOEL in females was 500 mg/kg/ day (Gilbert and Yano, 1995a).

344 rats were fed cloransulam-methyl at dosages of 10– 325 mg/kg/day. Body weight gain was decreased at the highest dosage. Treatment-related histologic effects were limited to the kidneys and thyroid. Hypertrophy of a population of renal collecting duct epithelial cells identified as -intercalated cells was reported in males and females fed 325 mg/kg/day. (A similar histologic change noted in rats, mice, and dogs following exposure to florasulam will be discussed later in this chapter.) Vacuolation of the proximal tubules (consistent with fatty changes) in males fed cloransulam-methyl at 325 mg/kg/day, and females fed 75 or 325 mg/kg/day, and an increase in the incidence of mineralization of the renal pelvis in males fed 75 or 325 mg/kg/day also were present. Thyroid changes were confined to the high-dosage males (325 mg/kg/day) and consisted of hyperplasia and hypertrophy of follicular cell epithelium. The NOEL in this study was 10 mg/kg/day (Jeffries et al., 1995a). B6C3F1 mice were fed diets containing cloransulammethyl at dosages of 10–1000 mg/kg/day for 2 years. As was seen in mouse subchronic studies, the liver was the primary target organ, with effects also noted in the kidneys. Increases in liver weights in males at 100 mg/kg/ day and females at 1000 mg/kg/day, and centrilobular hypertrophy in males at 100 mg/kg/day were the only treatment-related effects noted in the liver. Kidney weights were decreased in males at 1000 mg/kg/day and females at 100 mg/kg/day. In the kidneys, depletion of the normal epithelial cytoplasmic vacuoles, and decreases in the incidence of renal mineralization and renal tubular degeneration were noted in males at 100 mg/kg/day. All of these histologic changes were interpreted to be either incidental or adaptive-physiologic responses to the test material rather than adverse toxic effects. The NOEL in mice following chronic exposure was 10 mg/kg/day (Jeffries et al., 1995b). There was no evidence of a tumorigenic or carcinogenic response in either mice or rats following long-term exposure. In a 1-year chronic toxicity study, beagle dogs were fed dosages of 5–50 mg/kg/day. The only treatment-related effects were in the liver and consisted of a slight-to-moderate increase in accumulation of pigment in Kuppfer cells and hepatocytes, and slight centrilobular and midzonal hepatocellular hypertrophy at 10 mg/kg/day, with changes in hepatic-related serum chemistry parameters at 50 mg/kg/day (Szabo and Davis, 1994). The U.S. EPA considered 10 mg/kg/day the NOEL in this study (U.S. EPA, 1997a).

87.2.2.3  Chronic Toxicity and Carcinogenicity

In a battery of tests, cloransulam-methyl showed no evidence of genotoxic potential. These tests included a bacterial reverse mutation assay (Ames test), an in vitro cytogenetic assay in Chinese hamster ovary cells (CHO/HGPRT

The chronic toxicity of cloransulam-methyl has been evaluated in rats, mice, and dogs. In a 2-year study, Fischer

87.2.2.4  Mutagenicity

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assay), an in vitro chromosomal aberration assay in rat lymphocytes, and an in vivo cytogenetic assay in mouse bone marrow cells (U.S. EPA, 1997a, b).

87.2.2.5  Neurotoxicity The neurotoxic potential of cloransulam-methyl was evaluated in specialized studies. No neurotoxicologic effects were observed in rats following acute gavage exposure of up to 2000 mg/kg (highest dose tested). A complete battery of neurologic tests including functional observations (handheld and open field observations, grip strength, and landing foot splay), motor activity, and detailed neurohistopathology was conducted following 13-week exposure via the diet to dosages up to 1000 mg/kg/day. No treatmentrelated neurotoxic effects were observed in any of these measures (Shankar et al., 1993; Spencer et al., 1995).

87.2.2.6  Reproductive Toxicity Cloransulam-methyl had no effect on reproduction or embryofetal development. In a multigeneration reproduction study in Sprague-Dawley (SD) rats, dosages of 100 mg/kg/day and above produced kidney and thyroid effects in the adults consistent with effects seen in subchronic and chronic studies. The NOEL for parental animals was 10 mg/kg/day. No effects on reproductive performance or neonatal survival were seen even at the high dosage of 500 mg/kg/day. In a developmental tox­icity study in CD rats, gavage dosages of up to 1000 mg/kg/day (limit test) on gestation days 6–15 produced no maternal or developmental toxicity. In a developmental toxicity study in New Zealand White rabbits administered gavage dosages of 0, 30, 100, or 300 mg/kg/day on gestation days 7–19, maternal weight gain and feed consumption were affected only at 300 mg/kg/day. No adverse embryonal or fetal effects were noted at any dose level (Vedula et al., 1992; Zablotny et al., 1993, 1994).

87.2.2.7  Absorption, Distribution, Metabolism, and Excretion Metabolism studies were conducted with 14C-radiolabeled cloransulam-methyl in the F344 rat using dose levels of 5 or 1000 mg/kg. At 5 mg/kg, over 90% of either a single dose or repeated (15 days) doses was absorbed. At 1000 mg/kg, only 28–30% of a single dose was absorbed. Urinary elimination was rapid in both cases with half-lives of approximately 6–9 h. A higher percentage of the 5-mg/ kg dose was excreted in the urine by females (68–80%) than by males (40–50%) and these sex-dependent differences in disposition of the 5-mg/kg dose were attributed to more efficient elimination of unchanged cloransulammethyl in the female versus male kidney. Analyses of urine and fecal extracts indicated that parent cloransulam-methyl

Hayes’ Handbook of Pesticide Toxicology

accounted for the majority of the excreted radiolabeled material. The only metabolite present at amounts greater than 5% was identified as the 4-OH phenyl derivative of cloransulam-methyl. Other minor metabolites included a hydroxylation of the pyrimidine ring, though the position of hydroxylation was not identified, and an N-acetyl cysteine conjugate of the parent material. Due to rapid elimination, cloransulam-methyl has little potential to accumulate upon repeated administration (Domoradzki et al., 1995; Nolan et al., 1995).

87.2.3  Toxicity to Humans No studies are available on intentional human exposure. However, the risk to humans from exposure to cloransulam-methyl following normal use patterns is low. No detectable residues were found either in soybeans or, in most cases, in soybean forage or hay at a limit of detection of 0.005 ppm, and accumulation is unlikely based on plant and animal data. Tolerance levels of 0.02 ppm in soybean, 0.1 ppm for soybean forage, and 0.2 ppm for soybean hay have been established (U.S. EPA, 1997a). Using conservative estimates that assume 100% of crops contain the tolerance level, and a reference dose (RfD) of 0.10 mg/kg/ day (based on the NOEL from the chronic dog study), the calculated maximum potential average daily dose from all sources indicates use of 0.2% of the RfD in the subgroup with the highest aggregate exposure (nonnursing infants). The U.S. EPA estimated the margin of exposure (MOE) for occupational exposure to cloransulam-methyl to be between 2500 and 14,000, based on the use of a NOEL of 10 mg/kg/day (U.S. EPA, 1997a).

87.3  Diclosulam 87.3.1  Identity, Properties, and Uses 87.3.1.1  Chemical Name The IUPAC name for diclosulam is 2,6-dichloro-5-ethoxy7-fluoro[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonanilide; the CAS name is N-(2,6-dichlorophenyl)-5-ethoxy-7-fluor o[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide.

87.3.1.2  Structure See Figure 87.1 and Table 87.1.

87.3.1.3  Synonyms Diclosulam is also known as XR-564 or XDE-564 and is sold primarily in the United States and South America under the trade names including Strongarm, Spider, and Crosser herbicides. The CAS registry number is 145701-21-9.

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

87.3.1.4  Physical and Chemical Properties The empirical formula of diclosulam is C13H10Cl2FN5O3S, with a molecular weight of 406.2. Diclosulam is a solid, with a burnt vanilla odor, though the vapor pressure is low (5  1015 mmHg at 25°C). The water solubility is pHdependent and increases with increasing pH, from 117 mg/l at pH 5 to 4290 mg/l at pH 9, and the log Kow values are 0.047 at pH 7 and 0.448 at pH 9.

87.3.1.5  Uses Diclosulam is a soil-applied, preplanting broadleaf herbi­ cide for use in soybeans and peanuts at maximum label rates 35 and 26 g/ha, respectively.

87.3.2  Toxicity to Laboratory Animals 87.3.2.1  Acute Exposure The acute toxicity of diclosulam is low. The acute oral LD50 in the rat was greater than 5000 mg/kg, the dermal LD50 in the rabbit was greater than 2000 mg/kg, and the 4-h inhalation LC50 in the rat was greater than 5.0 mg/l of air. Diclosulam produced no indications of dermal irritation in rabbits or sensitization in the guinea pig, and only very slight transient eye irritation in the rabbit following acute exposure (Cieszlak and Clements, 1993; Gilbert, 1993a,b, c,d,e; U.S. EPA, 1998).

87.3.2.2  Repeated Exposure The primary target organs identified in dietary toxicity studies were the kidneys (rat) and the liver (rat, mouse, and dog). In the F344 rat, dosages of 500 and 1000 mg/kg/day for 2 weeks resulted in increased liver weights and enlarged ceca in males with no histopathologic changes. The NOEL was 100 mg/kg/day in males and 1000 mg/kg/day in females. Rats were given dietary dosages of 50–1000 mg/kg/day for 13 weeks. At 500 and 1000 mg/kg/day, body weights were decreased, and kidney and liver weights were increased. Very slight to moderate treatment-related hepatocellular hypertrophy was observed in males at 100 mg/kg/day and above, and in females at 1000 mg/kg/day. Kidney changes characterized as slightly to moderately decreased intracellular protein in the proximal tubule epithelium were seen in male rats at 500 mg/kg/day and above, secondary to slightly lower feed consumption in these animals. Slight decreases in red blood cell parameters were noted at 100 mg/kg/day and above. The NOEL from this study was 50 mg/kg/day (Stewart et al., 1992a; Szabo and Davis, 1993a). Dietary exposure of B6C3F1 mice to dosages of 100– 1000 mg/kg/day for 2 weeks resulted in slightly decreased kidney weights in both males and females at the high-dose level (1000 mg/kg/day) and slightly decreased hepatocellular vacuolation (consistent with decreased glycogen content) in

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females at 500 mg/kg/day and above. The NOEL was 500 mg/ kg/day in males and 100 mg/kg/day in females. Dosages of 100–1000 mg/kg/day were given to B6C3F1 mice for 13 weeks. Significant body weight effects were seen in males at 1000 mg/kg/day and in females at 500 and 1000 mg/kg/day, and slight-to-moderate hepatocellular hypertrophy was the primary histopathologic change noted at 500 and 1000 mg/kg/ day in males and females, respectively. Kidney weights were lower in males and females at 500 mg/kg/day and above, but there were no correlative changes in clinical chemistry or histopathologic parameters. The NOEL for subchronic ex­posure in the mouse was 100 mg/kg/day (Grandjean and Szabo, 1993; Stewart et al., 1993). Beagle dogs were given diclosulam at dosages of 50– 500 mg/kg/day for 2 weeks. Dosages of 250 mg/kg/day and above were unpalatable and resulted in severely decreased weight gain or weight loss, degenerative changes in the kidneys, and hepatocellular necrosis. A dosage of 50 mg/kg/ day produced microfocal hepatocellular necrosis in males, but no effects in females. In a subchronic study, dogs were given dosages of 0, 5, 25, or 100 mg/kg/day for 13 weeks. Slight, diffuse centrilobular hepatocellular hypertrophy was observed at 25 mg/kg/day. Higher dosages proved to be unpalatable, with secondary toxicity associated with inanition superimposed on the effects of diclosulam on the liver. The subchronic NOEL for the dog was 5 mg/kg/day (Swaim and Szabo, 1992; Szabo and Rachunek, 1992). In a 21-day repeated dermal application study in rabbits, no dermal or systemic effects were seen at 1000 mg/kg/day, the highest dosage tested (Redmond and Kociba, 1996).

87.3.2.3  Chronic Toxicity and Carcinogenicity Chronic studies in rodents with diclosulam produced adaptive changes in the kidney as the primary effect. In a 2-year study in Fischer 344 rats, decreased body weight and weight gain were observed at 400 mg/kg/day, along with changes in hematology, clinical chemistry, and urinalysis parameters associated with the decreased body weight. Histologically, a slight alteration in tubular morphology, mostly within the corticomedullary junction, was observed in the kidneys at 100 and 400 mg/kg/day. This subtle change in the cytologic character and architecture was considered a slight alteration of the normal physiologic state, rather than a pathologic effect indicative of a toxic injury. No effects were noted in rats at 5 mg/kg/day (Minnema, 1996a). Chronic dietary exposure of B6C3F1 mice to dosages of 50–500 mg/kg/day diclosulam for 2 years produced no treatment-related effects on survival, body weight, feed consumption, or clinical observations. The primary histologic change noted in male mice was a reduced vacuolation of the kidney tubular epithelium at all dose levels at the interim and terminal sacrifices, which correlated with decreased absolute and relative kidney weights. In female mice, minimal focal dilation with hyperplasia of the lining

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epithelium of renal cortical tubules was seen at 100 mg/kg/ day and above. In males, this same focal dilation was seen spontaneously across all groups, including controls. There appeared to be no biologic or toxicologic significance to these microscopic changes. The no-observed-adverse-effect level (NOAEL) in mice following chronic exposure was 50 mg/kg/day (Minnema, 1996b). There was no evidence of tumorigenicity or carcinogenicity in either mice or rats. In beagle dogs fed dosages of 2–25 mg/kg/day diclosulam for 1 year, only slight elevations in mean alkaline phosphatase and creatinine levels in dogs given 25 mg/kg/ day were observed. These slight elevations, however, were considered reflective of the normal variability in this species, and 25 mg/kg/day was the NOEL (Walker, 1996).

87.3.2.4  Mutagenicity In a battery of tests, diclosulam showed no evidence of genotoxic potential. These tests included a bacterial reverse mutation assay (Ames test), an in vitro cytogenetic assay in Chinese hamster ovary cells (CHO/HGPRT assay), an in vitro chromosomal aberration assay in rat lymphocytes, and an in vivo cytogenetic assay in mouse bone marrow (U.S. EPA, 1998).

87.3.2.5  Neurotoxicity No neurotoxicologic effects were noted in rats following acute gavage exposure to up to the limit dose of 2000 mg/kg or in a complete battery of neurologic tests including detailed histopathologic examination following 1 year of exposure via the diet to dosages up to 400 mg/kg/day (Mattsson et al., 1996; Minnema, 1996c).

87.3.2.6  Reproductive Toxicity Treatment with diclosulam had no effect on reproduction or fetal development. In a multigeneration reproduction study in Sprague-Dawley rats at dietary dosages up to 1000 mg/ kg/day, no indications of parental or reproductive toxicity were seen. Gavage dosages of up to 1000 mg/kg/day to pregnant Sprague-Dawley rats on gestation days 6–15 produced no maternal or developmental toxicity. In New Zealand White rabbits, no developmental effects were noted even at gavage dosages up to 650 mg/kg/day on gestation days 7–19, which severely affected maternal feed consumption and weight gain. The maternal NOEL in rabbits was 65 mg/ kg/day, whereas the developmental NOEL was 650 mg/kg/ day (Morseth, 1994; Zablotny, 1996; Zablotny et al., 1996).

87.3.2.7  Absorption, Distribution, Metabolism, and Excretion Metabolism studies conducted with 14C-diclosulam in the F344 rat using dose levels of 5 or 500 mg/kg revealed that

Hayes’ Handbook of Pesticide Toxicology

approximately 80% of a single or repeated (15 days) low dose was absorbed by both males and females. At 500 mg/kg, only 15–20% of a single dose was absorbed. Urinary elimination was rapid in both cases with half-lives of approximately 7–12 h. A higher percentage of the 5-mg/kg dose was excreted in the urine by females (62–68%) than by males (39–43%), with the remainder of the absorbed dose eliminated in the feces. At 500 mg/kg, the majority of the administered dose (82–85%) was found in the feces, with only 6–12% eliminated via the urine in both males and females. Within 72 h, less than 3% of the dose remained in the tissues and carcass in all dose groups. The primary urinary and fecal excretion products were identified as unchanged diclosulam and an OH-phenyl oxidation product. In addition, the N-acetyl cysteine conjugate of diclosulam, and the S-oxide of the N-acetyl cysteine conjugate were excreted in the urine of males and females, whereas the sulfate and/or glucuronide conjugate of the OH-phenyl metabolite was seen only in the urine of male rats. Based on rapid elimination, diclosulam has little potential to accumulate upon repeated administration (Stewart et al., 1996).

87.3.3  Toxicity to Humans No studies are available on intentional human exposure. However, risk assessments using conservative assumptions indicate high margins of safety with diclosulam. Residue studies indicated no detectable residues at a limit of detection of 0.003 ppm, and no likelihood for accumulation. A tolerance level of 0.02 ppm, based on a limit of quantitation of 0.01 ppm, and a reference dose of 0.05 mg/kg/day based on the lowest NOEL (5 mg/kg/day from the chronic rat study) have been proposed (U.S. EPA, 1998). Calculation of a maximum potential average daily dose assuming 100% of proposed crops with residues equal to the tolerance level indicates theoretical exposure to only 0.1% of the RfD in the population with the highest potential expos­ ure (nonnursing infants under 1 year old). The MOE for occupational exposure to diclosulam, calculated using exposure estimates from the U.S. EPA Pesticide Handlers Exposure Database (PHED), is estimated to be greater than 1000 based on the NOEL from the chronic dog study and assuming 100% absorption.

87.4  Florasulam 87.4.1  Identity, Properties, and Uses 87.4.1.1  Chemical Name The IUPAC name for florasulam is 2,6,8-trifluoro-5-me thoxy[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonanilide; the CAS name is N-(2,6-difluorophenyl)-8-fluoro-5-methoxy [1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide.

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

87.4.1.2  Structure See Figure 87.1 and Table 87.1.

87.4.1.3  Synonyms Florasulam is also known as XR-570, XDE-570, or DE570 and is sold either alone or in combination under registered trade names including Primus, Derby, Kantor, and Mustang herbicides. The CAS registry number is 145701-23-1.

87.4.1.4  Physical and Chemical Properties Florasulam is a light-colored solid with an empirical formula of C12H8F3N5O3S and a molecular weight of 359.3. It has a low vapor pressure (7.5  108 mmHg at 25°C) and decomposes at 193.5–230.5°C. The solubility increases with increasing pH, ranging from 84 mg/l at pH 5 to 9400 mg/l at pH 9. Florasulam is highly soluble in acetone (123 g/l) and acetonitrile (72 g/l), but substantially less soluble in octanol (0.18 g/l) and xylene (0.23 g/l). It has a pKa of 4.54 and log Kow values ranging from 1.00 at pH 4 to 2.06 at pH 10.

87.4.1.5  Uses Florasulam is a highly effective postemergence broadleaf herbicide for use in cereals, grassland, and turf. Maximum label use rate for the various crops range from 5 to 10 g per hectare.

87.4.2  Toxicity to Laboratory Animals 87.4.2.1  Acute Exposure Florasulam was essentially nonhazardous by the oral, dermal, and inhalation routes, was nonirritating to skin and eyes, and did not induce delayed contact hypersensitivity in either a modified Buehler test or a Magnusson and Kligman maximization study. The oral LD50 was greater than 6000 mg/kg in the rat and 5000 mg/kg/day in the mouse, the dermal LD50 in the rabbit was greater than 2000 mg/kg, and the 4-h inhal­ ation LC50 in the rat exceeded 5 mg/l (Brooks, 1997; Clements and Cieszlak, 1995; Gilbert, 1995a,b,c,d; Gilbert and Yano, 1995b; Johnson, 1996).

87.4.2.2  Repeated Exposure In dietary studies of 2- to 13-week duration, the kidney was identified as a target organ in rats, mice, and dogs, whereas the liver was also a target organ in dogs. In F344 rats, subacute exposure to dosages of 500 mg/ kg/day and above was associated with karyomegaly and anisokaryocytosis in proximal tubular epithelial cells in males and females, and tubular degeneration with regeneration in females. Individual proximal tubular cell necrosis

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was also seen in both sexes at 1000 mg/kg/day. The NOEL was 100 mg/kg/day. Subchronic studies were conducted in F344 and Sprague-Dawley rats at dosages up to 1000 mg/ kg/day. Dosages of 500 or 1000 mg/kg/day produced necrosis with regeneration in descending proximal tubules and a marginally increased incidence of degeneration with regeneration of renal tubules in females. Papillary mineralization (tubular debris) and papillary necrosis were reported at the highest dosages (800 mg/kg/day). Other highdosage effects included acidic urine (males only), increased kidney weight, perineal soiling, reduced body weight gain and feed consumption (due, at least in part, to reduced palatability of the diet), and reduced red blood cell indices. With the exception of mineralized debris in renal papillae and degeneration and regeneration of cortical tubules, all effects partially or completely resolved by the end of a 4-week recovery period. The subchronic NOEL in rats was 100 mg/kg/day (Liberacki et al., 1996; Redmond and Johnson, 1996a; Szabo and Davis, 1993b). In B6C3F1 mice, subacute exposure to dosages up to 1000 mg/kg/day was without effect (Szabo and Davis, 1992). The only response to subchronic exposure to dosages up to 1000 mg/kg/day was hypertrophy of renal collecting duct epithelial cells at 500 mg/kg/day and above. The subchronic NOEL in mice was 100 mg/kg/day (Redmond and Johnson, 1996b). In Beagle dogs, subacute exposure to a nominal dosage of 450 mg/kg/day was associated with reduced body weight gain and reduced feed consumption (due, at least in part, to reduced palatability of the diet). Hepatic changes characterized by increased liver weight and bile duct hyperplasia in males and females, and bile stasis and hep­ atocellular necrosis in males were observed in this group. At 150 mg/kg/day, the effects were limited to increased liver weight and bile duct hyperplasia. Serum alkaline phosphatase (AP) activity, probably of hepatic origin, was elevated at all dosages, including the low dosage of 50 mg/ kg/day. The increase in serum AP at the low dosage was without histopathological correlate. Therefore, 50 mg/kg/ day was considered a subacute NOAEL. Liver effects were not exacerbated by an extended treatment period, but renal hypertrophy (not seen after 4 weeks of exposure) similar to that reported in rats and mice was evident in dogs after subchronic exposure to 50 mg/kg/day (Sullivan and CroninSingleton, 1995; Sullivan and Singleton, 1995). Repeated dermal exposure to dosages up to 1000 mg/ kg/day for 4 weeks produced only transient dermal irritation in rats during the last week of treatment, with no systemic effects (Scortichini and Kociba, 1997).

87.4.2.3  Chronic Toxicity and Carcinogenicity In chronic dietary studies (1- to 2-year duration), hyper­trophy of a population of renal collecting duct cells identified as -intercalated cells remained the most sensitive ­morphological

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effect in all species. Hypertrophy was present at 50 mg/kg/day in dogs, 125 and 250 mg/kg/day and above in male and female rats, respectively, and 500 mg/kg/day and above in mice. In F344 rats, 2-year dietary exposure to dosages between 10 and 500 mg/kg/day identified the kidney as the only target organ. At the high-dose level (250 mg/kg/day in females and 500 mg/kg/day in males), very slight to slight hyper­trophy of renal collecting duct epithelial cells was evident after 1 year. After 2 years, this change had progressed to a moderate degree in some males. Other effects at this dose level included reduced body weight gain, reduced urinary pH, perineal soiling, reduced red blood cell indices, and renal changes similar to effects seen following subchronic expos­ ure to high-dose levels. At the next dose level (125 mg/kg/ day in females and 250 mg/kg/day in males), very slight to slight hypertrophy of renal collecting duct epithelial cells was evident in males after 1 year, and in males and females after 2 years. Reduced body weight gain, renal papillary mineralization (males), decreases in spontaneous chronic renal disease, reduced urinary pH, and perineal soiling were also seen in these animals. No effects occurred at 10 mg/kg/ day and there was no treatment-related effect on tumor formation in this study (Johnson et al., 1997). In B6C3F1 mice, 2-year dietary exposure to dosages up to 1000 mg/kg/day identified the kidney as the only target organ with hypertrophy of intercalated cells, decreased renal epithelial cell cytoplasmic lipid-like microvacuoles, and a decreased incidence of spontaneous chronic renal disease at 1000 mg/kg/day. Reduced body weights accompanied by minor changes in serum cholesterol and triglycerides were also present at 1000 mg/kg/day. At 500 mg/kg/day, very slight hypertrophy of renal collecting duct epithelial cells occurred in most males and females along with decreases in cytoplasmic lipid-like microvacuoles and spontaneous chronic disease (females only) of renal tubules. No effects occurred at 50 mg/kg/day, and there was no treatment-related tumorigenic or carcinogenic response at any dose level (Quast et al., 1997). In Beagle dogs, 1-year dietary exposure to florasulam revealed kidneys, liver, and adrenal glands as target organs. In the subchronic study, renal hypertrophy and modest elevations of serum alkaline phosphatase and liver weight were the only treatment-related effects seen at 100 mg/kg/day. However, treatment beyond 13 weeks resulted in reduced food consumption and body weight gain in some animals, and significant elevations in serum enzyme activities associated with liver toxicity. The original high dosage of 100 mg/ kg/day was therefore reduced to 50 mg/kg/day on Day 105. Thereafter, food consumption and body weight gain improved and red blood cell indices in females and serum transaminases in both sexes returned to normal. Serum alkaline phosphatase remained elevated to the end of the study. Slight hypertrophy of renal collecting duct epithelial cells and slight vacuolization of the zona reticularis and zona

Hayes’ Handbook of Pesticide Toxicology

fasciculata of adrenal glands were detected histologically in this high-dosage group. The fatty change in the adrenals of dogs represented a slight exacerbation of a spontaneous lesion, not associated with inflammation, necrosis, or clinical chemistry changes, and was considered of uncertain toxicological importance. No histopathological lesions were evident in the liver. The NOEL was 5 mg/kg/day (Stebbins and Haut, 1997). Histological and ultrastructural evaluation of the affected renal collecting duct cells characterized the hypertrophy as a mitochondrial proliferation of -intercalated cells, which functionally are involved in acid-base regulation and contain high levels of H-ATPase and HK-ATPase in the apical membrane (Brown et al., 1988; Garg, 1991; Hamm and Hering-Smith, 1993; Madsen and Tisher, 1986; Stokes, 1993; Verlander et al., 1991). Hypertrophy of intercalated cells has been reported as a physiological response to several factors affecting acid-base homeostasis, including respiratory acid­osis, metabolic acidosis, hypokalemia, and altered serum ad­renal mineralocorticoid levels (Ahn et al., 1996a,b; DeFronzo, 1980; Eiam-ong et al., 1994; Hansen et al., 1980; Madsen et al., 1991; Tsuruoka and Schwartz, 1996a,b; Verlander et al., 1994; Weiner and Wingo, 1997; Wingo and Cain, 1993). However, none of these factors was found to be adversely affected by florasulam (Weiner, 1997). The lack of any adverse sequelae associated with this change suggests that it is an adaptive rather than an adverse response to florasulam.

87.4.2.4  Mutagenicity In a battery of tests, florasulam showed no evidence of genotoxic potential. These tests included an in vitro bacterial reverse mutation assay (Ames test), an in vitro cytogenetic assay in Chinese hamster ovary cells (CHO/ HGPRT assay), an in vitro chromosomal aberration assay in rat lymphocytes, and an in vivo cytogenetic assay in mouse bone marrow cells (Lawlor, 1995; Lick et al., 1995; Linscombe et al., 1995a,b).

87.4.2.5  Neurotoxicity Acute gavage and chronic (1-year dietary) neurotoxicity studies in Fischer 344 rats revealed only nonspecific findings. In both acute and chronic neurotoxicity studies with florasulam, perineal urine staining at the highest dosages was the only treatment-related effect. No other effects were seen following an extensive battery of neurologic tests and neurohistopathological examinations (Mattsson and McGuirk, 1997; Shankar and Johnson, 1996).

87.4.2.6  Reproductive Toxicity In developmental toxicity studies, there were no adverse effects on intrauterine development or prenatal survival

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

in rats or rabbits administered gavage dosages as high as 600–750 mg/kg/day. Maternal effects on survival, feed consumption, and/or weight gains occurred at these high dosages. In SD rats, the embryo-fetal NOEL was 750 mg/ kg/day, whereas the maternal NOEL was 250 mg/kg/day. In New Zealand White rabbits, the NOEL for both maternal and embryo-fetal effects was 500 mg/kg/day. In a twogeneration dietary reproduction study in SD rats at dosages of 10–500 mg/kg/day, parental effects (weight gain, feed consumption, renal changes) were seen only at the highest dosage, with no effects on any reproductive parameter. Transient decreases in neonatal body weights, secondary to decreases in maternal feed consumption, were seen at 500 mg/kg/day. The parental NOEL was 100 mg/kg/day, whereas the NOEL for reproductive effects was 500 mg/kg/ day (Liberacki and Carney, 1997; Liberacki et al., 1997; Zablotny and Carney, 1997).

87.4.2.7  Absorption, Distribution, Metabolism, and Excretion In metabolism studies in F344 rats, single oral doses of 10–500 mg/kg 14C-florasulam were readily and extensively absorbed (90% of a 10-mg/kg dose within 24 h) and rapidly eliminated (plasma t1/2  8–10 h) primarily in the urine (85% of administered dose). The feces contained small amounts of the administered radioactivity (5–17%) depending on the dose. More than 75% of the 14C activity in urine was found to be unchanged florasulam. Two minor metabolites, identified as a free and a conjugated (sulfated) hydro­xyphenyl derivative of florasulam, were found. Feces contained unchanged florasulam and the free hydroxyphenyl-derivative. There was no evidence of hydrolysis of the sulfonamide bridge based on the metabolites found in the urine and feces. The rapid elimination of florasulam from tissues indicated no potential to accumulate upon repeated administration (Dryzga et al., 1996; Hansen, 1997). Absorption following in vivo dermal expos­ ure of rats to a concentrated suspension formulation containing 14C-florasulam was minimal (mean 0.5% over 72 h). Results obtained from in vitro studies were similar to those obtained from the in vivo study (Bounds, 1997; Perkins and Billington, 1998).

87.4.3  Toxicity to Humans No studies are available on intentional human exposure. Risk assessment calculations for the general population and for pesticide handlers indicate a low-risk estimate. Residue studies have indicated no detectable levels in cereal gains at the limit of quantitation. Maximum residue levels (MRLs) of 0.01 ppm in grains, and 0.05 ppm whole plants and straw based on the limit of detection, and a reference dose of 0.05 mg/kg/day on the basis of a chronic NOEL of 5 mg/kg/day in dogs have been proposed. The

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theoretical dietary intake of florasulam from all routes has been estimated to account for 0.1% of the ADI even in children 1–2 years of age, the most highly exposed population subgroup. Margins of Exposure (MOE) for pesticide handlers were determined to be significantly greater than 100 and thus florasulam does not pose a risk for occupational exposure (U.S. EPA, 2007a).

87.5  Flumetsulam 87.5.1  Identity, Properties, and Uses 87.5.1.1  Chemical Name The IUPAC name for flumetsulam is 2,6-difluoro-5-meth yl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonanilide; the CAS name is N-(2,6-difluorophenyl)-5-methyl[1,2,4]triazolo[1, 5-a]pyrimidine-2-sulfonamide.

87.5.1.2  Structure See Figure 87.1 and Table 87.1.

87.5.1.3  Synonyms Flumetsulam (also known as XRD-498) is the generic name for this material which is sold globally under registered trade names including Broadstrike, Python, Preside, and Scorpion herbicides. The CAS registry number is 98967-40-9.

87.5.1.4  Physical and Chemical Properties The empirical formula of flumetsulam is C12H9F2N5O2S, with a molecular weight of 325.3. Flumetsulam is a lightcolored powder at room temperature, with a melting point of 252.9°C, a vapor pressure of 2.8  1015 mmHg at 25°C, a Kow of 1.62 at pH 3.44, and a pKa of 4.60. Flumetsulam is soluble in water at 5.65 g/l at pH 7 and 25°C, but solubility decreases with decreasing pH, and it is less soluble in organic solvents.

87.5.1.5  Uses Flumetsulam is a broad-spectrum, season-long herbicide used in the control of broadleaf weeds in soybeans, corn, and other major crops. Flumetsulam is applied as a soilincorporated preplanting, preemergence, or postemergence herbicide depending on the formulation, at a maximum use rate of 80 g per hectare.

87.5.2  Toxicity to Laboratory Animals 87.5.2.1  Acute Exposure Flumetsulam is essentially nontoxic following acute exposure, with an acute oral LD50 greater than 5000 mg/kg, a dermal LD50 greater than 2000 mg/kg, an acute 4-h

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inhalation LC50 above the highest attainable aerosol concentration of 1.2 mg/l of air. It is a slight, transient eye irritant and produced no signs of dermal irritation in rabbits following acute exposure, nor was there any evidence of dermal sensitization in guinea pigs (Mizell et al. 1988a,b, c,d,e; U.S. EPA, 1993).

levels, with no histopathologic correlates, were reported in both males and females. A dosage of 500 mg/kg/day was considered the NOAEL in females (Cosse et al., 1989). Repeated dermal exposure to dosages of 100 mg/kg/day for 21 days produced very slight epidermal hyperplasia, but no indications of any systemic effects (Stebbins et al., 1990).

87.5.2.2  Repeated Exposure

87.5.2.3  Chronic Toxicity and Carcinogenicity

Subchronic toxicity studies in rats, mice, and dogs indicated a low degree of toxicity following repeated oral exposure. In rats, exposure to dietary concentrations of up to 5% (approximately 6000 mg/kg/day) for 2–4 weeks identified the kidney as the primary target organ. Effects in the kidney consisted of focal necrosis and inflammation of the papilla(e), and tubular epithelial cell degeneration and regeneration, with secondary effects on urinalysis parameters at the highest dosage. The only effect reported at 1000 mg/kg/day was cecal enlargement in males. However, the ceca were histologically normal, and 1000 mg/kg/day was considered the NOAEL following 2–4 weeks of exposure. Rats fed diets containing flumetsulam at dosages of 250–2500 mg/kg/day for 13 weeks exhibited dose-dependent changes similar to those seen after 4 weeks of exposure. The NOAEL in rats following subchronic exposure was 25 mg/kg/day (Yano et al., 1987, 1988; Zempel et al., 1988). In B6C3F1 mice fed flumetsulam for 2 weeks, decreased kidney weights were reported in males at dietary concentrations of 1.5 and 3.0% and in females given 3.0%, which corresponded to dosages 3500 mg/kg/day. The NOEL in mice was 0.5% (approximately 1150–1365 mg/kg/day). B6C3F1 mice given dosages of 100–5000 mg/kg/day for 13 weeks displayed only a minimal increase in centrolobular-to-midzonal hepatocellular eosinophilia at the highest dose level and decreased vacuolation of renal proximal tubular epithelium, which is of doubtful toxicologic significance. The NOEL for mice was 1000 mg/kg/day, and 5000 mg/ kg/day was considered a NOAEL (Bond et al., 1987; Stott et al., 1986). In both rats and mice, the increases in the size and weight of the cecum, observed only at high dosages and unassociated with any histologic changes, were considered adaptive in nature, most likely secondary to the effects of flumetsulam on the microenvironment within the cecum. Beagle dogs were fed flumetsulam at nominal dosages of 100–1000 mg/kg/day (males) or 1500 or 2500 mg/kg/day (females) for 2 weeks. In females, degeneration and regeneration of the renal tubular epithelial cells and lymphocytic infiltration of hepatic sinusoids were reported. The NOEL in dogs was approximately 800 mg/kg/day (nominally 1000 mg/kg/day). In dogs given a dosage of 1000 mg/kg/day for 13 weeks, degenerative microscopic changes in the renal papilla, slight biliary stasis, and hep­atocellular necrosis were observed. At 500 mg/kg/day, slight renal papillary degeneration was noted microscopically in males, and increases in serum AP and globulin levels and decreased serum albumin

Flumetsulam was fed to F344 rats and B6C3F1 mice for 2 years at dosages of 100–1000 mg/kg/day. No treatmentrelated adverse effects were noted in mice (Bond et al., 1991). In rats, atrophy of the renal papilla(e) with secondary hyperplasia and/or mineralization of the pelvic epithelium were noted in males given 1000 mg/kg/day, but not in females, and the NOEL was 500 mg/kg/day (Stott et al., 1991). As was noted following subchronic exposure, cecal enlargement with no accompanying histopathologic changes was observed in both rats and mice following chronic exposure. There was no evidence of a tumorigenic or carcinogenic response in either rats or mice at dosages up to 1000 mg/kg/day. The dog appeared to be the most sensitive species to long-term exposure to flumetsulam. Administration of dosages of 500 mg/kg/day in the diet for 1 year produced inflammatory and atrophic changes in the kidney, accompanied by calculi in females. At 100 mg/kg/day, only increased alkaline phosphatase activity and decreased serum albumin were reported, with no histologic changes in any organs. The chronic NOEL in dogs from this study was 20 mg/kg/day, whereas the NOAEL was 100 mg/kg/day (Yano et al., 1991).

87.5.2.4  Mutagenicity In a battery of tests, flumetsulam showed no evidence of genotoxic potential. Flumetsulam was negative in an in vitro bacterial reverse mutation assay (Ames test), an in vitro cytogenetic assay in Chinese hamster ovary cells (CHO/HGPRT assay), an in vitro rat hepatocyte unscheduled DNA synthesis (UDS) assay, and an in vivo cytogenetic assay in mouse bone marrow cells (U.S. EPA, 1993).

87.5.2.5  Reproductive Toxicity Flumetsulam did not affect development or reproduction in either rats or rabbits. No evidence of maternal toxicity, embryo-fetotoxicity, or teratogenicity was observed in rats following exposure of pregnant females to 1000 mg/kg/day in the diet, though the weights of the ceca were increased, consistent with effects noted in previous dietary studies. No parental toxicity or alterations in reproductive performance occurred in rats given up to 1000 mg/kg/day over two generations. Gavage administration of flumetsulam to pregnant rabbits at dosages of 500–700 mg/kg/day produced dose-related episodes of anorexia, with sequelae

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secondary to the altered nutritional status (deteriorated clinical condition, mortality, stomach erosions, etc.), but no embryo-fetotoxicity or teratogenicity accompanied these maternal effects. The maternal NOEL from this study was 100 mg/kg/day, whereas the NOEL for embryo-fetal development was 700 mg/kg/day (Hanley, 1989; Zempel et al., 1990; Zielke et al., 1988).

sulfonanilide; the CAS name is N-(2,6-dichloro-3-methylphenyl)-5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidine-2sulfonamide.

87.5.2.6  Absorption, Distribution, Metabolism, and Excretion

87.6.1.3  Synonyms

Flumetsulam was rapidly, though incompletely, absorbed in mice and rats, with absorption half-lives of less than 1 h following oral administration of doses of either 5 or 1000 mg/kg. Excretion was also rapid, with a urinary halflife of approximately 5–7 h. Following oral administration of 14C-flumetsulam, approximately 50–75% of the administered radiolabel was excreted in the urine primarily as unchanged parent material, though two minor (20% of urinary radiolabel) metabolites, believed to be conjugates of parent flumetsulam, were found in the urine of mice. Approximately 20–35% of the dose was found in the feces, which represented apparently unabsorbed flumetsulam (based on almost total elimination in the urine of an intravenous dose to rats) and tissue levels of 14C accounted for less than 1.5% of the administered dose. There were no differences in absorption, distribution or elimination based on sex, though slight differences were seen with increasing dose (Pottenger et al., 1991; Timchalk et al., 1988).

87.5.3  Toxicity to Humans No studies are available on intentional human exposure. Risk assessment indicates a low potential risk from normal use of flumetsulam. Residue tolerances of 0.05 ppm have been set by the U.S. EPA for soybeans and for corn grain, fodder, and forage. A reference dose of 1 mg/kg/day was established on the basis of a NOAEL of 100 mg/kg/ day from a 1-year study in dogs. Dietary risk evaluation assuming 100% of crops are treated and residues are at the established tolerance levels indicates only 0.2% of the RfD is used even by the highest exposed subgroup, children 3–5 years (U.S. EPA, 2008). Based on the use rates and the NOEL from the chronic dog study, the MOE for worker exposure is greater than 100 and as such is deemed not to represent a risk to humans.

87.6  Metosulam 87.6.1  Identity, Properties, and Uses 87.6.1.1  Chemical Name The IUPAC name for metosulam is 2,6-dichloro-5,7dimethoxy-3-methyl[1,2,4]triazolo[1,5-a] pyrimidine-2-

87.6.1.2  Structure See Figure 87.1 and Table 87.1.

Metosulam is also known as methoxsulam, XRD-511, XDE-511, and DE-511, and is sold either alone or in combination, under a variety of registered trade names including Tacco, Sansac, Eclipse, Atol, Kompal, and Sinal herbicides. The CAS number is 139528-85-1.

87.6.1.4  Physical and Chemical Properties Metosulam is a cream- to tan-colored powder with a low vapor pressure (7.5  1015 mmHg at 25°C). The empir­ ical formula is C14H13Cl2N5O4S, and the molecular weight is 418.3. The solubility of metosulam in water at 20°C and pH 7 is 700 mg/l. Given a pKa of 4.8, the solubility is pHdependent, with values of 100 mg/l at pH 5 and 5600 mg/l at pH 9 (at 20°C), and the log Kow is 2.12 at pH 5.

87.6.1.5  Uses Metosulam is a broad-spectrum, postemergence broadleaf herbicide intended for use in cereals, maize, pasture, alfalfa, and rice. Maximum label use rates for the various crops range from 5 to 30 g per hectare.

87.6.2  Toxicity to Laboratory Animals 87.6.2.1  Acute Exposure Metosulam has very low acute toxicity. The oral LD50 in Fischer 344 rats and CD-1 mice is greater than 5000 mg/kg. No toxicity, including histopathological changes of eyes and kidneys, was evident in beagle dogs given one to five daily doses of 2000 mg/kg, by gelatin capsule. The dermal LD50 in the rabbit was greater than 2000 mg/kg. The 4-h inhal­ ation LC50 in the rat was greater than the highest attainable concentration of 1.9 mg/l of air. Metosulam, when applied to the intact skin of rabbits, produced no signs of irritation. Following instillation into rabbit eyes, slight conjunctival redness developed within 1 h of treatment, but all treated eyes were normal within 1 day of treatment. There was no indication of contact sensitization in guinea pigs exposed to metosulam using either a Magnusson and Kligman maximization test or a modified Buehler topical patch method (Lockwood, 1989a,b,c; Lockwood and Szabo, 1989a,b; UK Ministry of Agriculture, Fisheries, and Food, 1996).

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87.6.2.2  Repeated Exposure

87.6.2.3  Chronic Toxicity and Carcinogenicity

Repeated exposure toxicity studies were conducted with metosulam in rats, mice, dogs, rabbits, and nonhuman primates (cynomolgus monkeys). In rats, dietary administration of dosages of 500–5000 mg/kg/day for 2 weeks to Sprague-Dawley rats resulted in lower body weights associated with unpalatability and the NOEL was 100 mg/kg/ day. No significant effects were reported in Long Evans rats administered dosages of up to 2000 mg/kg/day for 2 weeks. Following subchronic exposure, the kidney was identified as the major target organ. In a 13-week dietary study, the primary toxicological effects were renal alterations characterized as hypertrophy and nuclear pleomorphism of cells lining the proximal convoluted tubules at 100 mg/kg/day and above. After 4 weeks on control diet, hypertrophy of renal tubular cells had resolved, and nuclear pleomorphism was markedly decreased. The NOEL for subchronic dietary administration of metosulam in rats was 10 mg/kg/day. CD-1 mice administered metosulam in the diet at 100– 5000 mg/kg/day for 2 weeks exhibited centrilobular hepatocellular necrosis and decreased vacuolation in the liver only at 2000 mg/kg/day and above. The NOEL was 1000 mg/kg/ day. The only effect observed in a 13-week dietary study at dosages up to 2000 mg/kg/day, was mild hepatocellular hypertrophy and the NOEL was 250 mg/kg/day. The kidney was identified as the most sensitive target organ in the dog (similar to the rat). In addition, ocular toxicity in the form of retinal damage unique to this species was also observed. Dietary administration of metosulam to Beagle dogs at dosages of 100–1000 mg/kg/day for 14 days produced dose-related decreases in feed consumption and body weights; retinal degeneration, necrosis, and detachment; and degeneration or focal necrosis of distal renal collecting tubules and collecting ducts. Metosulam was fed to dogs at dosages of 5, 25, and 50 mg/kg/day for 13 weeks. Clinical signs of blindness occurred as early as 6 weeks in all dogs administered 50 mg/kg/day. Microscopic examination of the eyes from these dogs showed retinal degeneration with detachment. Choroidal structures (tapetum lucidum, pigmented epithelium, and choroidal blood vessels) and other ocular structures were normal. Ocular tissues from dogs administered 5 mg/kg/ day were normal. In the kidneys, very slight to moderate degeneration of the distal convoluted tubules and collecting ducts of dogs administered 25 mg/kg/day and above was reported, and the NOEL was 5 mg/kg/day. Male and female cynomolgus monkeys exposed to oral dosages of 0 or 100 mg/kg/day for 6 weeks showed no renal or ocular toxicity after detailed examination which included an extensive histopathologic evaluation. Repeated dermal exposure of New Zealand White rabbits to dosages of up to 1000 mg/kg/day for 21 days produced no signs of dermal irritation or systemic effects (UK Ministry of Agriculture, Fisheries, and Food, 1996).

Following chronic (2-y) exposure in Sprague-Dawley rats at dosages of 5–100 mg/kg/day, the primary effects were confined to the kidneys, consistent with the findings following subchronic exposure, and the effects were more severe in male than in female rats. At 100 mg/kg/day, nuclear pleomorphism and hyperplasia of cells of the proximal tubules as well as basophilic adenomas and adenocarcinomas of the renal cortex were observed. At 30 mg/ kg/day, nuclear pleomorphism of proximal tubular cells was present and only a single renal cortical adenocarcinoma, which was within the historical control incidence for this tumor (Charles River Breeding Laboratories, 1987), was observed in this group. Short-term exposure studies demonstrated the presence of mitotic figures and nuclear pleomorphism in the renal cortex of male rats following as little as 1 week of dietary exposure to 100 mg metosulam/ kg/day. Increased mitotic activity measured by BrdU incorporation correlated with the renal tubular epithelial changes noted histologically (UK Ministry of Agriculture, Fisheries, and Food, 1996). This suggested a nongenotoxic mechanism of repeated injury as described by Dietrich and Swenberg (1991) as the probable origin of the renal tumors in the chronic study with metosulam. In CD-1 mice fed dose levels of metosulam of up to 1000 mg/kg/day for 18 months, there was no evidence of any increase in tumor incidence and no effects were noted in any other parameter. Metosulam administered to Beagle dogs at dosages of 3–37.5 mg/kg/day for 12 months produced effects in the eyes and kidneys consistent with the findings of the subchronic study. At 37.5 mg/kg/day, variable retinal degeneration with detachment, beginning with diminished or absent pupillary light reflex, increased tapetal reflectivity, and progressive retinal deterioration, were observed. Degenerative lesions of the distal convoluted tubules and collecting ducts were also observed at 37.5 mg/kg/day. No effects were observed at lower levels, and the NOEL was 10 mg/kg/day (UK Ministry of Agriculture, Fisheries, and Food, 1996). The sensitivity of the dog eye to metosulam appears to be unique to this species. The pathology involved the loss of the photoreceptor layer and its nuclei, together with a collapse of the outer and inner plexiform layers and inner nuclear layer. No retinopathy was associated with the pigmented epithelial layer or in the tapetal cells. It is important to note that retinal pathologies were not detected with metosulam in any other species. Dosages of 300 mg/kg/day for 12 days in rabbits, and up to 1000 and 2000 mg/kg/ day for 13 weeks in rats and mice, respectively, were not associated with any retinal changes. Exposure of mice to 1000 mg/kg/day for 18 months or rats to 100 mg/kg/day for 2 years likewise induced no retinal pathology. Significantly, dosages of 100 mg/kg/day for 6 weeks in the nonhuman primate, the species most closely resembling human ocular

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

anatomy and physiology, also produced no evidence of retinal toxicity. Pharmacokinetic studies using radiolabeled metosulam indicated metosulam localized over the outer layer of the retina in the dog, but no selective localization was detected in the rat or mouse (see below) (UK Ministry of Agriculture, Fisheries, and Food, 1996).

87.6.2.4  Mutagenicity In a battery of tests, metosulam showed no evidence of genotoxic potential. These tests included an in vitro bacter­ ial reverse mutation assay (Ames test), an in vitro mammalian forward mutation test in Chinese hamster ovary cells (CHO/HGPRT assay), a mammalian cytogenetics test in rat lymphocytes, an in vitro unscheduled DNA synthesis (UDS) assay, and an in vivo mouse bone marrow micronucleus test (UK Ministry of Agriculture, Fisheries, and Food, 1996).

87.6.2.5  Reproductive Toxicity Metosulam did not produce any adverse reproductive or developmental effects when tested in rats and rabbits. There were no effects on maternal or developmental parameters in a conventional teratogenicity study in the SD rat at dietary levels up to 1000 mg/kg/day. In New Zealand White rabbits, maternal effects were noted at oral gavage dosages of 100 or 300 mg/kg/day and the maternal NOEL in rabbits was 30 mg/kg/day, but there was no indication of developmental effects at 300 mg/kg/day. In a two­generation reproduction study in Sprague-Dawley rats at dosages of 5–100 mg/kg/day, renal toxicity was observed among the parental rats at 100 mg/kg/day, consistent with the effects noted following chronic exposure, but reproductive performance was unaffected. The NOEL for parental toxicity from this study was 30 mg/kg/day, whereas the NOEL for reproductive effects was 100 mg/kg/day (UK Ministry of Agriculture, Fisheries, and Food, 1996).

87.6.2.6  Absorption, Distribution, Metabolism, and Excretion The metabolic fate of 14C-metosulam in rats, mice, and dogs following single or multiple oral administrations was evaluated. 14C-Metosulam was absorbed rapidly (t1/2  1 h) in all three species, though the extent of absorption was significantly higher in the rat (70%) than in the dog and mouse (20%). The rate of 14C elimination in mice and rats was comparable (t1/2  54–60 h), whereas the elimin­ ation rate in dogs was slightly slower (t1/2  73 h). In all three species, 14C-metosulam and metabolites were excreted in the urine. HPLC analysis of urine samples revealed extensive metabolism in both mice and rats, but much less pronounced metabolism in dogs. Analysis of 14C activity of the dog eyes indicated that this organ, a target for toxicity in the dog, exhibited an affinity for the radiotracer not seen in other species. Histoautoradiographic sections

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of dog eyes revealed radioactivity localized regionally over the outer layer of the retina, whereas analysis of tissues from rats and mice for 14C activity and histoautoradiography indicated a lack of selective affinity for any ocular tissues (Timchalk et al., 1996). The major metabolites were an oxidation product of the 3-methyl moiety of the phenyl ring and a demethylation of the 3-methoxy moiety of the pyrimidine ring. In studies conducted in male rats with 14Cmetosulam labeled in either the phenyl or pyrimidine ring, no evidence of cleavage of the sulfonamide bridge was seen (UK Ministry of Agriculture, Fisheries, and Food, 1996). In vitro dermal penetration studies using rat (SpragueDawley) and fresh human skin demonstrate that less than 1% of the applied metosulam actually penetrated the skin (UK Ministry of Agriculture, Fisheries, and Food, 1996).

87.6.3  Toxicity to Humans No studies are available on intentional human exposure. Risk assessment calculations for the general population and for pesticide handlers indicate acceptable risk estimates. Residue studies have indicated no detectable levels in cereal grains at the limit of quantitation. Maximum residue levels of 0.1 ppm in grains based on the limit of quantitation, and an acceptable daily intake (ADI) of 0.01 mg/kg/day on the basis of the chronic NOEL of 5 mg/ kg/day in rats and a conservative safety factor of 500 have been proposed. Using these values, the maximum theoretical dietary intake (MTDI) of metosulam from all routes of exposure has been estimated to account for 5% of the ADI. An acceptable operator exposure level (AOEL) of 0.04 mg/kg/day has been calculated using a NOEL of 10 mg/kg/day and a safety factor of 250 (UK Ministry of Agriculture, Fisheries, and Food, 1996).

87.7  Penoxsulam 87.7.1  Identity, Properties, and Uses 87.7.1.1  Chemical Name The IUPAC name for penoxsulam is 3-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin2-yl)-,,-trifluorotoluene-2-sulfonamide; the CAS name is 2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c] pyrimidin-2-yl)-6-(trifluoromethyl)benzenesulfonamide.

87.7.1.2  Structure See Figure 87.1 and Table 87.1.

87.7.1.3  Synonyms Penoxsulam is also known as XR-638, XDE-638, and DE-638 and is sold either alone or in combination, under

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a variety of registered trade names including: Citadel, Granite, Ricer, and Rainbow herbicides. The CAS number is 219714-96-2.

87.7.1.4  Physical and Chemical Properties Penoxsulam is an off-white-colored solid. It has a vapor pressure of 1.87 3 10216 mmHG at 20°C. The empirical formula is C16H14F5N5O5S and the molecular weight is 483.37. The solubility of penoxsulam in water at 19°C and pH 7 is 0.408 g/l. At pH 5, solubility is 0.00566 and 1.46 at pH 9.0. The log Kow in unbuffered water at 19°C is 0.354.

87.7.1.5  Uses Penoxsulam is a broad-spectrum systemic broadleaf herbi­ cide with pre- and postemergent uses. It was originally developed as a rice herbicide and is also registered for use on turf, trees, and vines and control of aquatic vegetation. Due to low use rates (17.535 g per hectare) and favorable environmental and human health profiles, penoxsulam has been designated as a reduced-risk pesticide for these uses by the U.S. EPA.

87.7.2  Toxicity to Laboratory Animals 87.7.2.1  Acute Exposure Penoxsulam has very low acute toxicity by the oral, dermal, and inhalation routes. It caused mild eye irritation and only very slight dermal irritation and was not a skin sensitizer (Magnusson and Kligman maximization method). The oral and dermal LD50 values were each greater than 5000 mg/kg in rats while the 4-h inhalation LC50 was 3.50 mg/l, the highest technically attainable aerosol concentration (Bonnette, 2000a,b,c,d,e; Hoffman, 1999).

87.7.2.2  Repeated Exposure The liver and/or kidneys were identified as target organs in rats, mice and dogs following dietary exposure for 4- and 13-week treatment intervals. In a 4-week study in Fischer 344 (F344) rats, urine soiling was noted in females at 500 and 1000 mg/kg/day. Body weight gains were reduced in both sexes at doses 500 mg/ kg/day. Liver (males and females) and kidney (females) weights were also increased at 500 mg/kg/day and multi­ focal hyperplasia and inflammation of the renal pelvic epithelium was observed in females at the same dose levels. Based on these findings, the NOAEL was established at 100 mg/kg/day. Subchronic studies were conducted in Fischer 344 and Sprague-Dawley (CD) rats at doses up to 1000 mg/kg/day. Doses 250 mg/kg/day caused decreased body weight gain and feed consumption, alterations in red

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blood cells and clinical chemistry parameters (males), perineal soiling, and increased liver weights. Slight centrilobular hepatocellular hypertrophy was observed in high-dose males, while very slight to slight mineralization of the renal pelvic epithelium and very slight to slight hyperplasia of the pelvic epithelium was seen in high-dose females. This observation was consistent with an irritant effect of the mineralized material. Similarly, hyperplasia of the transitional epithelium of the renal pelvis was observed in male and female CD rats at 250 mg/kg/day. The NOAEL was established at 50 mg/kg/day in F344 and 100 mg/kg/day in CD rats (Crissman and Dryzga, 2000; Johnson and Baker, 2000; Stebbins et al., 1998). Similar findings were observed in 4-week and 90-day studies in CD-1 mice. Treatment-related increases in absolute and relative liver weights as well as hepatocellular hypertrophy were observed at doses 100 mg/kg. Based on these findings, the NOAEL was determined to be 10 mg/ kg/day (Crissman and Zablotny, 1998; Yano et al., 2000). In a 4-week study, Beagle dogs received up to 0.90% (highest palatable concentration) in the diet. Body weights and feed consumption were reduced in high-dose males and females. Variable but treatment-related increases in serum enzyme activities (ALT, AST, and AP) were observed at doses  0.45%. Treatment-related increases in liver and thymic weights were also observed and histopathological liver and kidney effects (all dose levels in females) were also noted, and alterations in hematological parameters were observed. A NOAEL was not established in female dogs; it was 29 mg/kg/day in males. Similar effects on the liver and kidneys were observed in a subchronic study in which the NOEL was 0.045% or 18 and 20 mg/kg/day in males and females, respectively (Stebbins and Baker, 1998, 2000). In a 28-day percutaneous toxicity study, there were no treatment-related effects at a limit dose of 1000 mg/kg/day (Stebbins et al., 2000).

87.7.2.3  Chronic Toxicity and Carcinogenicity A combined chronic toxicity and carcinogenicity study was conducted in the Fischer 344 (F344) rat and a second species carcinogenicity study was conducted in the CD-1 mouse. Consistent with the subchronic studies, treatmentrelated histopathologic changes were observed in the kidneys and urinary bladder in chronic toxicity studies. In Fischer 344 rats, chronic exposure to penoxsulam at dosages of 50 and 250 mg/kg/day resulted in an increase in severity of chronic progressive glomerulonephropathy, a common lesion in aging rats. Effects observed at 250 mg/ kg/day included increased hyperplasia of the pelvic epithelium, crystals in the urinary bladder lumen, and hyperplasia of the urinary bladder mucosa. These and similar findings in

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

other repeat-dose studies are likely attributable to an irritant effect of precipitated penoxsulam, which was identified as the main component of the urinary bladder crystals (Dryzga and Markham, 2006). Body weights were negatively affected at the top dose in both males and females, while liver and kidney weights were increased. The only neoplasm with a statistically identified increase was large granular lymphocytic (LGL) leukemia in male rats. This was considered unrelated to treatment since a dose response was not present, the incidence was within the historical range reported by the National Toxicology Program (NTP), and it is a common tumor of male F344 rats. This opinion was supported by an independent Pathology Working Group review and various agency evaluations of the data, including the U.S. EPA, which accordingly has not performed a quantitative cancer risk assessment for penoxsulam. Penoxsulam was noncarcinogenic in an 18-month dietary study in CD1 mice; there were no adverse effects at the highest dose tested (Hardisty, 2002; Johnson et al., 2002; U.S. EPA, 2009; Yano and Day, 2002).

87.7.2.4  Mutagenicity In a battery of tests, penoxsulam showed no evidence of genotoxic potential. These tests included an in vitro bacterial reverse mutation assay (Ames test), an in vitro forward mutation test in Chinese hamster ovary cells (CHO/HGPRT assay), a mammalian cytogenetics test in rat lymphocytes, and an in vivo cytogenetic assay in mouse bone marrow cells (micronucleus test) (Day and Shabrang, 1999; Lawlor, 1999; Linscombe et al., 1999a,b).

87.7.2.5  Neurotoxicity Acute and chronic (1-year) neurotoxicity studies were conducted in Fischer 344 rats and did not reveal any specific neurotoxic effect. The NOEL in the acute study was 2000 mg/kg/day, while it was 250 mg/kg/day in the chronic study (Marable et al., 2002; Spencer and Johnson, 2000).

87.7.2.6  Reproductive Toxicity In oral gavage developmental toxicity studies, there were no effects on intrauterine development or prenatal survival at doses as high as 1000 mg/kg/day in CD rats and 75 mg/ kg/day in New Zealand White rabbits. Some maternal body weight effects and clinical signs were seen at the high-dose levels. In rats, the embryo-fetal NOEL was 1000 mg/kg (highest dose tested), while the maternal NOEL was 500 mg/kg/day. In rabbits, the NOAEL for embryo-fetal effects was 75 mg/kg/day (highest dose tested), while the maternal NOAEL was 25 mg/kg/day. In a multigeneration reproduction study in CD rats, dosages ranged from 30 to 300 mg/kg/day. There were no effects on reproduction at the highest dose tested (reproductive NOEL  300 mg/kg/day).

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The parental NOEL was 30 mg/kg/day based on hyperplasia of the renal pelvic epithelium at 100 mg/kg/day. The NOEL for offspring was also 30 mg/kg/day based on slight delays in preputial separation likely secondary to decreased body weight (Carney et al., 2000, 2002; Marty et al., 2001).

87.7.2.7  Absorption, Distribution, Metabolism, and Excretion In metabolism studies in F344 rats, a single oral gavage dose of 5 mg/kg of 14C-penoxsulam was readily and extensively absorbed and rapidly eliminated. Saturation of absorption was evident at a higher dose of 250 mg/kg/day. At the low dose, excretion of radiolabeled penoxsulam was primarily through the fecal route in males, while females eliminated radioactivity primarily in the urine and to a lesser extent in the feces. At the higher dose, elimination was primarily through the feces for both genders, likely representing saturation of absorption. The majority of the radioactivity (86% in urine) was excreted as unchanged penoxsulam; however, penoxsulam is biotransformed to 36 different metabolites. Metabolites identified in excreta were generally detected at low levels, although some fecal metabolites were shown to account for 5% of the administered dose. Penoxsulam was shown to be metabolized primarily through demethylation and ring hydroxylation with subsequent conjugation to glucuronic acid or glutathione. The rapid absorption and excretion of penoxsulam indicate no potential for bioaccumulation. Absorption at 24 h following dermal exposure of F344 rats was approximately 2% for undiluted and 0.4% for a spray dilution (0.03 g/l) of penoxsulam (Mandrala et al., 2002).

87.7.3  Toxicity to Humans No studies are available on intentional human exposure. Risk assessment calculations for the general population and for pesticide handlers indicate a low-risk estimate. Maximum residue levels (MRLs) of 0.01–0.02 ppm for crops have been established based on the analytical limit of detection and a reference dose of 0.147 mg/kg/day resulting from the chronic dog study (U.S. EPA, 2007c).

87.8  Pyroxsulam 87.8.1  Identity, Properties, and Uses 87.8.1.1  Chemical Name The IUPAC name for pyroxsulam is N-(5,7-dimethoxy[1,2, 4]triazolo[1,5-a]pyrimidin-2-yl)-2-methoxy-4-(trifluorom ethyl)pyridine-3-sulfonamide; the CAS name is N-(5,7-di methoxy[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-2-methoxy4-(trifluoromethyl)-3-pyridinesulfonamide.

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87.8.1.2  Structure See Figure 87.1 and Table 87.1.

87.8.1.3  Synonyms Pyroxsulam is also known as BAS 770H, X666742, XR742, XDE-742, DE-742; its CAS number is 422556-08-9. It is sold in plant protection products, either alone or in combination with other herbicide active ingredients, under a variety of registered trade names including: Crusader, Pallas, Perun, and Simplicity herbicides.

87.8.1.4  Physical and Chemical Properties Pyroxsulam has an off-white color. It has a vapor pressure 7.5  1010 Pa at 20°C. The empirical formula is C14H13F3N6O5S and the molecular weight is 434.4. The solubility of pyroxsulam in water at 20°C and pH 7 is 3.20 g/l.

87.8.1.5  Uses Pyroxsulam is a broad-spectrum systemic grass and broadleaf postemergence herbicide.

87.8.2  Toxicity to Laboratory Animals 87.8.2.1  Acute Exposure Pyroxsulam has very low acute toxicity by the oral, dermal, and inhalation routes. It caused slight, transient skin and eye irritation and was identified as a skin sensitizer in guinea pigs by the Magnusson and Kligman maximization method. The oral and dermal LD50 values were each greater than 2000 mg/kg in rats while the 4-h inhalation LC50 was 5.0 mg/L (Gamer and Leibold, 2003a,b, 2004; Kaufmann and Leibold, 2003a,b; Lowe, 2007).

87.8.2.2  Repeated Exposure Short-term dietary studies in rats, mice, and dogs and a short-term dermal study in rats were conducted. The liver was identified as the primary target organ in all species; serum total cholesterol was also universally increased. In a 4-week study in Fischer 344 (F344) rats, there were no adverse effects at doses up to 1150 mg/kg bw/ day (Stebbins and Day, 2001). In a 13-week study, body weight gain was reduced in females at 1000 mg/kg bw/day. There was evidence for a slight treatment-related effect on the liver of males at 1000 mg/kg bw/day: increased relative liver weight (and serum cholesterol) but without associated histopathological change it was not considered an adverse finding. By 28 days post-treatment, there was complete recovery of liver weight and a marked recovery of the cholesterol level (Stebbins et al., 2003). Similarly,

Hayes’ Handbook of Pesticide Toxicology

in a 13-week study in CD-1 mice the only effects were increased liver weight and serum cholesterol at a limit dose of 1000 mg/kg bw/day (Johnson et al., 2003). In a 13-week study in Beagle dogs, apparent effects comprised reduced body weight gain, increased liver weight, hepatocellular hypertrophy, and increased serum cholesterol at a high dose of 884 and 1142 mg/kg bw/day in males and females, respectively (Stebbins and Baker, 2003). In a 1-year study, increased liver weight without hepatocyte hypertrophy was seen in both sexes at the top dose of approximately 600 mg/kg bw/day. There was also evidence of increased serum cholesterol and alkaline phosphatase activity, which might reflect minimal cholestasis (Stebbins and Dryzga, 2004). Based on these studies, the overall short-term NOAEL was 90 mg/kg bw/day based on the minor effects seen at high-dose levels in the range of approximately 600–1000 mg/kg bw/day. In a 14-day dermal toxicity study in F344 rats, there were no treatment-related effects at the limit dose of 1000 mg/kg bw/day (Kaspers, 2004).

87.8.2.3  Chronic Toxicity and Carcinogenicity A combined chronic toxicity and carcinogenicity study was conducted in the Fischer 344 (F344) rat and a second species carcinogenicity study was conducted in the CD-1 mouse. In the F344 rat study, dose levels of 0, 10, 100, and 1000 mg/kg bw/day were administered via the diet for 2 years (Stebbins and Brooks, 2005). Body weight gain and feed consumption were slightly reduced in high-doselevel females. Slight increases in serum total cholesterol and liver weight, in the absence of any other associated changes including histopathologically, were considered nonadverse, adaptive changes. There were no treatmentrelated increases in neoplasms in male or female rats at any dose level, indicating that pyroxsulam did not have an carcinogenic potential under the conditions of this study up to and including a limit dose. The CD-1 mouse study dose levels of 0, 10, 100 and 1000 mg/kg bw/day were administered via the diet for 18 months (Johnson et al., 2005). The only treatmentrelated effects were in the liver of high-dose males and comprised increased liver weight, increased incidence of foci of altered hepatocytes, and a possible marginal increased incidence and number of hepatocellular adenomas and carcinomas. Doubt exists over an association with treatment as there was no dose–response relationship and differences from concurrent controls were not statistically identified but values for adenomas were just outside historical control ranges. Toxicokinetic data demonstrated systemic AUC values were less than dose proportional at the high-dose level but increased 30-fold from 10 to 1000 mg/kg bw. Pyroxsulam did not to cause any ­genotoxicity effects in a

Chapter | 87  Toxicology of Triazolopyrimidine Herbicides

complete battery of tests, including an in vivo mouse liver UDS assay conducted with a limit dose of 2000 mg/kg bw. Under the conditions of this study, the NOAEL was 100 and 1000 mg//kg bw/day for male and female mice, respectively.

87.8.2.4  Mutagenicity In a battery of tests, pyroxsulam showed no evidence of genotoxic potential. These tests comprised an in vitro bacterial reverse mutation assay (Ames test; Engelhardt and Leibold, 2003), an in vitro mammalian cytogenetics test in CD rat lymphocytes (Schistler, 2006), an in vitro mammalian forward mutation test in Chinese hamster ovary cells (CHO/HGPRT assay; Schisler and Grundy, 2006), and two in vivo assays in the CD-1 mouse: a cytogenetic assay in bone marrow cells (micronucleus test; Spencer and Grundy, 2004), and an unscheduled DNA synthesis (UDS) assay (Beevers, 2006), both of which involved oral gavage dosing at the limit dose of 2000 mg/kg bw.

87.8.2.5  Reproductive Toxicity Developmental studies in CD rats and NZW rabbits and a two-generation reproduction study have been completed for pyroxsulam. In the reproduction study in the CD rat, dose levels of 0, 10, 100, and 1000 mg/kg bw/day were administered via the diet (Carney et al., 2005). There was no evidence of systemic toxicity or adverse effects on any parameter of reproductive function of the parental animals, nor on survival, growth, or development of F1 or F2 offspring. The rat developmental toxicity study assessing dose levels of 0, 100, 300, and 1000 mg/kg bw/day via gavage produced no treatment-related maternal toxicity and no indications of embryo-fetal toxicity or teratogenicity (Carney and Tornesi, 2005). In the rabbit, doses of 0, 30, 100, and 300 mg/kg bw/day were also administered by gavage (Sloter, 2005). The high-dose level was based on body weight effects at 300, 600, and 1000 mg/kg bw/day in a probe dose-range finding study. However, no maternal toxicity, or effects on embryo-fetal survival and development, occurred in the main study.

87.8.2.6  Absorption, Distribution, Metabolism, and Excretion Studies of absorption, distribution, metabolism, and excretion (ADME) have been conducted in rats and mice. The study in male Fischer 344 (F344) rats was of typical regulatory test guideline design and investigated two dose levels, a low and a high dose of 10 and 1000 mg/kg body weight, respectively (Hansen et al., 2005). The mouse study was conducted to provide toxicokinetic information to aid investigation of the possible mechanism and relevance

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for human risk assessment of apparent treatment-related liver tumors in high-dose-level male mice following lifetime exposure to pyroxsulam (Hansen et al., 2006). The study investigated the three dose levels used in the mouse carcinogenicity study (10, 100, and 1000 mg/kg bw) in males; limited data were also generated for females. In rats, a single oral gavage dose of 10 mg/kg of 14Cpyroxsulam was readily and extensively absorbed (ca. 75%) and rapidly eliminated. Absorption was slightly lower at the limit dose of 1000 mg/kg. Excretion of radiolabeled pyroxsulam was rapid and virtually complete within 48 hours, primarily through the urine but with a small biliary component (ca. 17% of the total) identified from animals dosed by the intravenous route. The majority of the radioactivity (85%) was excreted as unchanged pyroxsulam and only one substance, the 2-desmethyl metabolite, representing at least 5% of the administered dose. There were no differences in the findings between single or repeat, 15-day, dosing and no evidence for metabolic induction (no alteration in metabolism of pyroxsulam). The rapid and extensive excretion with very low levels in carcass at 48 h after dosing (1% of administered dose) indicate no potential for accumulation. The investigatory study in the mouse revealed results similar to those outlined for the rat. Absorption was rapid and extensive and plasma elimination was rapid (t½ of 2–3 h) and excretion was primarily in urine.

87.8.3  Toxicity to Humans No studies are available on intentional human exposure. Risk assessment calculations for the general population and for pesticide handlers indicate a low-risk estimate. A reference dose of 1 mg/kg/day was established on the basis of a NOAEL of 100 mg/kg/day from the carcinogenicity study in mice. Dietary risk evaluation assuming 100% of crop are treated and residues are at the established tolerance level (0.01 ppm) indicates that the chronic dietary risk estimates for the U.S. population and all population subgroups utilize 0.1% of the RfD (U.S. EPA, 2007b). Risk assessment calculations for the general population and for pesticide operators, field workers, and bystanders indicate exposures significantly below proposed reference doses for pyroxsulam.

Conclusion In conclusion, the triazolopyrimides are a structurally-related class of herbicides which act to inhibit the enzyme acetolactate synthase (ALS) in plants. This target does not exist in animals and accordingly this class of chemicals exhibits low toxicity in mammals. The triazolopyrimidine herbicides have been comprehensively evaluated in guideline and GLP compliant toxicity studies required for the registration and

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authorization of pesticides in various geographies throughout the world. In general, they exhibit very low mammalian toxicity as assessed through acute, short-term, long-term (chronic), genotoxicity, reproduction, developmental, and neurotoxicity studies. In repeat-dose toxicity studies, the liver and kidneys have been identified as target organs with effects that were often adaptive in nature generally observed only at excessively high-dose levels. In addition, the triazolopyrimidines were shown to be rapidly absorbed and excreted, have a low potential for bioaccumulation, and in general are not extensively metabolized.

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