MEMORANDUM

SUBJECT : Review of Chlorsulfuron Studies Published Since Publication of the Reregistration

Eligibility Decision for Chlorsulfuron (U.S. EPA, 2005)

DATE : April 24, 2013

FROM : Jocelyn Hospital

Toxicologist Analytical Support Branch

TO : Daniel Bushman

TRI Petitions Coordinator Analytical Support Branch

I have reviewed all relevant studies on chlorsulfuron that have been published since the Reregistration Eligibility Decision for Chlorsulfuron (U.S. EPA 2005). This memo randum includes brief summaries o f the following studies: Mylchreest, 2005; Rudaya and Zhminko, 2009; Rudaya et al., 2010; and Rakitsky and Beloyedova, 2009

.

I. STUDY SUMMARIES

Mylchreest, 2005 : In a 2

- generation reproduction study, Mylchreest (2005) administered chlorsulfuron (97.6%) to 30 Crl:CD®(SD)IGS BR rats via the diet in an EPA guideline

- and GLP

- compliant study. The administered dose levels were 0, 100, 500, 2500, and 7500 ppm (average daily do ses of 0, 6, 30, 151, 456 mg/kg

- day in mal es and 0, 7, 39, 188, 591 mg/kg

- day in females) throughout the 10

- week premating period and throughout gestati on and lactation.

There was one set of litters per generation. P generation animals were randomly assigned to test groups (ranked by mo st recent body weight). The 100, 500, and 2500 ppm dose levels were selected because they were expected to produce no or minimal toxic effects based on a previous reproduction study ( Alvarez , 1981). The 7500 dose level was selected because it was expected to produce some systemic toxicity without mortality and provide additional reproductive data at a hig her dose than previ ously tested.

Each female was housed with a randomly

- selected, non

- sibling male from the same dose group. During cohabitation, females were examined daily for evidence of copulation (either the presence of an intravaginal copulation plug or sperm in vagin al lavage sampl es). On the day of copulation (d ay 0 of gestation) or when two weeks had elapsed, the females were returned to individual cage housing. P parental animals were given test diets for at least 70 days before mating, while F1 parental animals we re not mated until at least 70 days after they were selected from the F1 litters. Selection of F1 parental animals was random and occurred when the pups were 21 days of age and the mated animals were approximately 18 (P1) and 13 (F1) weeks of age at mating

. Feed formulations were prepared weekly or every two weeks when refrigerated stability of the test material was confirmed. Batches of feed were used within the established stability period and then discarded. Samples were analyzed to verify homogeneity and concentration of the test material in the diets. Duplicate samples from each dose level were taken and used to verify concentration at 3

- month interval s during the study. Test substance intake was also analyzed, and the authors reported that dams inbo th generations consumed approximately twice as much test material during the first two weeks of

lactation as they did during the premating and gestation periods.

Body weights, food consumption, and reproductive and developmental endpoints were observed. Ovarian follicular counts (a quantitative evaluation of primordial and growing follicles) were conducted on all lactating F1 females from control and high

- dose groups. All surviving P1 and F1 adult rats were sacrificed and subject to a gross pathological e xamination. The uteri of all cohabited females were examined for the presence and number of implantation sites. The reproductive organs and tissues of all rats with suspected impaired reproductive performance (e.g., failure to mate, conceive, sire, or deliver healthy offspring) were evaluated microscopically. The include : ovaries, uterus with oviducts and cervix, vagina, adrenal gland, pituitary glan d, testes, epididymides, prostate, seminal vesicles with coagulating glands and fluids, and lesions. All of these organs and tissues were also weighed except for the vagina and lesions. Additionally, r eproductive indices (e.g., mating index, fertility inde x, gestation index, implantation efficiency) were calculated from breeding and parturition records of P1 and F1 animals in the study. Offspring viability indices (pups born alive, 0

– 4 day viability index, lactation index, litter survival) were calculated from lactation records of litters in the study.

The authors observed no treatment

- related effects on mortality, clinical signs, organ weights, or gross/microscopic findings in adult rats from both the P and F1 generations. During the premating period, ho wever, the authors reported changes in body weight and body weight gain in males and females from both generations. A s light reductions in body weight (94

– 96% of control group) and body weight gain (77

– 89% of control group) in P 1 males during some week s in the highest dose group were accompanied by decreased in food efficiency (80

– 90%), but there was no treatment

- related body weight gain over the duration of the premating period (day 0

– 105)

. P 1 females in the highest dose group exhibited a significant decrease in body weight gain (74% of control group) in the first week of the study accompanied with decreased food efficiency (72% of control group) , but all body weight measures reverted to normal over the course of the premating period

. A sli ght reduction in body weight (94% of control group) and body weight gain (93

– 94% of control group) was observed in F1 males in the highest dose group at the end of the premating period (day 70). Similar reductions in body weight (92% of control group) an d body weight gain (91% of control group) were observed in the F1 females in the highest dose group at the end of the premating period (day 70) accompanied by decreased food efficiency throughout the study (week 0

– 10)

. Body weight and body weight gain, h owever, were comparable among all animals during gestation and lactation in both generations except slight decreases in body weight seen in F1 females in the highest dose group o n day 0 of gestation and day 7 o f lactation. The authors attributed slight inc reases in relative organ weight ( e.g., kidney, testes, epididymis) observed in both adult generations to decreased body weights and not to the test material

. The authors reported a parental LOAEL of 456 mg/kg

- day in males and 498 mg/kg

- day in females (note: the administered dose of 591 mg/kg

- day was adjusted for decreased intake in females during gestation) based on changes in body weight and food efficiency.

No treatment

- related effects were reported in litter size, live birth index, number born de ad, viability and lactation indices, clinical examinations, sex ratio, sexual maturation, organ weights, and gross or microscopic observations. The F1 sex ratio was significantly higher in the highest dose group (55% versus 45% males in the control group), but the authors did not consider this a treatment

- related effect because it fell within the historical control range (45

– 59%). Lower offspring body weights were observed in the highest dose group in both generations, but these differences were not consi dered adverse because the magnitude of body weight changes was slight (5

– 7%) and the potential effect of larger litter size on pup weight . The authors reported an offspring NOAEL of 456 mg/kg

- day in males and 498 mg/kg

- day in females (note: the administered dose of 591 mg/kg

- day was adjusted for decreased intake in females during gestation) , the highest dose tested.Lastly, there were no treatment

- related effects on ovarian follicles counts in F1 females, spe r m

an d estrous cycle parameters in P and F1 adults, mating, precoital interval, fertility, gestation length, number of implantation sites, and implantation efficiency in either generation. As such, the authors reported a reproductive NOAEL of 456 mg/kg

- day in males and 498 mg/kg

- day in female s (note: the administered dose of 591 mg/kg

- day was adjusted for decreased intake in females during gestation) , the highest dose tested.

These results demonstrate that chlorsulfuron did not cause any treatment

- related reproductive toxicity and its effects on parental body weight and food efficiency indicate moderately low to low toxicity.

Rudaya and Zhminko , 2009: In an acute oral toxicity study, Rudaya and Zhminko (2009) examined the effect of chlorsulfuron potassium salt administered intragastrically in male and female non

- pedigreed white rats, male and female mice, and male rabbits of the Chinchilla line. The authors did not report further details on animal speci es or strain . Chlorsulfuron potassium salt was administered once as an aqueous OP

- 7 (alkylaryl polyglycol ether emulsifier) at doses ranging from 1000 to 7000 mg/kg ( n = 6 rats per dose group ; sample sizes for the mice and rabbit tests were not reported ). The body weights of the study animals were not reported, but the authors reported that body weights ranged from 180

– 190g in the rats, 20

– 22g in the mice, and 2.10

– 2.30 kg in the chinchilla rabbit. The age of rats, mice, and rabbits at the time of the study were not reported.

The authors used behavior, outward appearance, intoxication onset time and type, and date of death as the main measures of toxicity. Mean lethal doses (LD 50 ) were calculated using the least

- squares method for probit analysis of mortality curves. In addition, the nature of toxic effects after a single oral dose of chlorsulfuron potassium salt (2790 mg/kg) was examined in male rats. Behavior (motor activity, orientation reaction, and exploration activity) and the prese nce and onset time of clinical signs of intoxication (no definitions of these signs were noted in the study ) were observed daily in these male rats. Other physiological and biochemical factors were observed at days 1, 3, 5, 10, and 15. These other factors included hematology, measures of liver function, measures of carbohydrate, measures of renal function, measures of homeostasis, measures of mineral metabolism, measures of thyroid function, and measures of adrenocortical function.

The authors concluded th at the LD 50 was 5580 ± 1002 mg/kg for male rats, 5500 ± 729 mg/kg for female rats, 2050 ± 367 mg/kg for male mice, 2460 ± 312 mg/kg for female mice, and 3900 ± 451 mg/kg for male rabbits. Significant behavioral changes (reduction of orientation reaction, d ecreased exploratory activity, and decreased motor activity) were observed in the day after administration of chlorsulfuron potassium salt in male rats, but no significant difference was found between the experimental and the control groups later in the st udy (i.e., days 3, 5, 10, and 15). Similar transient differences between the experimental and the control groups were found for a multitude of physiological and biochemical factors including: hematological parameters, measures of liver function, measure of homeostasis, electrolyte concentrations, and hormone concentrations in blood serum (including thyroid hormones, cortisone, and insulin). It is difficult to draw conclusions about the study’s findings , however, due to the lack of basic information provided by the authors on study design, analytical methods, and results. As such, findings from this study were not considered in EPA’s determination of hazard for chlorsulfuron.

Rudaya et al., 2010: In a recent chro nic oral toxicity study , Rudaya et al. (2010) examined the effect of chlorsulfuron potassium salt administered intragastrically in male white rats on various health endpoints. The authors did not report further details on animal species or strain

. Chlorsul furon potassium salt was administered as an aqueous OP

- 7 (alkylaryl polyglycol ether emulsifier) at dose levels of 0, 0.558, 5.58, and 55.8 mg/kg ( 6 rats/ dose group) over 9 months

. The body weights of the rats at the time of the study were not reported, but the authors reported that body weights ranged from 130

– 140 g. The age of rats at the time of the study was not reported.Physiological and biochemical factors were observed at 1, 3, 6, and 9 months. These factors

included intoxication symptoms ( no d efinitions of these signs were noted in the study ) , body weight (no body weight tables were presented in the study ) , behavior, hematology , measures of liver function,

measu res of carbohydrate metabolism, measures of renal function, measures of homeostasis, measures of mineral metabolism , measures of thyroid function

, and gross pathology of selected organs

. The authors reported no increases in the mortality rate, incidence of intoxication symptoms , or significant changes in body weight gain. Measured hematol ogical values did not indicate treatment

related effects of chlorsulfuron on white and red blood cell counts or on hemoglobin content.

The authors reported a multitude of effects of chlorsulfuron potassium salt on the liver, kidneys, heart, and thyroid gland, but due to the lack of information on study design, methods, and results, it is unclear whether these effects are statistically or biologically significant . The authors did not report whether some of these findings represented statistically signific ant changes as compared to the control group (e.g., protein and urea content in the urine, urine pH) and many of the measured parameters demonstrated only transient effects in the treatment groups (e.g., blood serum concentrations of alanine aminotransfera se, aspartate aminotransferase , cholesterol). Additionally, some of the measured parameters demonstrated multidirectional changes throughout the study period. For example, the authors noted that Na + and K + concentrations in the urine significantly increase d compared with the control group in month 1 and decreased compared with the control group in month 3, 6, and 9 in the highest dose group. In another example, t he authors reported a statistically significant increase in blood serum concentrations of glucos e in the highest dose group (55.8 mg/kg) in months 1 and 3 and reported a statistically significant decrease in blood serum concentrations of glucose in the highest dose group in months 6 and 9

. The authors concluded that chlorsulfuron potassium salt cause d hyperglycemia at the beginning of the study period and hypoglycemia at the end of the study period due to the acti on of sulfonylurea derivatives (such as chlorsulfuron potassium salt ) on hydrocarbon metabolism.

The authors observed transient effects of chlorsulfuron treatment on behavior

. There was a significant decrease in central nervous system function in the highest dose group (55.8 mg/kg ) in month 1 as measured by orientation response (the number of squares crossed per minute) and hole

- board exploratory behavior (the number of holes investigated in 3 minutes). The reported changes at month 1 were 74.7% for orientation response and 56.4% for exploratory behavior. The authors also reported a significant decrease (55.4%) in explora tory behavior in this dose group in month 9, but they did not report any changes in the highest dose group in month 3 or 6 of the study. Additionally, the authors observed a significant decrease (30.1%) in exploratory behavior in the second highest dose gr oup (5.58 mg/kg ) in month 1 and a significant decrease (54.4%) in month 3. H owever, no changes were reported in this dose
group in month 6 or 9.

The authors reported several statistically significant changes in liver function in the highest dose group (55 .8 mg/kg). These changes included an increase in lipid peroxidation intensity (500%), an increase in amidopyrine N

- dem e thylation activity in the post

- mitochondrial fraction of the liver (74.5%), and a decrease in NAD content (5.3%) compared with the contro l group at the end of the study period. The authors noted that these findings are indicator s of monooxygenase induction and intensification of lipid peroxidation in the liver.

Morphological changes were reported in the highest dose group (55.8 mg/kg ) based on the findings from pathomorphological examination of various organs. All of the organs assessed in this dose group displayed signs of physiological stress (microcirculatory disturbances and lymphoproliterative

rea c tions), while more substantial c hanges were observed in the lungs (e.g., perivascular edema), kidneys ( e.g., atrophy of individual tufts, cellular micronecrosis), thyroid gland (e.g., small follicle structure, proliferation of interfollicular cells), and adrenal glands (e.g., disorganiza tion of cortical cells, focal heter otrophy of zona glomerulosa, nodular degeneration of reticular zone cells ).Based on these findings, the authors concluded that the no

- effect dose of chlorsulfuron potassium

salt is 0.558 mg/kg

. It is difficult to draw co nclusions about the study’s findings, however, due to the lack of basic information provided by the authors on study design, analytical methods, and results. As such, findings from this study were not considered in EPA’s determination of hazard for chlorsulfuron.

Rakitsky and Beloyedova, 2009 : In an acute and chronic study of several sulfonylurea herbicide s, Rakitsky and Beloyedova (2009) administered chlorsulfuron to rats, mice, dogs, and rabbits. Neither species nor strains were reported by t he authors. No details on dosing (including specific method of administration) were provided by the authors and it is not apparent that a concurrent control group was used . The authors measured central nervous function, liver, kidney, and hematologic funct ion. The authors reported an oral LD 50 in rats of 5,545

– 6,293 mg/kg in males and females, respectively, and a dermal LD 50 of 2,500 mg/kg in rabbits. The authors also reported an LC 50 (4 hours) of > 5,900 mg/kg in rats. In the chronic studies, the authors performed testing at 0.5, 3, 6, 9, and 12 months after exposure. They reported general changes in body weight, nervous system (summation threshold index, hole exploratory behavior), hematology, liver function, kidney function, and morphological changes fo r the 10 sulfonylurea herbicides studied, but they provide no further description of the results nor do they include chemical

- specific findings. The authors did note that chlorsulfuron exposure increased the frequency of benign testicular tumors in rats sl ightly exceeding historical controls. The authors reported a chronic no

observed

- effec t level (NOEL) of 0.2

– 5 mg/kg

- day for rats, 108 mg/kg

- day for mice, and 60.6 mg/kg

- day for dogs, but they did not indicate from which health endpoints these NOELs were derived. Again, it was unclear what other doses were tested in these animals. It is difficult to draw conclusions about the study’s findings due to the lack of basic infor mation provided by the authors on study design, analytical methods, and results. As such, findings from this study were not considered in EPA’s determination of hazard for chlorsulfuron.

II. CONCLUSION

No additional a cceptable studies that strongly suggest ei ther acute or chronic toxicity of chlorsulfuron were identified in the literature

. A relatively recent guideline studies (Mylchreest, 2005) was not able to replicate findings one of the studies upon which the addition of chlorsulfuron to the list of toxic chemicals subject to reporting requirements of section 313 of the Emergency Planning and Community Right to Know Act (EPCRA) was based ( Wood , 1981). The reported f indings from the other additio nal studies (Rudaya and Zhminko , 2009; Rudaya et al., 2010 ; Rakitsky and Beloyedova, 2009 ) were not considered in EPA’s determination of hazard for chlorsulfuron due to the study deficiencies outlined prev iously. As such, the re is no compelling evidence of the acute toxicity, carcinogenicity, reproductive or developmental toxicity, genotoxicity , or other chronic toxicity of chlorsulfuron

. While

treatment

- related body weight changes wer e observed in the Mylchreest (2005) study

, t hese changes were observed at a relatively high dose level (close to 500 mg/kg

- day) and were observed in the absence of any other treatment

- related effects

. Therefore , chlorsulfuron is not reasonably anticipated to cause serious or irreversible acute or chronic toxicity in humans.

III. R EFERENCES

Mylchreest, E. 2005. Chlorsulfuron (DPX

- W4189) Technical: Multigeneration Reproduction Study in Rats. DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE, Laboratory Project ID: DUPONT.13495,14601,904. September 11, 2003

– June 4, 2004. MRID 46493201. Unpublished. 

### Data Evaluation Record: Taylor, Linda (2007), MRID 46493201.

Rakitsky, V.N. and Beloyedova, N.S. 2009. Toxicity and Hazardousness of Sulfonylurea Herbicides. Toxicology Herald 4: 25

– 30 . Translated from Russian

.Rudaya, P.L., and Zhminko , P.G. 2009. Toxic Properties of Chlorsulfuron Potassium Salt Herbicide

Administered Once Orally to Mammals. Modern Problems of Toxicology 2: 59

– 65. Translated from Ukra i nian

. Rudaya, P.L., Zhminko, P.G., Povyakel, L.I., and Reshavska, O.V. 2010. Toxicod ynamics of Chlorsulfuron Potassium Salt Given Orally In Long

- Term Experiment on White Rats. Modern Problems of Toxicology 1: 59

– 63. Translated from Ukra i nian

.