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 memorandum includes brief 

summaries of 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 doses of 0, 6, 30, 151, 456 

mg/kg-day in males and 0, 7, 39, 188, 591 mg/kg-day in females) throughout the 10-week premating 

period and throughout gestation and lactation. 

There was one set of litters per generation. P generation animals were randomly assigned to test 

groups (ranked by most 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 higher dose than 

previously 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 vaginal lavage samples). On the day of copulation (day 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 were 

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 

intervals during the study. Test substance intake was also analyzed, and the authors reported that dams in 
both 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 examination. 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 gland, 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, reproductive indices (e.g., mating index, fertility index, 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, 

however, the authors reported changes in body weight and body weight gain in males and females from 

both generations. A slight reductions in body weight (94 – 96% of control group) and body weight gain

(77 – 89% of control group) in P1 males during some weeks 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). P1 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 slight 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) and 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, however, 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 on day 0 of gestation and day 7 of lactation. The authors attributed 

slight increases 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 dead, 

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 considered 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, sperm 

and 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 females (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 species 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 (LD50) 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 presence 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 that the LD50 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, decreased 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 study (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 chronic 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. Chlorsulfuron 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 definitions 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, 

measures 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 hematological values did not indicate treatmentrelated 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 significant 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 

aminotransferase, 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 increased 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, the authors reported a statistically significant increase in blood serum concentrations 

of glucose 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 caused hyperglycemia at the beginning of the 

study period and hypoglycemia at the end of the study period due to the action 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 exploratory 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 group (5.58 mg/kg) in 

month 1 and a significant decrease (54.4%) in month 3. However, 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-demethylation activity in the post-mitochondrial fraction of the liver (74.5%), 

and a decrease in NAD content (5.3%) compared with the control group at the end of the study period. 

The authors noted that these findings are indicators 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 

reactions), while more substantial changes 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., disorganization of cortical cells, focal 

heterotrophy 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 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.

Rakitsky and Beloyedova, 2009: In an acute and chronic study of several sulfonylurea herbicides, 

Rakitsky and Beloyedova (2009) administered chlorsulfuron to rats, mice, dogs, and rabbits. Neither 

species nor strains were reported by the 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 function. The 

authors reported an oral LD50 in rats of 5,545 – 6,293 mg/kg in males and females, respectively, and a 

dermal LD50 of 2,500 mg/kg in rabbits. The authors also reported an LC50 (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 for 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 slightly exceeding historical controls. The authors reported a chronic noobserved-effect 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 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.

II. CONCLUSION

No additional acceptable studies that strongly suggest either 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 findings from the other 

additional 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 previously. As such, there 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 were observed in the Mylchreest (2005) study, these 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. REFERENCES

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 Ukrainian.

Rudaya, P.L., Zhminko, P.G., Povyakel, L.I., and Reshavska, O.V. 2010. Toxicodynamics of 

Chlorsulfuron Potassium Salt Given Orally In Long-Term Experiment on White Rats. Modern 

Problems of Toxicology 1: 59 – 63. Translated from Ukrainian.