Chlorimuron ethyl

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7800

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-({[(4-chloro-6-methoxypyrimidin-2-yl)carbamoyl]amino}sulfonyl)benzoate

EC Number:

618-690-2

Cas Number:

90982-32-4

Molecular formula:

C15H15ClN4O6S

IUPAC Name:

ethyl 2-({[(4-chloro-6-methoxypyrimidin-2-yl)carbamoyl]amino}sulfonyl)benzoate

Test material form:

solid

Remarks:

White

Details on test material:

95 to >99% purity

Specific details on test material used for the study:

97.5% purity

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

female

Details on test animals or test system and environmental conditions:

The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at DuPont Haskell. By using the Crl:CD(SD) rat, immunotoxicity studies were conducted in the same strain that was used for other toxicology studies.

Animals were housed in pairs in solid bottom caging with Shepherd's™ Cob + PLUS™ as enrichment.

Animal rooms were maintained at a temperature of 18-26ºC (64-79ºF) and a relative humidity of 30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle.

All rats were provided tap water ad libitum. All rats were fed PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum. During the test (exposure) period, test substance was incorporated into the feed at concentrations specified by study design.

Upon arrival at DuPont Haskell, all rats were housed in quarantine. The rats were: Quarantined for 6 days, identified temporarily by either the presence or absence of a colored tail mark and cage identification, weighed 3 times during quarantine, observed with respect to weight gain and any gross signs of disease or injury.

The rats were released from quarantine based on acceptable body weights and clinical signs of all rats.

Rats, selected based on adequate body weight gain and freedom from any clinical signs of disease or injury, were distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there were no statistically significant differences among group body weight means. The weight variation of selected rats did not exceed ± 20% of the mean weight.

At grouping, each rat was assigned an animal number/cage identification number. The animal number/cage identification number was tattooed on the tail of each rat and included on the cage label.

At study start (test day 0) the rats were approximately 8 weeks of age.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet Sample Preparation

Acetonitrile (50 mL) was added to an aliquot (5 ± 0.05 g) of each diet sample. The mixtures were placed in an ultrasonic bath and sonicated for approximately 60 minutes, and swirled every 15 minutes. Each extract was filtered (VWR Syringe filter, Glass Fiber Acrodisc 1.0 µm, 25 mm HPLC certified) and aliquots of the filtrates were either analyzed by HPLC without farther dilution or diluted with the 0 ppm control diet extract before HPLC analysis.

Recovery Sample Preparation

Concurrent with diet analyses, recovery of the test substance from spiked control diet was tested at the low concentration level (approximately 125 ppm) and at the high concentration level (approximately 2500 ppm) to confirm the analytical method performance. Aliquots from a stock solution of the test substance prepared in acetonitrile were added to and mixed with the control diet for the low concentration level. The acetonitrile from the applied solution was allowed to evaporate or blown with N2 to dryness before processing. The test substance was weighed and mixed directly with control diet for the high concentration level. The samples were then processed and analyzed in the same manner as diet samples at the similar concentrations.

Chromatographic Conditions

Concentrations of the test substance in diet extract samples were determined by HPLC with UV detection, details are as follows:

Instrument: Agilent 1100 liquid chromatograph
Column: Agilent Zorbax SB-Cl8, 5 µm, 150 x 2.1 mm
Flow Rate: 0.400 mL/min
Stoptime: 7.50 minutes
Mobile phase: 55% Acetonitrile/45% 3.1 mM phosphoric acid
Detection: UV absorbance at 230 nm
Injection Volume: 5.00 µ
Column Temperature: 30.0°C

Calibration and Quantitation

The analytical standard of 98.8% purity test substance was used to make a stock solution in acetonitrile. The stock solution was further diluted with the control diet extract to make a set of standard solutions that covered the targeted concentrations of the sample extracts. Peak area counts from the HPLC analysis of these standard solutions were used to construct a calibration curve by Agilent's ChemStation software. Measured concentrations for the samples were determined by applying the peak area counts from replicate injections of each sample to the calibration curve.

Homogeneity of the test substance in the samples was evaluated by calculating the relative standard deviation (RSD = standard deviation/average x 100%) of the measured concentrations of the top, middle, and bottom samples for each concentration level. A RSD of equal to or less than 10% is the standard criterion at Haskell laboratory for acceptable distribution of the test substance throughout the diet.

Concentration verification of the test substance in the samples was evaluated by the average results of the top, middle, and bottom or single sample

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 ppm

Doses / concentrations 2

Dose / conc.:

500 ppm

Doses / concentrations 3

Dose / conc.:

1 250 ppm

Doses / concentrations 4

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

10 female rats

Control animals:

yes, plain diet

Details on study design:

The purpose of this study was to evaluate the potential of the test substance to suppress the primary humoral immune response to sheep red blood cells (sRBC) when incorporated into nutritionally adequate diet and fed to female rats for at least 28 days. The dietary route of administration was selected because it is a potential route of human exposure. Groups of 10 female rats were administered the test substance at dietary levels of 0, 125, 500, 1250, or 2500 ppm. Body weights, food consumption measurements, and clinical observations were recorded during the in-life period. Prior to sacrifice, the immune system was stimulated by injecting sRBC on test day 23 and blood samples were collected from each rat on test day 28. The serum samples were assayed for their concentrations of sRBC-specific IgM antibodies to provide a quantitative assessment of humoral immune response. Serum from animals similarly challenged with a positive control immunosuppressive agent was analyzed concurrently to provide confirmation that the assay performance was acceptable for detection of immunosuppression. At sacrifice, each animal was examined grossly and selected organs were weighed (brain, spleen, and thymus).

Samples of the test diets were analyzed, and the results indicated that the test substance was at the targeted concentrations, homogeneously mixed, and stable under the conditions of the study.

Examinations

Observations and clinical examinations performed and frequency:

Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least once daily throughout the study.

At every weighing, except on the day of sacrifice, each rat was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior. Any abnormal clinical signs noted were recorded.

Sacrifice and pathology:

At the end of the exposure period, the animals were euthanized by isoflurane anesthesia and exsanguination and underwent a gross evaluation. The order of sacrifice for scheduled deaths was stratified across groups.

The maximum volume of blood was collected at sacrifice from the abdominal vena cava, while the animal was under isoflurane anesthesia. Blood was placed in a tube with no anticoagulant and processed to serum for humoral immune function.

Following gross examination, the spleen, thymus, and brain from all animals in were weighed. The spleen, thymus, femur, and sternum were placed in 10% buffered formalin but further processing to slide and evaluation was not necessary to support experimental findings.

Relative organ weights (relative to final body weight; relative to brain weight) for weighed organs were calculated. Final body weights determined just prior to necropsy were used in the assessment of organ weight changes.

Humoral immunity examinations:

On test day 23, animals were injected intravenously in the lateral tail vein with 0.5 mL of 4 x 10(8) sheep red blood cells (sRBC) per mL (Lampire Biological Laboratories, Pipersville, Pennsylvania, U.S.A.) Following the intravenous injection of sRBC, rats in the positive control group were injected intraperitoneally for 5 consecutive days with 25 mg/kg/day of the known immunosuppressive agent, cyclophosphamide monohydrate in deionized water, at a dose volume of 10 mL/kg body weight.

Five days after sRBC injection (test day 28), the animals were euthanized by isoflurane anesthesia and exsanguination. Blood was collected from each rat, from the abdominal vena cava while the animal was under isoflurane anesthesia, processed to serum, and frozen at ≤-60ºC until analyzed. Humoral immune function was evaluated by analyzing sera from individual control and test-substance treated animals for sRBC-specific IgM levels with an enzyme-linked immunosorbent assay (ELISA). The serum from each animal was assayed as 2-fold serial dilutions, with 1 replicate per dilution. The optical density (OD) of the serum samples was measured at 450 nm and the log2 of the mean result of the serial diluted serum sample was reported.

Serum collected from rats injected with cyclophosphamide monohydrate was run concurrently with the study samples as a positive control.

Positive control:

25 mg/kg/day Cyclophosphamide

Statistics:

Method of Statistical Analysis

Parameter: For Body Weight, Body Weight Gain, Food Consumption, Food Efficiency, Humoral Immune Function Data*, and Organ Weights: Preliminary Test: Levene’s test for homogeneity and Shapiro-Wilk test for normality.

If preliminary test is not significant, One-way analysis of variance followed by Dunnett's test.

If preliminary test is significant, Transforms of the data to achieve normality and variance homogeneity were used. The order of transforms attempted was Log, square-root, and rank-order. If the log and square-root transforms failed, the rank-order was used.

* sRBC-specific serum IgM antibody data were transformed to Log2 to achieve normality or homogenous variances.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean weekly body weights of the 2500 ppm group were consistently lower throughout the study but were not statistically significant. Because of the limited magnitude these changes were not considered adverse. There were also no test substance-related adverse effects on body weight in female rats at any other concentration or in the positive control group. At test day 28, mean body weight for rats fed 125, 500, 1250, and 2500 ppm was 100%, 96%, 100% and 94% of the control, respectively.

The overall body weight gain (test day 0-28), when compared to the control group for female rats fed 0, 125, 500, 1250, and 2500 ppm, was 95%, 87%, 94%, and 79% of the control, respectively. Although the 21% reduction in body weight gain for the 2500 ppm group was statistically significant, it only corresponded to a 6% reduction in mean body weight and was not considered adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on food consumption in female rats at any concentration. A statistically significant decrease in mean daily food consumption during test interval days 14-21 and an increase during test interval days 21-28 was observed in female rats fed 500 or 1250 ppm, respectively. Neither change was in a dose-response relationship, nor was there any significance observed in these groups at the overall test interval of 0-28 days. Mean overall daily food consumption (test day 0-28) in the 2500 ppm group was 97% of the control.

The overall mean daily intake of test substance was calculated to be 0, 9.5, 37, 95 and 184 mg/kg bw/day for the 0, 125, 500, 1250, and 2500 ppm concentrations, respectively.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on food efficiency in female rats at any concentration. Mean overall daily food efficiency (test day 0-28) in the 2500 ppm group was 81% of the control.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on humoral immune function in female rats at any concentration. A statistically significant increase was observed in group mean IgM for rats fed 1250 ppm (118% of control). This increase did not occur in a dose-response manner. Decreases rather than increases are indicative of an immunotoxic effect; therefore, this increase was considered to be unrelated to treatment and non-adverse.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

All rats survived until the scheduled day of sacrifice. There were no statistically significant changes on organ weights in female rats at any concentration.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The group mean IgM result from rats dosed with the positive control, cyclophosphamide monohydrate, was 73% of the vehicle control group. This reduction in immune response indicates the test system was valid for evaluating immunosuppression.

Statistically significant decreases in absolute and relative (% body weight and % brain weight) spleen and thymus weights were observed in the positive control group receiving cyclophosphamide monohydrate. Since cyclophosphamide monohydrate is a known immunosuppressant, these decreases occurred as expected.

Specific immunotoxic examinations

Cell viabilities:

not examined

Humoral immunity examinations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on humoral immune function in female rats at any concentration. A statistically significant increase was observed in group mean IgM for rats fed 1250 ppm (118% of control). This increase did not occur in a dose-response manner. Decreases rather than increases are indicative of an immunotoxic effect; therefore, this increase was considered to be unrelated to treatment and non-adverse.

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No immunotoxic effects at the highest dose tested

Applicant's summary and conclusion

Conclusions:

The test substance is not considered to be an immunotoxicant.

Executive summary:

The purpose of this study was to evaluate the potential of the test substance to suppress the primary humoral immune response to sheep red blood cells (sRBC) when incorporated into nutritionally adequate diet and fed to female rats for at least 28 days (OPPTS 870.7800).

The dietary route of administration was selected because it is a potential route of human exposure. Groups of 10 female rats were administered the test substance at dietary levels of 0, 125, 500, 1250, or 2500 ppm. Body weights, food consumption measurements, and clinical observations were recorded during the in-life period. Prior to sacrifice, the immune system was stimulated by injecting sRBC on test day 23 and blood samples were collected from each rat on test day 28. The serum samples were assayed for their concentrations of sRBC-specific IgM antibodies to provide a quantitative assessment of humoral immune response. Serum from animals similarly challenged with a positive control immunosuppressive agent was analyzed concurrently to provide confirmation that the assay performance was acceptable for detection of immunosuppression. At sacrifice, each animal was examined grossly and selected organs were weighed (brain, spleen, and thymus).

 

Samples of the test diets were analyzed, and the results indicated that the test substance was at the targeted concentrations, homogeneously mixed, and stable under the conditions of the study.

 

The overall mean daily intake of test substance was calculated to be 0, 9.5, 37, 95, and 184 mg/kg/day for the 0, 125, 500, 1250, and 2500 ppm concentrations, respectively.

 

There were no adverse effects on body weight or nutritional parameters in female rats fed 0, 125, 500, 1250, and 2500 ppm of the test substance. No clinical signs of systemic toxicity were observed.

 

No test substance-related effects were observed on 1) gross pathology; 2) absolute and relative brain, spleen, and thymus weights; or 3) humoral immune response.

Under the conditions of this study, the humoral immune response and systemic toxicity no-observed-adverse-effect level (NOAEL) was 2500 ppm, the highest concentration tested. This concentration is equivalent to 184 mg/kg/day in female rats. The NOAEL is based on only minimal reductions in mean body weight and weight gains that were considered non-adverse and the absence of adverse test substance-related effects on any other in-life or anatomic pathology parameter or on the humoral immune response in female rats fed up to 2500 ppm.

 

The test substance is not considered to be an immunotoxicant.

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