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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August - 23 November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: 871302lEEC; Part B; Subchronic Oral Toxicity Test
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Japan MAFF, 59NohSan, No. 4200: Subchronic Oral Toxicity Test
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
The test substance, identified as RH-573 Technical, Lot Number B-1103, contained 97.5% of the following active ingredient (a.i.): N-methyl isothiazolone.

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
On August 03, 1999, one hundred (100) Crl:CD BR rats (50 males and 50 females) were received at approximately 4 weeks of age from Charles River Laboratories, Kingston, NY. Upon arrival, all animals were placed directly into a study room and were weighed and examined for physical abnormalities. The animals were acclimated to the study room for approximately two weeks prior to the initiation of treatment. Standard quarantine procedures of the Laboratory Animal Services Unit were in effect for the first week of acclimation. All animals were identified by a unique number tattooed on their tail.

The animals were housed individually in suspended stainless steel cages (18 cm x 34 cm x 18 cm) with wire mesh fronts and bottoms. Clean cage banks were supplied approximately every 2 weeks. Cages were suspended above absorbent paper liners which were changed at least 3 times a week.

The animals were housed in an environmentally controlled room with controls set to maintain temperature at approximately 23°C with a relative humidity of 30-70%. Temperature and relative humidity were monitored 24 hours a day and the light cycle was automatically controlled, 12 hours on and 12 hours off. The average daily temperature in the animal room ranged from 20°C to 22°C over the course of the study. During this same time period, the average daily relative humidity ranged from 28% to 74%. The average relative humidity was above the protocol specified range on two days and below the range on one day. This deviation from the protocol was judged to have had no adverse impact on the study outcome. The temperature and relative humidity ranges during this study were in general compliance with those recommended by the "Guide for the Care and Use of Laboratory Animals" (ISBN No. 0-309-05377-3, Revised 1996), and the minor excursions that occurred from these ranges were not considered to have had any adverse effects on the conduct or results of this study.

During the acclimation and treatment phases of the study, all rats had free access to PMI Certified Rodent Diet 5002(M) (Purina Mills Inc., Richmond, IN). During the acclimation period, water purified by reverse osmosis (RO water) via water bottles was provided ad libitum. During the treatment period RO water (control or treated) via water bottles was available ad libitum.

Animal Selection and Randomization
One week prior to initiation of treatment (i.e., first day of pretest), all rats were weighed and observed for clinical abnormalities. Apparently healthy animals were selected and randomly assigned to one of four groups (10 rats/sex/group) using a computerized stratified randomization procedure based on these body weights. There were no statistically significant differences in body weights at the beginning of pretest among groups of rats of the same sex.

Each rat's cage was labeled with a cage card indicating the unique animal number, protocol number, sex, test substance, and dose level of test substance administered. At the start of dosing, animals were approximately 6 weeks old. Extra untreated animals were returned to the Laboratory Animal Services Unit.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
1% Solutions were prepared from this lot of RH-573 Technical. These 1% solutions were assigned Lot Numbers BJE 4174 and BJE 4196, and Rohm and Haas Toxicology Department (TD) Sample Numbers 99-055 and 99-091, respectively. These samples of RH-573 Technical solution were colorless liquids containing approximately 1 % active ingredient.

An insufficient amount of 1% solution (Lot Number BJE 41 74, TD Number 99-055) was originally submitted for testing. Additional 1% RH-573 Technical solution (Lot Number BJE 4196, TD Number 99-091) was prepared from the same lot of RH-573 Technical (Lot Number B- 1103) as the original 1 % solution. The a.i. concentration of both 1 % solutions was analytically verified to contain approximately 1% RH-573 Technical as active ingredient.

Dose Preparation and Analysis
The appropriate amount of RH-573 Technical was used to prepare sufficient quantities of drinking water at concentrations of 75, 250, and 1000 ppm a.i.. The test substance was weighed in a hood and diluted in RO water. Group 1 (control) received RO water only. Solutions were prepared each week and animals were given fresh water bottles once every 7 days or as required. Solutions were stored at room temperature during the week.

Any treated water remaining in the water bottles or unused at the end of a dosing period was discarded as hazardous waste.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of RH-573 Technical in RO water, when stored at room temperature for 8 and 15 days, was determined from the first water preparation. Homogeneity was determined by analyzing top, middle and bottom samples from the first dose preparation. Samples of water prepared for Weeks 1, 4, 8, and 12 were analyzed for active ingredient content to determine proximity to target concentrations.

HPLC Analysis. The analyses reported here were performed on a Hewlett Packard Series 1 100 HPLC system. Instrument control and data acquisition was performed using Hewlett Packard HP ChemStation Software Version A.6.03. Chromatographic Conditions are described below.

Chromatographic Conditions
Column..........................................Supelcosil LC ABZ, 25 cm x 4.6 mm, 5 um
Mobile phase.................................50% Methanol / 50% Deionized Water
Column Temperature..................25 C
Separation.....................................Isocratic
Injection Volume..........................5 uL
Detection.......................................UV, 254 nm
Flow Rate.......................................1 mL/min.
Approximate Retn. Time.............2.9 - 3.1 min.
Run Time.......................................10 min

RH-573 Technical solutions were submitted on six occasions (8/18/99, 8/26/99, 9/2/99, 9/8/99, 10/6/99, and 11/3/99) for proximity to target analysis. Homogeneity was also confirmed for the samples submitted on 8/18/99. Stability was assessed on samples submitted on 8/18/99, 8/26/99, and 9/2/99. The samples were either analyzed on the same day they were received or they were stored at room temperature until they could be analyzed.

Homogeneity. Top, middle, and bottom samples from each dosing solution prepared on 8/18/99 were submitted in order to confirm the homogeneity of the solution preparation. The analytical results confirmed that the samples were homogeneous. The amount detected for the 75 ppm samples ranged from 64.4 ppm to 65.7 ppm. The amount detected for the 250 ppm samples ranged from 239.7 ppm to 243.3 ppm. The amount detected for the 1000 ppm samples ranged from 912.9 ppm to 935.2 ppm.

Stability. Room temperature storage stability was investigated over a period of 15 days. Samples from each dosing concentration were analyzed on Days 0, 8, and 15. The average proximity to target for the 75 ppm solutions ranged from 86.8% to 95.9% with an overall average of 92.6 +/- 5.4%. The average proximity to target for the 250 ppm solutions ranged from 93.7% to 96.6% with an overall average of 95.3 +/- 1.5%. The average proximity to target for the 1000 ppm solutions ranged from 92.2% to 95.1% with an overall average of 93.8 +/- 1.6%. The variability in the data is within the expected variation of the assay. Therefore, the samples were stable for 15 days.

Proximity to Taraet. Dosing solution samples submitted on 8/18/99 (Sample Nos. 99P-135-01 through 99P-135-010) contained 86.8% to 96.6% of the target concentration of RH-573 active ingredient. No RH-573 was detected in the solvent control sample, 99P-135-01.

Dosing solution samples submitted on 8/26/99 (Sample Nos. 99P-135-19 through 99P-135-22) contained 95.0% to 95.5% of the target concentration of RH-573 active ingredient. No RH-573 was detected in the solvent control sample, 99P-134-19.

Dosing solution samples submitted on 9/2/99 (Sample Nos. 99P-135-31 through 99P-135-34) contained 93.7% to 95.9% of the target concentration of RH-573 active ingredient. No RH-573 was detected in the solvent control sample, 99P-134-31.

On 9/2/99, an aliquot of the test material, 1% RH-573 Technical Solution, TD No. 99-091, Lot No. BJE 4196, was also submitted for analysis to verify the RH-573 active ingredient concentration. This sample contained an average of 9369.1 ppm of RH-573 active ingredient. This amount represents 93.7% of the target concentration (1% or 10,000 ppm) of RH-573.

Dosing solution samples submitted on 9/8/99 (Sample Nos. 99P-135-39 through 99P-135-42) were analyzed on 11/3/99 and contained 102% to 110% of the target concentration of RH- 573 active ingredient. These samples were originally analyzed on 9/10/99 but it was later observed that the analytical standards that were used for the analysis had expired. Therefore, the samples were reanalyzed on 11/3/99. The retained solvent control sample, 99P-135-43, was submitted for analysis on 11/3/99 because the original solvent control sample, 99P-135-39, was inadvertently discarded. No RH-573 was detected in the solvent control sample, 99P-134-43.

Dosing solution samples submitted on 10/6/99 (Sample Nos. 99P- 135-59 through 99P- 135-62) contained 106% to 107% of the target concentration of RH-573 active ingredient. No RH-573 was detected in the solvent control sample, 99P-134-59.

Dosing solution samples submitted on 11/3/99 (Sample Nos. 99P-135-79 through 99P-135-82) contained 104% to 1 10% of the target concentration of RH-573 active ingredient. No RH-573 was detected in the solvent control sample, 99P-134-79.
Duration of treatment / exposure:
3 months (August 19 - November 23, 1999)
Frequency of treatment:
Continuous
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 225, 1000 ppm
Basis:
nominal in water
No. of animals per sex per dose:
10 males and 10 females/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
See Table 1 below
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
Clinical Observations
Each rat was observed in its cage at least once each day for detection of morbidity, mortality, or clinical signs of toxicity throughout the study. Cage liners were inspected daily for abnormal appearance of urine or feces and excessive spillage of feed and/or water. Detailed Clinical Observations (DCO) were performed weekly outside the home cage, in a standard arena, beginning one week prior to the start of dosing. The DCOs noted changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., selfmutilation, walking backwards) were recorded if present.

Body Weight, Feed Consumption, and Water Consumption
The weight of each rat and the quantity of feed and water consumed were determined weekly for all animals beginning one week prior to the initiation of dosing and continuing through the 13th week of treatment. Compound intake was calculated using the following formula:

Compound Intake (mg a.i./kg of body weightlday) = [Concentration in drinking water (ppm) x Water Consumption (g/animal/day)]/Body weight (g) at end of week

A terminal body weight was also determined for each animal immediately prior to necropsy, after an overnight fast.

Ophthalmology
Indirect ophthalmoscopic examinations were performed on all animals prior to initiation of treatment and prior to termination of treatment.

Detailed Clinical Observations (DCO)
A detailed clinical observation (DCO) was performed on all rats weekly beginning one week prior to the start of dosing. Each evaluation started with an assessment of the animal's behavior in its home cage and each animal, in turn, was then transferred to an open arena. The animal was observed in the open arena for one minute. Following the 1-minute observation period, the pupillary response was assessed by shining a penlight in each eye. During the DCO, the animal's reflexes and response to stimuli were not evaluated except for the pupillary response to light. The DCOs were performed by the same individual (primary observer) throughout the study. A different individual (recorder) was present to record the observations.

The Detailed Clinical Observation (DCO) consisted of parameters 1-16 & 23.
The Functional Observational Battery (FOB) consisted of parameters 1-25:
1. Home cage behavior
2. Home cage involuntary behavior (convulsions or unusual postures or movements)
3. Reaction to removal from home cage
4. Reaction to handling (transporting rat to the arena)
5. Open arena - Record:
.....a. # of rears
.....b. presence or absence of fecal boli and condition of boli (e.g., normal, soft, or diarrhea)
.....c. # of urine pools (e.g., none, one to four, or five or more)
6. Open arena: activity/level of arousal
7. Open arena: involuntary behavior (convulsions or unusual postures or movements)
8. Open arena: gait pattern
9. Open arena: severity of gait abnormality
10. Open arena: palpebral closure
1 1. Open arena: prominence of the eyes
12. Lacrimation
13. Salivation
14. Piloerection
1 5. Respiration
16. General appearance
17. Response to approach (with blunt probe)
18. Tactile reaction (touch the rump with blunt probe, avoid rat's field of vision)
19. Auditory reaction (use of clicker to make sudden sound)
20. Pain response (tail pinch reaction)
2 1. Visual placing
22. Aerial righting reflex
23. Pupillary response
24. Grip strength (hindlimb and forelimb; 2 trials each)
25. Landing hind foot splay (2 trials)

Functional Observational Battery (FOB)
A Functional Observational Battery (FOB) was performed on all surviving rats during the 13th week of dosing. Each FOB evaluation started with an assessment of the animal's behavior in its home cage and each animal, in turn, was then transferred to the open arena. The animal was observed in the open arena for two minutes. Following the 2-minute observation period, the rat's response to approach by a visual stimulus was evaluated (i.e., a blunt probe was brought to approximately 3 cm of the rat's muzzle). The rat's response to a tactile stimulus was evaluated by avoiding the rat's field of vision and gently touching the rat's rump with a blunt probe. The rat's response to auditory stimulus was assessed using a 'clicker' placed approximately 5 cm above the back of the rat and making a sudden sound. The rat's response to a painful stimulus was assessed by pinching its tail with blunt forceps a approximately 3 cm from the tip of the tail. The visual placing response of each animal was evaluated by holding the base of the tail and lowering the animal forward toward the edge of a gray metal cart. Each rat was then held in a supine position and dropped from a height of approximately 30 cm onto a thickly padded surface to assess aerial righting reflex. Pupillary response was assessed by shining a penlight in each eye. Forelimb and hindlimb grip strength were assessed twice using a strength gauge. To measure landing hindlimb foot splay, the pad on each of the rat's hind feet was painted with tempera paint (Van Aken International, Rancho Cucamonga, CA) and the rat was dropped from approximately 40 cm onto a thickly padded surface. The distance between the paw prints was measured, the procedure was repeated and the mean distance reported. The remaining tempera paint was removed with water and a gauze pad and the rat was returned to its home cage.

For purposes of administering the FOBs, animals were divided into three replicate groups with approximately one third of the animals assessed on a given day during the 13th week of dosing. Dose groups and sexes were evenly distributed across the three replicate groups and the FOBs were conducted at approximately the same time each day across the three days. The FOBs were performed by a trained individual who was unaware of the animals1 dose groups (i.e., blind observations). Each animal in a given day's FOB group was assigned an observation code number 1 through 26 or 27 so that the animal's true identification number and dose group were not known to the observer. The FOBs were performed by the same individual (primary observer) across the three days. A different individual (recorder) was present to record the observations.

Motor Activity (MA)
Passive infrared motion sensors (Infrared Motion Activity System, Coulbourn Instruments, Allentown, PA) were calibrated and verified prior to conducting motor activity assessment for this study. Calibration and verification was conducted on November 01 & 02, 1999, following Standard Operating Procedures (Rohrn and Haas Company Toxicology Department, Section 28) for the sensors in cage positions 1-30.

The Infrared Activity System Calibrator (Coulbourn Instruments, Allentown, PA) was used for both the calibration and verification of each motor activity sensor. The calibrator consisted of a heat source which was mounted on a rotating post in the middle of the unit. The heat source alternated between large and small movements with stops in between each movement. The computer monitored the calibration procedure and determined that the sensors were within acceptable parameters to detect differences in no movement, small movement and large movement. Following calibration, the verification procedure was conducted on each sensor using the Infrared Activity System Calibrator. Each sensor was verified for two minutes while the computer recorded each stop (no movement), small movement (calibrator moved for less than 1 second) and large movement (calibrator moved for 1 second or more). Both the number of movements and time (in seconds) spent in movement were recorded and documented during verification.

The movement of each rat was monitored for 1.5 hours. Motor activity assessment was performed after all FOBs in a replicate were completed. Each rat was placed individually in a stainless steel cage (wire mesh front and bottom; 41 cm x 24 cm x 18 cm) with a passive infrared motion sensor mounted to the cage front.

The infrared sensors of each cage were connected to a computer. The computer calculated and recorded the number of movements and time spent in movement for each animal at 5-minute intervals. The computer recorded the rat's activity as either no movement, small movement (rat moved for less than one second), or large movement (rat moved for 1 second or more). Large and small movements were summed for purposes of data analysis.

At the beginning of each motor activity session, the sensors were monitored for a few minutes to ensure that each sensor was recording movement during the session. If movement was recorded, the sensors were considered to be operating properly.
Sacrifice and pathology:
Clinical Pathology
Hematology and clinical chemistry measurements were performed on all animals at terminal necropsy. Animals were fasted overnight and blood samples were collected just prior to the terminal necropsy. All samples were collected from the abdominal aorta of rats that had been anesthetized with sodium pentobarbital (NembutalB, 50 mg/ml, Abbott Laboratories, North Chicago, IL) administered intraperitoneally at approximately 0.1 ml/100 g body weight to effect. Blood samples were collected in an order that rotated through treatment groups (i.e., one animal from each treatment group was bled before a second animal from the same group). Once blood was collected, animals were exsanguinated via the abdominal aorta. Necropsy and blood sample collection were performed over two consecutive days (5 animals/sex/group were necropsied each day).

The following hematology and clinical chemistry parameters were measured on each sample:

Hematology
Hematocrit (HCT)
Erythrocyte Count (RBC)
Hemoglobin (HGB)
Total White Blood Cell Count (WBC) and Differential White Cell Count (DIFF)
Reticulocyte Count (RT)*
Platelet Count (PLT)
Mean Cell Volume (MCV)
Mean Cell Hemoglobin (MCH)
Mean Cell Hemoglobin Concentration (MCHC)
Prothrombin Time (PT)#
*Slides were prepared for reticulocyte counts but were not considered necessary to read by the Study Director, Program ManagerISponsor, and Pathologist.
#Blood samples from the first 19 animals necropsied were inadvertently not collected for the determination of Prothrombin Time (PT). This deviation from the protocol had no effect on the results of the study.

All hematology parameters excluding the white blood cell differential counts and prothrombin time were determined using a Baker@ 9000 Hematology Analyzer (Biochem Immunosystems (U.S.), Inc., Allentown, PA). White blood cell differential counts were determined by microscopic examination. Prothrombin time was determined using an IL ACL-1000 (Instrumentation Laboratories, Inc., Milan, Italy).

Clinical Chemistry
a. Alanine Aminotransferase (ALT)............................................... l. Albumin/Globulin (A/G) Ratio
b. Aspartate Aminotransferase (AST).......................................... m.Total Bilirubin (BILI)
c. Cholesterol (CHOL)..................................................................... n. Calcium (CA)
d. Blood Urea Nitrogen (BUN) ........................................................o. Sodium (NA)
e. Glucose (GLU)...............................................................................p. Potassium (K)
f. Alkaline Phosphatase (ALP).........................................................q. Chloride (CL)
g. Total Protein (TP)..........................................................................r. Inorganic Phosphorus (PHOS)
h. Triglycerides (TRIG).....................................................................s. Creatinine (CREA)
i. Gamma Glutamyl Transpeptidase (GGT)
j . Albumin (ALB)
k. Globulin (GLOB)

All clinical chemistry parameters were analyzed using a Hitachi 704 Random-Access Chemistry System (Roche Diagnostics Boehringer Mannheim Corporation, Indianapolis, IN).

Gross Pathology, and Organ Weights
At the end of the three-month treatment period, all surviving rats were anesthetized with an intraperitoneal injection of sodium pentobarbital, euthanized by exsanguination, and necropsied. All organs, tissues and body cavities of the euthanized animals were examined and gross abnormalities were recorded. The following organs were weighed from each rat:

adrenals (2)...............................ovary (2)
brain...........................................spleen
epididymides (2).......................testes (2)
heart............................................thymus
kidneys (2).................................uterus
liver

Relative organ weights were calculated as a percentage of terminal body weight.

Tissues and Organs Preserved
The following tissues fiom all rats were preserved in 10% neutral buffered formalin:
adrenals (2)
aorta
bone, femur & femoral-tibia1 joint
bone, sternum (with marrow)
bone marrow smear (femur)*
brain (including cerebrum, cerebellum, and pons)
cervix
coagulating glands
epididymides (2)
esophagus
exorbital lacrimal glands
eyes (optic nerve, retina)
gross lesions
heart
intestine
duodenum cecum
jejunum colon
ileum rectum
kidneys (2)
larynx
liver
lungs
lymph nodes (cervical and mesenteric)
mammary gland (females)
masses
muscle, skeletal
nerve, peripheral (sciatic)
nasal cavity
ovaries (2)
pancreas
parathyroid
pharynx
pituitary
prostate
salivary gland
seminal vesicles
skin
spinal cord (cervical, mid-thoracic, lumbar)
spleen
stomach (forestomach and glandular)
testes (2)
thymus (where present)
thyroid
trachea
urinary bladder
uterus
vagina
* Bone marrow smears were prepared from all animals but were not examined.

Histopathology
Histopathologic examinations were performed on all tissues listed above from animals in the high dose and control groups. Microscopic examinations on male and female rats from Groups 2 and 3 were limited to sections of the livers, lungs, and kidneys. All tissues having gross lesions were examined.
Other examinations:
No additional information available.
Statistics:
Routine Analysis: Distribution of body weight, feed consumption, water consumption, clinical chemistry, hematology, and organ weights were inspected for normality and homogeneity of variance across treatment and sex. Transformation of data was performed, if needed, to meet analysis of variance assumptions. Overall treatment effects on organ weights, feed consumption, water consumption, clinical chemistry, hematology, and body weight for each sex were evaluated by using one-way analysis of variance models. If a significant treatment effect was detected, pairwise comparisons between each dose group and control was made utilizing Dunnett's t-test. For those parameters where a pretest value was available, analysis of covariance was used. Significant treatment effects from the analysis of covariance was followed by pairwise comparison to the control group using Dunnett's t-test on covariance adjusted means. The criterion of statistical significance was p<0.05.

Functional Observational Battery and Motor Activity Analysis:
Parameters in the DCO or FOB using interval scales, such as the grip strength test and landing foot splay test, were analyzed using parametric methodologies when the assumptions of normality and homogeneity of variances were met. When the assumptions were not met, transformation to an alternative scale (i.e., logarithm, square root) was performed prior to further analysis. When transformation of the data failed to meet parametric assumptions, nonparametric rank procedures were used to assess overall compound effects.

Initially, analysis of variance models were used to assess the effects of replicate. Subsequent analysis of variance models were used to assess the presence or absence of an overall compound effect. Separate analyses were performed for males and females.

Continued below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased in 1000 ppm males and females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased in 1000 ppm males.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Decreased in 75 ppm males and 250 and 1000 ppm males and females.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Body Weight and Cumulative Body Weight Gain
There were no treatment-related effects on body weight in either sex at dose levels up to and including 250 ppm. Treatment-related decreases (5%-13%) in body weight were observed in Group 4 (1000 ppm) males throughout the course of treatment (statistically significant from Weeks 3-13). Treatment-related decreases (4%-10%) in body weight were also observed in Group 4 (1000 ppm) females throughout the course of treatment. However, these decreases were not statistically significant. Cumulative body weight gain was decreased (statistically significant) in Group 4 males from Week 1 through 13 (15%-22%). Cumulative body weight gain was also decreased in Group 4 females from Week 1 through 13 (17%-23%), but the decreases were statistically significant only during Weeks 6 and 7. The effects on body weight and cumulative body weight gain were judged secondary to decreased water consumption in both male and female animals.

Feed Consumption
There were no treatment-related effects on feed consumption in males at dose levels up to and including 250 ppm, or in females at any dose level. Statistically significant, treatment-related decreases (12%-20%) in feed consumption were observed in Group 4 (1000 ppm) males throughout the duration of treatment (Weeks 1-13). These effects most likely resulted from decreased water consumption.

Occasional, statistically significant decreases in feed consumption were observed in Group 3 (250 ppm) males (Weeks 5, 11, 12, and 13). These findings were considered incidental due to the small magnitude of the changes (9%-14%) and the lack of persistence throughout the study. There appeared to be a slight decrease in feed consumption in Group 4 (1000 ppm) females during the early stages of the study (statistically significant in Week 1 only). The relationship of these slight decreases to treatment was equivocal.

Water Consumption
There were no treatment-related effects on water consumption in female animals at a dose level of 75 ppm. Treatment-related decreases in water consumption (7%-21%) were observed in Group 2 (75 ppm) males throughout the study (statistically significant in Weeks 1,4, and 10). Statistically significant, treatment-related decreases in water consumption were observed in Group 3 (250 ppm) (18%-31%) and Group 4 (1000 ppm) (32%-46%) males throughout the entire treatment period (Weeks 1-13). Treatment-related decreases in water consumption were observed in Group 3 (250 ppm) (16%-43%) and Group 4 (1000 ppm) (24%-45%) females throughout the study (statistically significant in most weeks). The a decreases in water consumption were judged to be due to palatability, since there was a clear dose-response effect, and there were no corresponding changes in gross pathology or histopathology indicative of treatment-related irritation in the oral cavity, esophagus, or gastrointestinal tract.

Compound Intake
The average daily intake of RH-573 Technical over the entire treatment period was described in Table 2.

Hematology
No treatment-related effects on hematology parameters or white blood cell differential counts were observed in male or female animals at any dose level.

In Group 3 and 4 females, statistically significant decreases in red blood cell count (5% and 6%, respectively), hemoglobin (6% and 7%, respectively), and hematocrit (8% and 7%, respectively) were observed. These decreases were judged not to be treatment-related since there were no corresponding changes in histopathology (bone marrow or spleen) or other hematologic changes (i.e., no change in platelets), there was no evidence of a dose-response (the changes at 250 and 1000 ppm were approximately equivalent), the magnitude of the changes was small, and the changes were observed only in one sex.

Clinical Chemistry
There were no treatment-related changes in any clinical chemistry parameter in either sex at any dose level. Statistically significant decreases in total bilirubin (Group 4 males - 14%) and glucose (Group 4 females - 13%) were not considered biologically significant or treatment-related due to the small magnitude of the changes and the fact that they were only observed in one sex.

Ophthalmology
Ophthalmoscopic examination at termination of treatment indicated there was no treatment-related ocular disease in any RH-573 Technical-treated animals. The abnormalities observed occur commonly in laboratory rats of this strain and age and were judged to be unrelated to treatment.

Organ Weights
There were no treatment-related effects on organ weights in either sex at any dose level. Statistically significant increases in relative brain (14%) and relative kidney (10%) weights were observed in Group 4 males, as were increased relative kidney (15%) weights in Group 4 females. These changes in relative organ weights were not considered treatmentrelated but rather the result of the decreased terminal body weights seen in both sexes (12% and 9% in males and females, respectively). In addition, no corresponding changes in absolute organ weights were observed, and there were no correlative gross or histopathological findings with these organs.

Detailed Clinical Observations (DCO)
No treatment-related systemic or neurological effects were seen in any DCO parameters in either sex at any dose level. There was a statistically significant decrease in the number of rears in Group 4 (1000 ppm) females during Week 12. Since this decrease was observed in only one sex at one time point, it was considered incidental and not related to treatment.

Week 13 Functional Observational Battery (FOB)
No treatment-related systemic or neurological effects were seen in any FOB parameters in either sex at any dose level. There were no statistically significant findings in any FOB parameter, and there were no statistically significant or treatment-related effects in any of the animals' reflexes, responses to stimuli, grip strength, or landing hind foot splay.

Week 13 Motor Activity (MA)
No treatment-related systemic or neurological effects were seen in any MA parameters in either sex at any dose level. There were no statistically significant or treatment-related effects in either total time spent in movement or total number of movements.

Gross Pathology
No treatment-related gross pathological findings were observed in either sex at any dose level.

Histopathology
No treatment-related microscopic changes were observed in any of the organs or tissues specified for evaluation in male or female rats exposed to concentrations of 75, 250, or 1000 ppm a.i. of RH-573 Technical in the drinking water for three months. All microscopic changes that were observed in the various organs and tissues were typical of those that occur spontaneously and their type, incidence, or severity was not considered to have been influenced by exposure to RH-573 Technical.

Effect levels

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Dose descriptor:
NOAEL
Effect level:
19 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: Decreased cumulative body weight gain and decreased feed consumption in male 1000 ppm drinking water group.
Dose descriptor:
NOAEL
Effect level:
24.6 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: Decreased cumulative body weight gain in female 1000 ppm drinking water group.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Summary

Administration of RH-573 Technical in the drinking water for three months produced the following treatment-related effects in male and/or female rats:

75 ppm (6.51 mg- a.i./kg bw/day in males; 9.78 mg a.i./kg bw/day in females)

  • Decreased water consumption (males only)

250 ppm - NOAEL - (19.0 mg a.i./kg bw/day in males; 24.6 mg a.i./kg bw/day in females)

  • Decreased water consumption (both sexes)

1000 ppm (65.7 mg a.i./kg bw/day in males: 93.5 mg a.i./kg bw/day in females)

  • Decreased water consumption (both sexes)
  • Decreased body weight (both sexes)
  • Decreased cumulative body weight gain (both sexes)
  • Decreased feed consumption (males only)

Table 2 Average Daily Intake of RH-573 Technical

   Drinking Water     Compound Intake (mg a.i./kg bw/day
 Group  Concentration  Males  Females
 2  75 ppm  6.51  9.78
 3  250 ppm  19.0  24.6
 4  1000 ppm  65.7  93.5

Applicant's summary and conclusion

Conclusions:
RH-573 Technical, when administered in the drinking water to rats for three months at doses of 0, 75, 250, or 1000 ppm active ingredient (a.i.), had a No Observed Adverse Effect Level (NOAEL) of 250 ppm (equivalent to 19.0 and 24.6 mg a.i./kg of body weight/day in males and females, respectively). Effects on body weight and feed consumption were noted at 1000 ppm (65.7 and 93.5 mg a.i./kg of body weight/day in males and females, respectively). There was no evidence of systemic toxicity at any dose level tested.
Executive summary:

The toxicity of RH-573 Technical [Lot Number B-1103; 97.5% active ingredient (a.i.)] was assessed in Crl:CD®BR rats when administered in the drinking water for three months. RH-573 Technical was prepared as an aqueous solution and administered in the drinking water to four groups of 10 rats/sex at doses of 0, 75, 250, or 1000 ppm a.i.. Dose levels were selected based on range-finding studies which indicated that concentrations higher than 1000 pprn would have compromised animal health. All animals were observed at least once daily for mortality, signs of ill health or reaction to treatment. Body weight, water and feed consumption were determined weekly. Animals were examined weekly using Detailed Clinical Observations (DCO). During Week 13, animals were assessed using a Functional Observational Battery (FOB) and monitored for motor activity (MA). At the end of the treatment period, all rats were euthanized and necropsied. Blood samples were collected for hematology and clinical chemistry analysis from all rats at terminal necropsy. Selected organs were weighed and tissues were collected for histopathologic evaluation.

There were no mortalities in either sex at any dose level.

No treatment-related systemic or neurologic effects were seen in the daily clinical observations, the Detailed Clinical Observations or Functional Observational Battery parameters, or motor activity in either sex at any time at any dose level.

There were no treatment-related effects on water consumption in female animals at a dose level of 75 ppm. Treatment-related decreases in water consumption were observed in Group 2 (75 ppm) (7-21%), Group 3 (250 ppm) (18-31%), and Group 4 (1000 ppm) (32-46%) males throughout the entire treatment period. Treatment-related decreases in water consumption were also observed in Group 3 (250 ppm) (16- 43%) and Group 4 (1000 ppm) (24-45%) females throughout most of the dosing phase.

There were no treatment-related effects on feed consumption in males at dose levels up to and including 250 ppm, or in females at any dose level. Treatment-related decreases in feed consumption (12-20%) were observed in Group 4 (1000 ppm) males throughout the duration of treatment.

There were no treatment-related effects on body weight in either sex at dose levels up to and including 250 ppm. Treatment-related decreases in body weight were observed in Group 4 (1000 ppm) males (5- 13%) and females (4-10%). Cumulative body weight gain was also decreased in Group 4 males (15-22%) and females (17-23%). These decreases were observed throughout the entire treatment period.

There were no treatment-related changes in any hematology or clinical chemistry parameters in either sex at any dose level.

No treatment-related changes in organ weights were noted in either sex at any dose level.

No treatment-related gross or microscopic pathological findings were observed in either sex at any dose level.

Conclusion

RH-573 Technical, when administered in the drinking water to rats for three months at doses of 0, 75, 250, or 1000 ppm active ingredient (a.i.), had a No Observed Adverse Effect Level (NOAEL) of 250 ppm (equivalent to 19.0 and 24.6 mg a.i./kg of body weight/day in males and females, respectively). Effects on body weight and feed consumption were noted at 1000 ppm (65.7 and 93.5 mg a.i./kg of body weight/day in males and females, respectively). There was no evidence of systemic toxicity at any dose level tested.