Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 28, 2001 - August 30, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to guideline
Guideline:
other: 87/302/EEC; Part B; Subchronic oral Toxicity Test
Qualifier:
according to guideline
Guideline:
other: Japan MAFF, 59NohSan, No. 4200: Subchronic Oral Toxicity Test
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(3-oxazolidinyl)ethyl methacrylate
EC Number:
256-260-2
EC Name:
2-(3-oxazolidinyl)ethyl methacrylate
Cas Number:
46235-93-2
Molecular formula:
C9H15NO3
IUPAC Name:
2-(1,3-oxazolidin-3-yl)ethyl 2-methylprop-2-enoate
Test material form:
liquid
Remarks:
yellow

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Details on species / strain selection:
The rat was selected since it is a recommended rodent species for evaluating subchronic toxicity via oral administration. The Crl:CD®BR strain of rat was selected due to the extensive experience with and historical data available for this strain at this testing facility and at the supplier.
Sex:
male/female
Details on test animals or test system and environmental conditions:
On May 15, 2001, ninety (90) Crl:CD®BR rats (45 males and 45 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 the 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 21.1°C to 23.9°C over the course of the study. During this same time period, the average daily relative humidity ranged from 45% to 68%. 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 untreated PMI Certified Rodent Diet 5002 (M) (Purina Mills Inc., Richmond, IN) and water (purified by reverse osmosis) via an automatic watering system.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The compound was administered via oral gavage since oral gavage is a practical and reliable method of oral administration.
Vehicle:
corn oil
Details on oral exposure:
Rats in Groups 1, 2, 3, and 4 received OXEMA administered via oral gavage at dose levels of 0 (control), 25, 100, or 400 mg a.i./kg of body weight, respectively, for three months (5 days/week). Corn oil was used as the vehicle in all gavage preparations and as the control substance. All dose levels were administered at a constant volume of 5 ml/kg.

The appropriate amount of OXEMA was used to prepare sufficient quantities of gavage suspensions at concentrations appropriate to deliver dose levels of 25, 100, and 400 mg a.i./kg/day at a constant volume of 5 ml/kg. Dose levels were adjusted for active ingredient since the a.i. of the test substance was 93.4%. A detailed written procedure used for dose preparation is included in the raw data for this study. Briefly, the appropriate amount of OXEMA was weighed, and corn oil was added until the appropriate volume was achieved. A magnetic stir bar was added and the suspension was stirred on a magnetic stir plate for approximately 15 minutes. Group 1 (control) received corn oil only. Gavage suspensions were prepared every two weeks and stored refrigerated. Prior to gavaging animals each day, an aliquot of each concentration was placed in a small beaker with a magnetic stir bar and allowed to stir on a magnetic stir plate for approximately 15 minutes to warm to room temperature. Suspensions were kept stirring during dosing. Daily dosing suspensions and any treated gavage suspensions unused at the end of a dosing period were discarded as hazardous waste.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of OXEMA in corn oil, when stored refrigerated for 15 days, was determined from the first dose preparation. Homogeneity was determined by analyzing top, middle and bottom samples from the first dose preparation. Samples of dosing suspensions prepared during the middle and at the end of the study (Weeks 7 and 13) were also analyzed for active ingredient content to determine proximity to target concentrations. Analyses were conducted by a method which was previously verified and approved. Unused analytical suspensions were discarded as hazardous waste.

Homogeneity ranged from 104.1% to 113.7%.
After 15 days of refrigeration, the amount of OXEMA detected in the 25 mg a.i./kg samples was 91.2% of the amount detected in the samples on Day 0. The amount of OXEMA in the 100 mg a.i./kg samples was 96.0% of the Day 0 amount, and the amount of OXEMA in the 400 mg a.i./kg samples was 94.8% of the Day 0 amount.
The analytically confirmed concentrations were within an acceptable range of the target concentrations. The average proximity to target for the 25 mg a.i./kg dose level ranged from 95.0% to 106.1%, the 100 mg a.i./kg dose level ranged from 95.9% to 105.2%, and the 400 mg a.i./kg dose level ranged from 96.8% to 113.7%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes
yes, concurrent vehicle
Details on study design:
One week prior to initiation of treatment (i.e., first day of pretest), all rats were weighed and observed for externally visible 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.

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.

Body Weight and Feed Consumption
The weight of each rat and the quantity of feed consumed were determined weekly for all animals beginning one week prior to the initiation of dosing and continuing through the 13th week of treatment.
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 the initiation of treatment and prior to the termination of treatment (during the 13th week of dosing).

Detailed Clinical Observations (DCO)
Detailed Clinical Observations (DCO) were performed weekly outside the home cage, in a standard arena, beginning one week prior to the start of dosing. DCO, however, were not performed during the week when FOB were conducted (i.e., Week 12). During DCO, the animals were observed for 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, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded if present.
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 DCO were performed by one individual (observer) and a different individual (recorder) was present to record the observations.

The Detailed Clinical Observations (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 11. Open arena: prominence of the eyes 12. Lacrimation 13. Salivation 14. Piloerection 15. 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) 21. 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 12th 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 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 four replicate groups. One of the four replicate groups was evaluated on each of four days during the 12th week of dosing. Dose groups and sexes were as evenly distributed as possible across the four replicate groups and the FOBs were conducted at approximately the same time each day across the four days. The FOBs were performed by an individual who was unaware of the animals' dose groups (i.e., blind observations). Each animal in a given day's FOB group was assigned an observation code number 1 through 18 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 (observer) across the four days.

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 July 23, 2001, following Standard Operating Procedures (Rohm and Haas Company Toxicology Department, Section 28) for the sensors in cage positions 1-22.
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.

Clinical Pathology
Hematology and clinical chemistry measurements were performed on all surviving 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 (Nembutal®, 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, when possible). Once blood was collected, animals were exsanguinated via the abdominal aorta. Necropsy and blood sample collection were performed over three consecutive days.
The following hematology and clinical chemistry parameters were measured on each sample:
Hematology
a. Hematocrit (HCT) b. Erythrocyte Count (RBC) c. Hemoglobin (HGB) d. Total White Blood Cell Count (WBC)
and Differential White Cell Count (DIFF) e. Platelet Count (PLT) f. Mean Cell Volume (MCV) g. Mean Cell Hemoglobin (MCH) h. Mean Cell Hemoglobin Concentration (MCHC) i. Prothrombin Time (PT)
j. Reticulocyte Count (RET)*
*Slides were prepared for reticulocyte counts but were not considered necessary to read by the Study Director, Program Manager/Sponsor, and Veterinary Pathologist.
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) b. Aspartate Aminotransferase (AST) c. Total Cholesterol (CHOL) d. Blood Urea Nitrogen (BUN) e. Glucose (GLU) f. Alkaline Phosphatase (ALP) g. Total Protein (TP) h. Triglycerides (TRIG) i. Gamma Glutamyltransferase (GGT) j. Albumin (ALB) k. Globulin (GLOB) l. Albumin/Globulin (A/G) Ratio m. Total Bilirubin (BILI) n. Calcium (CA) o. Sodium (NA) p. Potassium (K) q. Chloride (CL) r. Inorganic Phosphorus (PHOS) s. Creatinine (CREA)
All clinical chemistry parameters were analyzed using a Hitachi® 704 Random-Access Chemistry System (Roche Diagnostics Boehringer Mannheim Corporation, Indianapolis, IN).
Sacrifice and pathology:
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 male and/or female rat:
adrenals (2) ovaries (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 from all male and/or female rats were preserved in 10% neutral buffered formalin:
adrenals (2); aorta; bone, femur & femoral-tibial joint; bone, sternum (with marrow); brain (including cerebrum, cerebellum, and medulla/pons); cervix; coagulating gland; epididymides (2); esophagus; eye (optic nerve, retina); gross lesions; heart; intestine (with Peyer's patches) - duodenum, cecum, jejunum, colon, ilium, rectum; kidneys (2); larynx; liver; lungs (2); lymph nodes (cervical/mandibular and mesenteric); mammary gland (females); masses; muscle, skeletal; nasal cavity (nose); nerve, peripheral (sciatic); ovaries (2); pancreas; parathyroid; pharynx; pituitary; prostate; salivary glands; seminal vesicles; skin; spinal cord (cervical, mid-thoracic, lumbar); spleen; stomach (forestomach and glandular); testes (2); thymus; thyroid; trachea; urinary bladder; uterus; vagina

Histopathology
Histopathologic examinations were performed on all tissues listed above from all animals found dead, euthanized moribund or for humane reasons, and all animals in the high dose (Group 4) and control (Group 1) groups. Since a number of animals in the high dose group (Group 4) did not survive the entire 13 weeks of dosing due to early deaths or euthanasia, all tissues were examined from all animals in the mid-dose group (Group 3). All tissues having gross lesions were examined.
Statistics:
Statistical Methods
Routine Analysis: Distribution of body weight, feed 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, 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 using Dunnett's t-test. Analysis of covariance was used for those parameters where a pretest value was available. Significant treatment effects from the analysis of covariance was followed by pairwise comparison to the control group using Dunnett's t-test on covariate adjusted means. The criterion of statistical significance or type I error rate was p=0.05.

Functional Observational Battery and Motor Activity Analysis: Parameters in the DCO or FOB using interval scales, such as grip strength and landing foot splay, and data from the motor activity test were analyzed using parametric methodologies. Analysis of variance was used to assess the presence or absence of an overall compound effect on observed responses. Separate analyses were performed for males and females. Pairwise comparisons of least square means between control and each dose level were evaluated using Dunnett's t-test. Statistical significance was indicated whenever a p-value of 0.05 or less (Dunnett's criterion) was obtained for the treatment effect.
Parameters in the FOB having graded or count scores, e.g., home cage behavior and involuntary behavior, and quantal data from the DCO or FOB, were analyzed using appropriate contingency table procedures.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related systemic or neurological effects were seen in any DCO parameters in females treated at 25 mg a.i./kg or in males at any dose level. Treatment-related, statistically significant decreases (25-45%) in the number of rears in Group 3 (100 mg a.i./kg) and/or Group 4 (400 mg a.i./kg) females occurred during Weeks 8, 9, 10, 11, and 13.
There were statistically significant differences in home cage behavior in Group 4 (400 mg a.i./kg) males at Week 2 and females at Week 10. These differences were considered incidental and unrelated to treatment since they were observed in only one sex at each of the two time points, and the behaviors noted were within normal limits for laboratory rats (e.g., awake, immobile, apparently normal posture versus apparently normal movement such as rearing, drinking, and/or grooming). A statistically significant difference in respiration (normal versus abnormal) was noted in Group 4 (400 mg a.i./kg) males at Week 10. This abnormal respiration was considered to have been caused by aspiration of the test substance which resulted from the animals’ struggling and resistance to dosing, and was not the result of systemic toxicity.
Mortality:
mortality observed, treatment-related
Description (incidence):
No treatment-related deaths were noted during the study in either sex at dose levels up to and including 100 mg a.i./kg. One male in Group 2 (25 mg a.i./kg) was humanely euthanized during Week 3, and one male in Group 3 (100 mg a.i./kg) was humanely euthanized during Week 11. These deaths were not considered treatment-related but rather due to inadvertent dosing trauma. The irritancy of the test substance seemed to have caused the rats to struggle during the dosing procedure. Gross necropsy of these animals revealed perforation of the esophagus, rib, or hard palate.
Five (5) males and three (3) females in Group 4 (400 mg a.i./kg) died or were humanely sacrificed during the course of the study. This early mortality was judged to be treatment-related and mainly due to the extensive ulceration of the gastric mucosa.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on body weight or cumulative body weight gain in females at any dose level or in males at dose levels up to and including 100 mg a.i./kg. Statistically significant treatment-related decreases (11-28%) in body weight were observed in Group 4 (400 mg a.i./kg) males from Week 2 through Week 13. Similarly, cumulative body weight gain was significantly decreased (27-44%) in Group 4 males throughout the entire duration of the study (Weeks 1-13). Terminal body weight, which was determined for each animal immediately prior to necropsy, after an overnight fast, was significantly decreased (28%) in Group 4 (400 mg a.i./kg) males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on feed consumption in females at any dose level or in males at dose levels up to and including 100 mg a.i./kg. Treatment-related decreases in feed consumption were observed in Group 4 (400 mg a.i./kg) males during all weeks of the study. Statistically significant decreases (13-25%) occurred during six different weeks of the study, while decreases that were not statistically significant (3-29%) occurred during all other weeks.
A statistically significant increase (15%) in feed consumption in Group 4 (400 mg a.i./kg) females during Week 11 was not considered toxicologically significant since it was an increase instead of a decrease and since it occurred at only one time point during the study.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination at termination of treatment indicated there was no treatment-related ocular disease in any OXEMA-treated animals. The abnormalities observed occur commonly in laboratory rats of this strain and age and were judged to be unrelated to treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects on hematology parameters were observed in male or female animals at the 25 mg a.i./kg dose level. No treatment-related effects on white blood cell differential counts were observed in either sex at any dose level.
Treatment-related decreases in red blood cell count (7-36%), hemoglobin (8-36%), and hematocrit (9-37%) were noted in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) animals of both sexes. These decreases were statistically significant for all parameters except Group 3 (100 mg a.i./kg) male red blood cell count. A statistically significant, treatment-related increase in platelets (17%) was observed in Group 4 (400 mg a.i./kg) males.
A statistically significant increase in white blood cell count in Group 4 (400 mg a.i./kg) males was considered incidental. A statistically significant decrease in mean cell volume in Group 3 (100 mg a.i./kg) females was not considered treatment-related since it was small in magnitude (7%) and since no decrease was seen in the high dose (400 mg a.i./kg) group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related changes in any clinical chemistry parameters in either sex at the 25 mg a.i./kg dose level. Treatment-related, statistically significant increases were seen in alkaline phosphatase in Group 3 (100 mg a.i./kg) (32%) and Group 4 (400 mg a.i./kg) (31%) males. In Group 4 (400 mg a.i./kg) males, there was also a treatment-related increase (36%) in aspartate aminotransferase, however the increase was not statistically significant. Treatment-related increases in blood urea nitrogen (BUN) were observed in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) animals of both sexes. Although these increases in BUN were statistically significant [except for Group 3 (100 mg a.i./kg) females], none of them were remarkable. For males, the control (0 mg a.i./kg) value for BUN was 12.6 mg/dl, while the value for Group 3 (100 mg a.i./kg) was 14.9 mg/dl and that for Group 4 (400 mg a.i./kg) was 15.9 mg/dl. For females, the control (0 mg a.i./kg) value for BUN was 14.4 mg/dl, while the value for Group 3 (100 mg a.i./kg) was 16.8 mg/dl and that for Group 4 (400 mg a.i./kg) was 18.7 mg/dl.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increases in albumin, total protein, and albumin/globulin ratio seen in Group 3 (100 mg a.i./kg) females were considered incidental since similar effects were not seen in Group 4 (400 mg a.i./kg). Similarly, a statistically significant increase in triglycerides in Group 3 (100 mg a.i./kg) males was judged not treatment-related since no effect was seen in Group 4 (400 mg a.i./kg). Statistically significant decreases in alanine aminotransferase (ALT) were seen in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) females. These were not considered treatment-related since the statistically significant differences most likely resulted from two female animals in the control (0 mg a.i./kg) group whose values for ALT were unusually high. In Group 4 (400 mg a.i./kg) animals, total bilirubin was statistically significantly increased in males and decreased in females. These total bilirubin changes were not considered treatment-related but rather due to biological variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related systemic or neurological effects were seen in any FOB parameters in either sex at any dose level. There was a statistically significant increase (23%) in hindlimb grip strength in Group 4 (400 mg a.i./kg) males. This was considered an incidental finding since it was seen in only one sex and since it was an increase instead of a decrease. There were no treatment-related effects in any of the animals’ other reflexes, responses to stimuli, grip strength, or landing hind foot splay.
No treatment-related effects were seen in any MA parameters in either sex at any dose level.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on organ weights in females treated at 25 mg a.i./kg or in males treated at dose levels up to and including 100 mg a.i./kg. Treatment-related, statistically significant increases in absolute (27%) and relative (74%) adrenal weight were observed in Group 4 (400 mg a.i./kg) males. Terminal body weights were significantly decreased (28%) in Group 4 (400 mg a.i./kg) males. Treatment-related, statistically significant increases in absolute kidney (24 and 30%), relative kidney (24 and 40%), absolute liver (20 and 56%), and relative liver (19 and 66%) were noted in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) females, respectively.
Statistically significant decreases in absolute spleen (25%), absolute epididymus (19%), and absolute heart (20%) weights in Group 4 (400 mg a.i./kg) males were considered secondary to the effects of treatment on body weights. Likewise, significantly increased relative brain (33%), relative heart (11%), relative kidney (31%), relative liver (43%), and relative testes (32%) weights seen in Group 4 (400 mg a.i./kg) males were judged to be secondary to the decreased terminal body weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related gross pathological findings were observed in either sex at dose levels up to and including 100 mg a.i./kg. In several of the high dose (400 mg a.i./kg) animals, the intestines, forestomach, and glandular stomach were distended. Also seen in this group were areas of foci in the forestomach. All other gross pathological findings were judged to be related to dosing error or incidental since they occurred spontaneously or at single or low incidences, and since there was no dose-response effect in the severity or incidence of findings.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No microscopic changes directly related to treatment with the test substance were observed in any of the male or female rats in Group 2 (25 mg a.i./kg).
Microscopic changes considered to be directly associated with treatment with OXEMA occurred in the liver of Group 4 (400 mg a.i./kg) and stomach of Groups 3 (100 mg a.i./kg) and 4 (400 mg a.i./kg) male and female rats.
The treatment-related effect in the liver consisted of minimal or mild centrilobular hepatocellular hypertrophy in several male and female Group 4 (400 mg a.i./kg) rats.
The most extensive microscopic changes occurred in the stomach of which the most common changes were hyperplasia and hyperkeratosis of the mucosa of the forestomach, erosions and/or ulcers of the gastric mucosa, edema and inflammation (inflammatory cell infiltrations) in the submucosa of the stomach (in the areas of erosions and ulceration) and areas of superficial mucosal hemorrhage. The lesions were most extensive in Group 4 (400 mg a.i./kg) rats, but hyperplasia and hyperkeratosis of the forestomach mucosa also occurred in most male and female rats in Group 3 (100 mg a.i./kg). The gastric changes described above are consistent with the irritating and corrosive characteristics of the test substance.
The other microscopic changes that were observed were considered to have been secondary to the moribundity in the non-surviving rats or related to the aspiration of the test substance into the nasal cavity as a result of the resistance of the rats to dosing. All other microscopic changes were considered to have occurred spontaneously and their type, incidence, or severity was not influenced by exposure to OXEMA.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Administration of OXEMA via oral gavage for three months (5 days/week) produced treatment-related effects in male and female rats in the mid- and high dose group. The results for each group are presented below as a comprehensive summary:

25 mg a.i./kg bw
• No observed effects

100 mg a.i./kg bw
• Decreased number of rears (DCO) - (females only)
• Decreased red blood cell count (both sexes)
• Decreased hemoglobin (both sexes)
• Decreased hematocrit (both sexes)
• Increased alkaline phosphatase (males only)
• Increased blood urea nitrogen (both sexes)
• Increased absolute kidney weight (females only)
• Increased relative kidney weight (females only)
• Increased absolute liver weight (females only)
• Increased relative liver weight (females only)
• Hyperplasia and hyperkeratosis of the forestomach mucosa (both sexes)

400 mg a.i./kg bw
• Decreased body weight (males only)
• Decreased cumulative body weight gain (males only)
• Decreased feed consumption (males only)
• Decreased number of rears (DCO) - (females only)
• Decreased red blood cell count (both sexes)
• Decreased hemoglobin (both sexes)
• Decreased hematocrit (both sexes)
• Increased platelets (males only)
• Increased alkaline phosphatase (males only)
• Increased aspartate aminotranferase (males only)
• Increased blood urea nitrogen (both sexes)
• Decreased terminal body weight (males only)
• Increased absolute adrenal weight (males only)
• Increased relative adrenal weight (males only)
• Increased absolute kidney weight (females only)
• Increased relative kidney weight (females only)
• Increased absolute liver weight (females only)
• Increased relative liver weight (females only)
• Distended intestines, forestomach, and glandular stomach (both sexes)
• Areas of foci in the forestomach (both sexes)
• Minimal or mild centrilobular hepatocellular hypertrophy (both sexes)
• Hyperplasia and hyperkeratosis of the forestomach mucosa (both sexes)
• Erosions and/or ulcers of the gastric mucosa (both sexes)
• Edema and inflammation in the submucosa of the stomach (in the areas of erosions and ulceration) (both sexes)
• Areas of superficial mucosal hemorrhage in the stomach (both sexes)

Effect levels

Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

See attached tables.

Applicant's summary and conclusion

Conclusions:
OXEMA, when administered via oral gavage to rats for three months (5 days/week) at doses of 0, 25, 100, or 400 mg active ingredient (a.i.)/kg body weight, yielded a No Observed Effect Level (NOEL) of 25 mg a.i./kg of body weight. 100 Mg a.i./kg of body weight was considered the LOEL (Lowest Observed Effect Level) on the basis of treatment-related effects on hematology (decreased red blood cell count, hemoglobin, and hematocrit – both sexes), clinical chemistry (increased alkaline phosphatase – males only; increased blood urea nitrogen – both sexes), organ weights (increased absolute and relative liver and kidney weight – females only), and hyperplasia and hyperkeratosis of the forestomach mucosa in both sexes.
Executive summary:

The toxicity of OXEMA was assessed in Crl:CD®BR rats when administered via oral gavage to four groups of 10 rats/sex at doses of 0, 25, 100, or 400 mg a.i./kg/day for three months. Rats received test material 5 days per week at a constant volume of 5 ml/kg in corn oil. All animals were observed at least once daily for mortality, signs of ill health or reaction to treatment. Body weight and feed consumption were measured weekly. Animals were examined weekly beginning one week prior to the start of dosing using Detailed Clinical Observations (DCO). During Week 12, animals were assessed using a Functional Observational Battery (FOB) and monitored for motor activity (MA). At the end of the treatment period, all surviving rats were euthanized and necropsied. Blood samples were collected for hematology and clinical chemistry analysis from all surviving rats at terminal necropsy. Selected organs were weighed, and the designated tissues were collected and submitted for histopathologic evaluation.

No treatment-related systemic or neurologic effects were seen in the daily clinical observations, the Functional Observational Battery parameters, or motor activity in either sex at any dose level. Treatment-related, statistically significant effects were seen in the weekly DCO. Decreases (25-45%) in the number of rears in Group 3 (100 mg a.i./kg) and/or Group 4 (400 mg a.i./kg) females occurred during Weeks 8, 9, 10, 11, and 13. There were no other treatment-related effects seen in the DCO parameters in either sex.

There were no treatment-related effects on body weight, cumulative body weight gain, or feed consumption in females at any dose level or in males at dose levels up to and including 100 mg a.i./kg. Statistically significant treatment-related decreases (11-28%) in body weight were observed in Group 4 (400 mg a.i./kg) males from Week 2 through Week 13. Similarly, cumulative body weight gain was significantly decreased (27-44%) in Group 4 males throughout the entire duration of the study (Weeks 1-13). Terminal body weight, which was determined for each animal immediately prior to necropsy, after an overnight fast, was significantly decreased (28%) in Group 4 (400 mg a.i./kg) males. Treatment-related decreases (3-29%) in feed consumption were observed in Group 4 (400 mg a.i./kg) males during all weeks of the study.

No treatment-related effects on hematology parameters were observed in male or female animals at the 25 mg a.i./kg dose level. No treatment-related effects on white blood cell differential counts were observed in either sex at any dose level. Treatment-related decreases in red blood cell count (7-36%), hemoglobin (8-36%), and hematocrit (9-37%) were noted in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) animals of both sexes. A treatment-related, statistically significant increase in platelets (17%) was observed in Group 4 (400 mg a.i./kg) males.

There were no treatment-related changes in any clinical chemistry parameters in either sex at the 25 mg a.i./kg dose level. Treatment-related, statistically significant increases were seen in alkaline phosphatase in Group 3 (100 mg a.i./kg) (32%) and Group 4 (400 mg a.i./kg) (31%) males. In Group 4 (400 mg a.i./kg) males, there was also a treatment-related increase (36%) in aspartate aminotransferase, however the increase was not statistically significant. Treatment-related increases in blood urea nitrogen (BUN) were observed in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) animals of both sexes.

There were no treatment-related effects on organ weights in females treated at 25 mg a.i./kg or in males treated at dose levels up to and including 100 mg a.i./kg. Treatment-related, statistically significant increases in absolute (27%) and relative (74%) adrenal weight were observed in Group 4 (400 mg a.i./kg) males. Treatment-related, statistically significant increases in absolute kidney (24 and 30%), relative kidney (24 and 40%), absolute liver (20 and 56%), and relative liver (19 and 66%) were noted in Group 3 (100 mg a.i./kg) and Group 4 (400 mg a.i./kg) females, respectively.

No treatment-related gross pathological findings were observed in either sex at dose levels up to and including 100 mg a.i./kg. In several of the high dose (400 mg a.i./kg) animals, the intestines, forestomach, and glandular stomach were distended. Also seen in this group were areas of foci in the forestomach.

No microscopic changes directly related to treatment with the test substance were observed in any of the male or female rats in Group 2 (25 mg a.i./kg). Microscopic changes considered to be directly associated with treatment with OXEMA occurred in the liver of Group 4 (400 mg a.i./kg) and stomach of Groups 3 (100 mg a.i./kg) and 4 (400 mg a.i./kg) male and female rats. The treatment-related effect in the liver consisted of minimal or mild centrilobular hepatocellular hypertrophy in several male and female Group 4 (400 mg a.i./kg) rats. The most extensive microscopic changes occurred in the stomach of which the most common changes were hyperplasia and hyperkeratosis of the mucosa of the forestomach, erosions and/or ulcers of the gastric mucosa, edema and inflammation (inflammatory cell infiltrations) in the submucosa of the stomach (in the areas of erosions and ulceration) and areas of superficial mucosal hemorrhage. The lesions were most extensive in Group 4 (400 mg a.i./kg) rats, but hyperplasia and hyperkeratosis of the forestomach mucosa also occurred in most male and female rats in Group 3 (100 mg a.i./kg).

The data in this report support the conclusion that OXEMA, when administered via oral gavage to rats for three months (5 days per week) at doses of 0, 25, 100, or 400 mg active ingredient (a.i.)/kg, yielded a No Observed Effect Level (NOEL) of 25 mg a.i./kg of body weight. 100 Mg a.i./kg of body weight was considered the LOEL (Lowest Observed Effect Level) on the basis of treatment-related effects on hematology (decreased red blood cell count, hemoglobin, and hematocrit – both sexes), clinical chemistry (increased alkaline phosphatase – males only; increased blood urea nitrogen – both sexes), organ weights (increased absolute and relative liver and kidney weight – females only), and hyperplasia and hyperkeratosis of the forestomach mucosa in both sexes.