Urea, polymer with ethanedial

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 06, 2013 - March 24, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Oligomerisation reaction products of glyoxal and urea.

EC Number:

610-945-6

Cas Number:

53037-34-6

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

Oligomerisation reaction products of glyoxal and urea.

Details on test material:

- Name of test material (as cited in study report): Nopcote 1661
- Physical state: Light yellow, clear liquid (solution)
- Analytical purity: 46.3% w/w (in water solution)
- Lot/batch No.: 79389
- Expiration date of the lot/batch: 11 July 2013
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%)
- Stability under test conditions: A non-GLP 24-hours stability of the test item in the vehicle assessed, CiToxLAB study code: 12/331-929AN

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories, Research Models and Services, Germany GmbH- Age at study initiation: Approx. 10 weeks at starting and 12 weeks at mating.- Weight at study initiation: Males: 362–391 g, Females: 195-225 g- Fasting period before study:- Housing: up to 5 animals per sex and cage type II and/or III polycarbonatem, with lignocel bedding.- Diet (e.g. ad libitum): Ad libitum- Water (e.g. ad libitum): Ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18.5-23.6 ºC- Humidity (%): 33-68 %- Air changes (per hr): 15-20 air chaiges per hour- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(distilled)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals. The amount of the dose volumes was formulated considering the density of the test item and then they were corrected with the purity of the test item in water solution.VEHICLE- Amount of vehicle (if gavage): 1.71 mL/kg bw- Lot/batch no. (if required): 3450611 / 4310612

Details on mating procedure:

- M/F ratio per cage: 1 female and one male of the same dose group (1:1).- Length of cohabitation: Until copulation occurred, for up to 5 days.- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy. Sperm positive females were caged individually. - After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulations for concentration and homogeneity was performed using validated spectrophotometric method (CiToxLAB study code 12/331-316AN). Control, top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyse and the other for back-up. No test item was identified in the control samples. The test item formulation appeared to be homogenous and had actual concentrations of 99-105% of the nominal concentrations, within the 100±10% acceptable range. These results were considered suitable for the study purposes.

Duration of treatment / exposure:

2 weeks before mating, during the mating, and continued up to and including the day before the necropsy.

Frequency of treatment:

Daily, 7 days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 animals per sex and per dose (at least 8 pregnant female/group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based the previous repeated dose range finding study in the rat (CiToxLAB study code12/331-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Test item administrated to Wistar rats at 62.5, 250 and 1000 mg/kg bw/day, daily for 7 consecutive days, was not associated with any overt adverse effects that could be clearly ascribed to test item administration other than a transient body weight and food intake effect in the first few days of treatment in males at the High dose. Based on the results, the dose levels selected for the main study were 0, 62.5, 250, 1000 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule:Twice daily for morbidity and mortality. Once daily for general clinical observations and behavious.On gestation day 13 or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat). DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once before the first exposure, and at least weekly.- Observations: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or 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), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.BODY WEIGHT: Yes- Time schedule for examinations: before treatment for randomization purposes, on Day 0, afterwards at least weekly and at termination. Parental females were weighed on gestation days 7, 14 and 20 and postpartal days 0 (24 h after parturition), 4 and 5.FOOD CONSUMPTION:- Animal food consumption was determined by re-weighing the non-consumed diet on Day 7 and then at least weekly.HAEMATOLOGY: Yes- Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Red Blood Cell (erythrocyte), White Blood Cell (leukocyte), Haemoglobin concentration, Haematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell (erythrocyte) volume, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, Large Unstained Cells, Activated Partial Thromboplastin Time, Prothrombin Time.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Blood sugar concentration, Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ration, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -activity, Bile acids.URINALYSIS: Yes- Time schedule for collection of urine: Prior to scheduled necropsy- Metabolism cages used for collection of urine: Yes (16 hours)- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Leukocyte, Nitrite, pH, Protein, Glucose, Urobilinogen, Bilirubin, Ketones, ERY Blood, Erythrocytes, Specific Gravity, Sediment, Volume, Colour/Appearance.NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: During the last exposure week (males on Day 24, females on PND 4).- Dose groups that were examined: 5 rats per sex and per dose (subgroup A)- Battery of functions tested: sensory activity / grip strength / motor activity.

Oestrous cyclicity (parental animals):

Estrous cyclicity was observed in all female parentals.

Sperm parameters (parental animals):

Parameters examined in all male parental: Spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

PARAMETERS EXAMINEDThe following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, runts (significantly smaller pups), postnatal mortality, presence of gross anomalies, weight gain (weighed on postnatal day 0 and 4), physical or behavioural abnormalities.OTHER PARAMETERS: Duration of gestation (calculated from day 0 of pregnancy).Dams were observed whether they form a nest from the bedding material and cover their new-borns or not. Efficiency of suckling.

Postmortem examinations (parental animals):

SACRIFICE- Male animals: All surviving animals, after at least 14 days post-mating period.- Maternal animals: All surviving animals, at post-natal day 5.GROSS PATHOLOGY: YesAll animals were necropsied. External appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corporea lutea was recorded in the females as applicable.ORGAN WEIGHTS: YesIn all animals, uterus (with and without cervix), vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands, brain. ovaries and pituitary were measured.For 5 rats per sex and group (subgroup B), heart, kidneys, liver, spleen, thymus and adrenals were also weighted. Paired and individual organ weights were summarised. Relative organ weight (to body and brain weight) were calculated.HISTOPATHOLOGY: YesThe weighed organs and all organs showing macroscopic lesions of all adult animals were preserved.For 5 rats per sex and group (subgroup B), the following organs and tissues were preserved:Gross findings, Adrenal glands, Animal identification, Aorta (thoracic and abdominal), Brain, Clitoral gland / Preputial gland, Epididymes, Eye with the optic nerve, Oesophagus, Femur with marrow incl. joint, Heart, Kidney, Large intestine, External lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph nodes, Larynx, Nasopharynx, Ovaries with oviduct, Pancreas, Pituitary, Prostate, Salivary glands, Sciatic nerve, Seminal vesicles (with coagulating glands), Skeletal muscle (quadriceps), Skin/subcutis with mammary, gland area, Small intestine, Spinal cord (cervical, lumbar, and thoracic levels), Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina.Detailed histological examination were performed in the control and high dose groups and all macroscopic findings from all animals.Special attention were paid to evolution of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and insterstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

SACRIFICEAll F1 offspring was terminated on Day 4 post-partum. GROSS NECROPSYGross necropsy consisted of external examination for gross abnormalities.

Statistics:

The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Reproductive indices:

Male mating index, female mating index, male fertility indey, gestion index.

Offspring viability indices:

Survival index, pre-implantation mortality, intrauterine mortality, total mortality, post-natal mortality, sex-ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs related to treatment.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the pre-mating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared to control, lower food consumption values, attaining statistical significance were noted for males at 1000 mg/kg (High dose) during the first week of the treatment. These changes were generally correlated with the body weight and body weight gain values. In addition, slightly lower food consumption was observed during the mating period.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant differences between the control and any of treated groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher bile acid concentration, attaining statistical significance were noted in High dose male and female animals. Compared to the control higher potassium and serum urea concentration (Urea) were observed in Mid and High dose female animals. No signs of kidney injury were observed during pathology evaluation.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urobilinogen and urinary bilirubin, calcium oxalate crystals as well as dark yellow discoloration of the urine was presented in high dose males. In high dose females and in one from the mid dose, calcium oxalate crystals were also presented in the urine. Dark yellow discoloration of the urine was observed in one mid dose female. Semi quantitative measurement of protein in the urine showed a slight elevation in high dose, when compared to the control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related ulcers of the non-glandular mucosa of the stomach were microscopically observed in 4/7 High dose males and 4/7 High dose females, and were in correlation with necropsy. There was no evidence of test item-related histological findings in the High dose animals or macroscopic observations from all groups in the reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogonic cells representing different phases of the development and differentiation of the spermatozoa were similar in control and high dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both control and high dose females.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the oestrus cycle or reproductive parameters.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogonic cells representing different phases of the development and differentiation of the spermatozoa were similar in control and high dose males.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation to the test item administration.Mating indices = 100% in all groups.Fertility indices = 83% in control, 92% in mid dose, and 100 in low and high doses. Gestation indices = 90% in control, 100% in low, mid and high doses.Test item administration did not have impact on the duration of mating period. There was no effect of treatment noted during gestation, parturition or the post-partal period. Mean duration of pregnancy was similar to control (22-24 days). All the parturitions were normal. Number of mean corporea lutea and implantation sites were similar in the treated group compared to control. There were no item related effects on pre/post-implantation, post-natal or mortality values.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

250 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical biochemistry

urinalysis

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Any adverse effect considered treatment related or toxicologically significant.No external abnormalities were detected at the clinical or external macroscopic examination.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Test item administration did not lead to mortality. The sex ratios were similar in the control and treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment on the offspring body weight or body weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no abnormalities at the external examination of all pups surviving to post-natal day.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormal behaviour of the pups was noted.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)Test item administration did not lead to mortality. The sex ratios were similar in the control and treated groups.CLINICAL SIGNS (OFFSPRING)Any adverse effect considered treatment related or toxicologically significant. No abnormal behaviour of the pups was noted. No external abnormalities were detected at the clinical or external macroscopic examination. BODY WEIGHT (OFFSPRING)There was no adverse effect of treatment on the offspring body weight or body weight gain.GROSS PATHOLOGY (OFFSPRING)There were no abnormalities at the external examination of all pups surviving to post-natal day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction / developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Reproductive ability assessment and indices

	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Paired males - females	12/12	12/12	12/12	12/12
Pregnant females	10/12	12/12	11/12	12/12
Pregnant females, with stillborn	1/10	0/12	0/11	0/12
Male-female Mating Index (%)	100	100	100	100
Male-female Fertility Index (%)	83	100	92	100
Gestation Index (%)	90	100	100	100

Evaluation of the gestation, parturition and post-partal period

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Number of pregnant females	10	12	11	12
Number of delivered dams	10	12	11	12
Duration of pregnancy (days, mean)	22.50	22.33	22.82	22.42
Number of corpora lutea / dams (mean)	19.50	20.17	18.82	19.25
Number of implantations / dams (mean)	17.10	16.25	14.82	16.58
Number of dams with live pups day 0	9	12	11	12
Number of dams with live pups day 4	9	12	11	12
Pre-implantation mortality (%)	12.10	18.56	22.31	13.82
Intrauterine mortality (%)	19.86	7.46	15.26	9.13
Post-natal mortality (%)	0.00	3.69*	1.56	6.88
Total mortality (%)	19.86	10.94	16.64	14.18

Mortality, clinical observations and survival of pups

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Number of liveborns (total) on PND0	141	181	145	181
Number of viable pups (mean) on PND0	14.10	15.00	13.18	14.75
Number of viable pups (mean) on PND4	14.10	14.50	12.91	14.33
Number of viable pups (total) on PND0	141	180	145	177
Number of viable pups (total) on PND4	141	174*	142	172*
Survival index PND0	90.00	99.54	100.00	96.67
Survival index PND4	100.00	96.31*	98.44	93.12
Sex ratio (%) on PND0	46.24	39.71	45.85	53.94
Sex ratio (%) on PND4	46.24	39.24	45.70	52.19

Applicant's summary and conclusion

Conclusions:

The NOAEL for parental toxicity after at least 28 days of oral exposure to test item was determined to be 250 mg/kg bw/day in rats (basis for effect: minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level) . The NOAEL for the reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day (basis for effect: no treatment related effects noted at the highest dose tested).

Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECD Guideline 422 following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels, 62.5, 250 and 1000 mg/kg bw/day. The control animals were treated with the vehicle only (distilled water). 12 Male and female Wistar rats per group were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Selected organs were subjected to histopathology examination. No mortality was observed during the study. There were no clinical signs related to treatment. There was no effect of treatment noted during evaluation of the functional observation battery, grip strength, foot splay or motor activity. In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the premating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control. There was no effect of treatment noted during evaluation of the reproductive parameters. There were no adverse effects on the F1 offspring viability, clinical signs or development. Single or multiple ulcers of the non-glandular mucosa of the stomach (4/12 High dose males and 4/12 High dose females) were observed as treatment-related macroscopic findings. There was no effect of treatment on organ weights. Test item related microscopic findings were found at 1000 mg/kg bw/day (High dose). In the stomach, ulcers of the non-glandular mucosa were microscopically observed in 4 High dose males and 4 High dose females. There were no microscopic findings related to treatment at 62.5 or 250 mg/kg bw/day. Lower food consumption values were noted for males at 1000 mg/kg during the first week of the treatment. These changes generally correlated with the body weight and body weight gain values. There was no effect of treatment on haematology and blood clotting parameters. Increases were noted in potassium and/or urea and bile acids in high dose males and females. Calcium oxalate crystals were noted in high dose males and females, with increases in urobilinogen and bilirubin in males.

Based on the minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level, the no adverse effect level (NOAEL) was determined to be 250 mg/kg bw/day in male and female rats after at least 28 days of test item treatment by oral gavage. Moreover, since no treatment related effects were noted during the evaluation of the reproductive paramenters and F1 offsprings, the NOAEL for the reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day.