Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study available as unpublished report, no restrictions, fully adequate for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, July 2000.
GLP compliance:
yes (incl. certificate)
Remarks:
Toxi-Coop Zrt., Pálya u. 2., H-2120 Dunakeszi, Hungary
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 85 - 90 days old
- Weight at study initiation: Males: 356 - 418 g; Females: 210 - 251 g
- Housing: Cage type: Type III polypropylene/polycarbonate; Size: 22 x 32 x 19 cm (width x length x height), Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany). Before mating: 2 animals of the same sex/cage, Mating: 1 male and 1 female / cage, Pregnant females: individually, Males after mating: 2 animals/cage
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water, as for human consumption, ad libitum
- Acclimation period: 40 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water in concentrations necessitated for this study.
- Concentration in vehicle: 20, 50 and 100 mg/mL.
- Amount of vehicle: 5 mL/kg bw
Details on mating procedure:
Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred. Three females that failed to mate within 14 days were re-mated with proven males of the same group due to unsuccessful mating (1/12, in control, 100 and 500 mg/kg bw/day groups, each). Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Petol PM 410-4N proved to be stable at room temperature and at 5 ± 3°C for 3 days. A separate analytical report has provided this data (Toxi-Coop Study no. 742.102.3922). Analytical control of dosing solutions (concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 12 mL of each formulation (20, 50 and 100 mg/mL) and five aliquots of 1 mL control substance (vehicle) were taken at the first and second occasions. Date of sampling: May 29 and July 09, 2013; Date of analytical control: May 30 and July 10, 2013; The real concentration of Petol PM 410-4N formulations administered to animals were in accordance with the acceptance limit at each set of sampling measurements. The measured concentrations were within a range of 97 and 108 % of nominal values at both analytical occasions.
Duration of treatment / exposure:
Males: 44 days (14 days pre-mating and 4 – 21 days mating plus a 26 – 9 days post mating period) and then they were sacrificed on Day 44.
Females: 14 days pre-mating, for up to 21 days mating period where it was neccessary, through gestation and for 4 or 8 days after delivery, with necropsy on the following day.
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 250, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with Petol PM 410-4N in rats (Study no. 742.439.4206, not reported). In this preliminary study the mid-dose of 300 mg/kg bw/day was well tolerated whereas the highest dose (1000 mg/kg bw/day on days 0-2, 750 mg/kg bw/day from day 4) caused weight loss, mortality and clinical signs of toxicity. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- All F1 offspring were euthanized on postnatal day 4.

Examinations

Parental animals: Observations and examinations:
- Mortality: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
- Clinical observations: General clinical observations were made once a day, after treatment at approximately the same time. More detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
- Body weight: All parental animals were weighed with an accuracy of 1 g. Parental males were weighed on the first day of dosing (day 0), weekly thereafter and at termination. Parental females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition) and 4 post-partum. The body weight of high dose treated animals (male and female) showing weight loss were measured occasionally daily during the first five weeks. Body weight of the female animals was additionally weighed on gestational days 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight was measured on day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).
- Food consumption: The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating phase (pre-mating days 7, 13 for male and female animals, gestation days 7, 14 and 21, lactation day 4 for female animals). Food consumption of male animals was also determined by weekly interval during post-mating period.
- Examination of placental sign: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If it was negative on day 13, the examination was repeated on day 14 of gestation.
- Clinical pathology: Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1 ml 9NC Microtube, 0.106 mol/L, Greiner Bio-One International AG, or equivalent), one for hematology (MiniCollect® EDTA tubes, spray-dried, 0.25 mL, Greiner Bio-One International AG, or equivalent), and the third one (VACUETTE® Serum Tube, 2.5 mL, Greiner Bio-One International AG, or equivalent) to obtain serum samples for clinical chemistry. Tubes for hematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 mL blood was collected, if possible into clinical chemistry tubes.
- Hematology: The hematology parameters were measured in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy) by SYSMEX XT-2000iV. Hematology parameters examined: White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential white blood cell count (Neutrophil, Lymphocyte, Eosinophil, Monocyte, Basophil).
- Blood coagulation: The blood coagulation parameters were determined by AMAX Destiny Plus. Blood coagulation parameters examined: Activated partial Thromboplastin Time, Prothrombin Time.
- Clinical chemistry: The clinical chemistry parameters were measured by Konelab 60i. Clinical chemistry parameters examined: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Gamma Glutamyltransferase activity, Alkaline Phosphatase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Bile acids, Inorganic phosphate concentration, Calcium concentration, Sodium concentration, Potassium concentration, Chloride concentration, Albumin concentration, Total Protein concentration, Albumin/globulin ratio.
Litter observations:
- Observation of the delivery process: Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded and were calculated from day 0 of pregnancy. The duration of gestation was recorded and calculated from day 0 of pregnancy. Dams were observed whether they made a nest from the bedding material and nurse their new-borns or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam. Each litter was examined as soon as possible after delivery (within 24 h of parturition), to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities. Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete), and day 4 post-partum with an accuracy of 0.1g. Any abnormal behavior of the offspring was recorded. All litters were checked and recorded daily for the number of viable and dead pups. Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.
Postmortem examinations (parental animals):
- Necropsy: Gross necropsy was performed on dead animals shortly after its death and on the remaining animals terminally (one day after the last treatment). Animals were euthanized by exsanguination after verification of Isofluran-narcosis. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded. The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries and pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected for blood collection from each group. Thyroid and parathyroid were preserved together with pharynx. List of organs to be preserved: adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with vagina), gross lesions, heart, kidneys, large intestines (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion;), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder.
- Organ weight: At the time of necropsy, body weight, brain weight and weight of the testes and epididymides of all parental male animals were determined. Relative organ weights (to body and brain weight) were calculated and reported. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed. Paired organs were weighed together; absolute organ weights were reported. Relative organ weights (to body and brain weight) were calculated and reported.
- Histopathology: Detailed histological examinations were performed on the ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. In addition, detailed histological examinations were performed on the ovaries, uterus, vagina, pituitary of not mated or non-pregnant females and pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland of males cohabited with. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups (n= 5 animals/sex/group). Histopathological examinations of the small intestines, kidneys and liver were extended to animals of the low and mid dose groups (n= 5 animals/sex/group) because treatment-related changes were observed in the small intestines and liver of examined animals in the high dose group at the histopathological examination. Tubulonephrosis in the kidneys of animals in the high-dose group was detected with a low total incidence, however the presence of this lesion in one surviving high-dose male (1/5) increases the likelihood that this finding was related to treatment as well, therefore a decision was made to examine kidneys in the low and mid dose treated animals.
Postmortem examinations (offspring):
Offspring found dead and offspring euthanized on post-natal day 4 were carefully examined for gross abnormalities. Stillborn and dead pups were subjected to necropsy. A lung flotation test was performed at the necropsy of pups just after birth to determine whether those were died intrauterine or after delivery. [The lung flotation test was negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.].
Statistics:
The statistical evaluation of appropriate data were 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 comparison was performed using Mann-Whitney U-test. Chi2 test was performed if feasible. Frequency of clinical observations, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
Mating Index, Fertility Index, Gestation Index
Offspring viability indices:
Pre-implantation mortality, Post-implantation mortality, Intrauterine mortality, Post-natal mortality, Total mortality, Survival Index, Sex ratio.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

- Mortality: Two male and two female animals of 500 mg/kg bw/day group were found dead on Days 14, 20, 25, and 49.
- Clinical observations: Clinical signs of the four animals that died prematurely included: Salivation (4/4), diarrhea (4/4), noisy breathing (2/4), piloerection (3/4), decreased activity (2/4), incoordination (1/4), narrow eye aperture (1/4), hunched back (1/4), swollen abdomen (1/4), dyspnea (1/4). Daily observations: Test item related clinical signs appeared in the surviving male and female animals of 500 and 250 mg/kg bw/day groups. Salivation (8/10 male and 8/11 female), diarrhea (6/10 male and 5/11 female), noisy breathing (4/10 male), piloerection (1/10 male and 2/11 female) and decreased body tone (2/10 male) were observed in 500 mg/kg bw/day group. Prone position (1/10 male) and nuzzling up the bedding material (1/10 male) as well as sanguineous fur around nose (1/10 male) were also noted for single animals of the high dose group during the premating, mating or post-mating period. Salivation (12/12 male and 8/12 female) and noisy breathing (2/12 male) were detected in 250 mg/kg bw/day group. One male animal of 100 mg/kg bw/day group (1/12) also salived between days 28 and 43. Alopecia on the right side of the back was detected of a single control female animal on the last day of the observation period. All surviving female animal was symptom-free during the gestation and lactation periods. Vaginal bleeding was observed in one dam of 500 mg/kg bw/day group for one day after the delivery as individual sign. Weekly observations: Diarrhea was observed in some male animals on Days 7, 13, 20 and 41 (5/12, 3/12, 1/11 and 1/10, respectively). Noisy breathing was also detected in male animals (4/12) of 500 mg/kg bw/day on Days 13. One male animal of 500 mg/kg bw/day group showed decreased body tone, piloerection, prone position and nuzzling up the bedding material on Day 13 but recovered thereafter and the behavior and physical condition were normal at the weekly observations from Day 20 up to the day of the necropsy. Decreased activity, diarrhea, incoordination, narrow eye aperture, piloerection and hunched back were noted for one male animal of 500 mg/kg bw/day group, which died shortly after the observation on Day 20. Noisy breathing was detected in male animals of the 250 mg/kg bw/day group on Days 13, 27 or 44 (1/12, each). In the female animals of 500 mg/kg bw/day group, diarrhea and piloerection were observed on Days 7 (3/12 and 2/12, respectively) and 20 (1/6, both). Alopecia on the back of one female control animal was detected on the day of the necropsy.
- Functional observation: Functional observations did not demonstrate any test item related changes in the behavior, physical condition and reactions to different type of stimuli of animals selected for examination (500, 250 or 100 mg/kg bw/day, control) at the end of the treatment period (Day 42).
- Body weight and body weight gain: The body weight development was reduced in male animals of 500 and 250 mg/kg bw/day group during the entire study. Significantly less body weight gain was detected in the male animals of 500 mg/kg bw/day during the first two weeks as well as between Day 0 and 41 (total body weight gain) with respect to the control. These less body weight gains resulted in less body weight with respect to the control (< -10%) from day 7 up to and including last day of the measurement. The mean total body weight gain of male animals of 250 mg/kg bw/day was significantly less than in the control group due to the reduced – statistically not significant – bodyweight gain with respect to control during the entire observation period. In the female animals, there were no test item related statistically significant differences in the mean body weight and body weight gain between the control and test item treated groups during the pre-mating period. However, the mean body weight decreased during the first week in comparison with the starting (Day 0) value due to the significant body weight decrease of some female animals (4/12) between Days 0 and 7. This body weight decrease of some high dose female animals was considered to be indicative of the test item influence, which however was transient as it had no significant influence on the body weight and summarized body weight gain because the body weight gain exceeded the control value on week 2. Statistical significances were noted for the higher mean body weight gain of female group receiving 500 mg/kg bw/day dose of the test item between Days 7 and 13, in 250 mg/kg bw/day group during gestation weeks 1 and 3, and for the total body weight gain (during the entire gestation period). This resulted in a higher mean body weight of pregnant animals of 250 mg/kg bw/day group on gestation day 21. Compared to the control group, statistical significances were detected at the slightly higher mean body weight gain of female animals of 500 and 100 mg/kg bw/day groups on the gestation week 1. Although being statistically different to the concurrent control group, these differences in the body weight gain (in 500, 250 and 100 mg/kg bw/day groups) and in the body weight (in 250 mg/kg bw/day group) were not considered to be of toxicologically relevant because those were due to the low values of the control group. (The control mean value was less than the historical control mean). The body weight of dams of 500 mg/kg bw/day was below the control value on lactation day 0, however the differences with respect to the control group was slight (-7 %) and therefore were judged to be toxicologically not significant.
- Food consumption: The mean daily food consumption of male rats in 500 and 250 mg/kg bw/day groups was statistically significantly less than in the control group in full correspondence with the body weight development between Days 0 and 7 and between Days 7 and 13. In male animals of 250 mg/kg bw/day group, the mean daily food consumption was also below the control value between Days 20 and 27. Similarly, the mean daily food consumption of female animals was slightly less comparing to control group at 500 mg/kg bw/day on week 1 (between Days 0 and 7). On the contrary, the mean daily food intake of 250 mg/kg bw/day female animals exceeded the control’s value between gestation days 14 and 21.
- Hematology and blood coagulation: Test item related changes were observed in percentages of neutrophil granulocytes and in the percentage of lymphocytes at 500 mg/kg bw/day (male and female) and at 250 mg/kg bw/day (male), and in white blood cell count of female animals in 250 and 500 mg/kg bw/day group. Compared to their control group, the mean percentages of neutrophil granulocytes (NEU) were higher, the mean percentage of lymphocytes (LYM) were lower in male animals of 500 and 250 mg/kg bw/day and in female animals of 500 mg/kg bw/day. The mean hematocrit value (HCT) of male animals in 100 mg/kg bw/day group was slightly below the control value however the value remained within the historical control range. The mean white blood cell count (WBC) of female animals received 500 or 250 mg/kg bw/day dose was statistically significantly higher than in the control group.
- Clinical chemistry: No pathologic test item effect was detected at the evaluation of clinical chemistry parameters. The mean activity of alanine aminotransferase (ALT) was slightly higher with respect to the control at 500 mg/kg bw/day (male and female animals) and 250 mg/kg bw/day (female) dose levels. The values remained within the historical control ranges and the histopathological findings and the respective organs weights did not indicate a test item effect on the liver therefore, this effect was considered to be of no toxicological relevance. Statistically significant differences were indicated between the control and test item treated groups of male animals for the less concentration of bile acids (BAC) and albumin (ALB) at 500 mg/kg bw/day and albumin:globulin ratio (A/G) at 250 mg/kg bw/day, for the less total bilirubin (TBIL) concentration of male animals and for a higher serum level of potassium (K+) in male and female animals of 100 mg/kg bw/day. These slight changes were judged to be of little or no biological significance as the mean values were well within the historical control ranges, occurred sporadically and were not related to doses.
- Necropsy: Necropsy observation did not reveal any test item related macroscopic findings in surviving animals at any dose level (500, 250 or 100 mg/kg bw/day). In dead male animals (2/12) of 500 mg/kg bw/day group, dark red liver (1/2), congenital absence of one side kidney, atrophied spleen (2/2) or thymus, (1/2), smaller than normal sex organs or accessory sex organs [testes (1/2), epididymides (2/2), prostate (1/2), seminal vesicles (2/2)], gas filled stomach (1/2) or intestines (2/2), yellowish gelatinous intestinal content (2/2), reddish colored duodenum (1/2) and undernourishment (1/2) were observed. In dead female animals of 500 mg/kg bw/day group (2/12), enlarged adrenal glands (2/2), white knots on the wall of the stomach (1/2), gas content in the stomach (1/2) or intestines (2/2), yellowish gelatinous intestinal content (1/2), reddish colored duodenum (1/2) were detected at the gross pathology. The fur was contaminated with blood around the nose (1/2) and with faces around the anus (1/2). In the uterus, of dead pregnant animal (1/2) early death and fetal death were observed. There were no macroscopic findings in the surviving male animals at any dose level (500, 250 or 100 mg/kg bw/day and control group) and in dams of 250 or 100 mg/kg bw/day groups. Hydrometra was detected in two dams (2/9) of 500 mg/kg bw/day group. The liver was pale, nutmeg-like patterned and enlarged in one female control dam (1/10). Alopecia on the back and early death of fetuses and dark-red placenta in the uterine horns were seen in the single not delivered pregnant control female animal (1/1). In non-pregnant females of 250 mg/kg bw/day group (2/4), hydrometra was observed. Hydrometra is a species specific finding in relation with the sexual cycle of animals and there were no histopathological findings referring to test item related toxic effects at 500 or 250 mg/kg bw/day dose levels. Hepatic observations, alopecia and early death, dark placenta, as individual changes were present in single control female animals. These alterations are commonly seen in untreated experimental rats of this strain, therefore were considered to be without any toxicological relevance.
- Organ weight: Slightly but statistically significant higher mean weights of spleen (relative to body and brain weights) were observed in female animals dosed with 500 mg/kg bw/day and although no histopathological effect was noted, it cannot be excluded that these findings are related to a test item effect on function of these organs. Statistical significances noted for higher mean testes and brain weights relative to body weight were due to the slightly but significantly less fasted body weight with respect to control in male animals of 500 and 250 mg/kg bw/day group. Consequently, the mean body weight relative to brain weight of these groups was less with respect to control. The mean heart weight relative to brain weight was slightly lower than in the control group in male animals of 500 mg/kg bw/day group. The toxicological significance of these observed organ weight changes are probably negligible, as they were well within the range of the historical controls.
- Histopathology: In all dead animals of 500 mg/kg bw/day (2/12 males and 2/12 females), histological examination revealed histiocytic infiltration in the mucous membrane of small intestines, bile duct hyperplasia in the liver, multifocal tubulonephrosis in the kidney, alveolar edema in the lungs, lymphoid atrophy in the spleen and thymus). These findings probably were in connection with the test item and explain the fatal death of these animals. In addition, in one of dead male animal, decreased intensity of spermatogenesis in the testis and decreased amount of secrete in the seminal vesicle, coagulating gland and prostate was observed. In one female dead animal, glandular dilatation in the wall of stomach occurred. The glandular dilatation in the wall of stomach and the decreased intensity of spermatogenesis, and the decreased amount of secrete in the sex glands were considered as individual disorders. In surviving animals of 500 mg/kg bw/day selected for histologic examination, histiocytic infiltration in the mucous membrane of small intestine (4/5 male and 4/5 female), and minimal or mild bile duct hyperplasia in the liver (2/5 male), and tubulonephrosis in the kidneys (1/5) were observed. Histiocytic infiltration occurred mainly in the villi, in the connective tissue (stratum villosum) of the mucous membrane. These histiocytes (macrophages) contained vacuoles in their cytoplasm and their appearance resembled on the “foamy cells”. They accumulated in the tissues and the villi, as consequence, were thickened. This finding was detectable mainly in the jejunum and lighter in the ileum, and was in connection with the effect of test item. Histiocytic infiltration in the mucous membrane of small intestine or bile duct hyperplasia in the liver was not detected in the animals selected for histological examination in the middle or low dose groups. In the surviving male animals, the investigated organs of reproductive system (testes, epididymides, sex glands) were histologically normal and characteristic on the sexually mature organism in all cases of control and all treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, seminal vesicles, coagulating gland and prostate and pituitary gland was normal in all cases as well. In the surviving female animals of control and high dose groups, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and 250 or 500 mg/kg bw/day treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation, the epithelial capsule and ovarian stroma was normal in all cases as well. The histological picture of uterus cervix, vagina and pituitary was normal. Lack of corpora lutea was detected in the ovaries of not mated female animal of 100 mg/kg bw/day group. Individual alterations were observed in some female animals. Perilobular vacuolization occurred in the liver of one control female animal and as well as hemorrhage in the uterus of one control female animal, which did not deliver. In some female rats dilatation of uterus was observed (2/9 dams of 500 mg/kg bw/day group and 2/4 non-pregnant females of 250 mg/kg bw/day group). Dilatation of uterus – without inflammation or other pathological lesion – is a physiological phenomenon in connection with the normal sexual cycle. No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) in the cardiovascular system, the hematopoetic system, the skeleton, or the central or peripheral nervous system was observed. The structure and the cell morphology of the endocrine glands was the same in the control and treated animals.
- Delivery Data of Dams: There was no evidence of test item related effect on delivery data of dams. Compared to the control group, statistical significances were detected at the slightly shorter duration of pregnancy at 250 and 500 mg/kg bw/day and at the less mean number of stillborns at 500, 250 and100 mg/kg bw/day groups, which were not considered to be of toxicologically relevant. The mean number of corpora lutea, implantation, mean number of pre- and post-implantation loss and mean number total intrauterine mortality were within the relevant historical control ranges. The mean number of total births, live-borns, and viable pups on day 0 were comparable in the control and all test item treated groups.
- Reproductive Performance: Test item related changes were not found in the reproductive performance of male or female animals. All examined parameters were similar in the control and 500 mg/kg bw/day groups except gestation index, which was higher in the high dose females than in the control group. Statistical significances were observed with respect to the appropriate control in the male animals of 250 mg/kg bw/day group (percentage of fertile males, i.e. fertility index) and 100 mg/kg bw/day group (in the percentage of not mated, mated or fertile animals, as well as in the copulatory or fertility indices). The number and percentage of non-pregnant animals was higher and the number and percentage of pregnant females were less at 250 mg/kg bw/day than in the control group. In the contrary, the number and percentage of non-pregnant animals was less and the percentage of pregnant females was higher at 100 mg/kg bw/day group, with respect to the control. The statistical significances noted for the copulatory (100 mg/kg bw/day), fertility (250 and 100 mg/kg bw/day), or gestation indices (500, 250 and 100 mg/kg bw/day), were not considered to be toxicologically relevant. These slight but statistically significant differences to the control data were considered to be indicative of the biological variation and not related to the test item. Similarly, the inadequate nourishing instinct of one dam of 500 mg/kg bw/day group causing total litter loss (5 missing and 5 dead offspring) was considered to be a species specific findings occurring also in not treated experimental rats.

Effect levels (P0)

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Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 250 mg/kg bw/day, clinical signs (male and female), slight changes in the body weight gain, food consumption and hematological parameters (NEU, LYM in male animals, WBC in female animals) were detected.
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The male and female reproductive performance (gonad function, mating behavior, conception) was normal in parental male and female animals.

Results: F1 generation

Details on results (F1)

- Mortality: There were no significant differences with respect to the control in the percent or litter means of live born pups, viable pups and dead pups on lactation days 0 and 4. One dam of 500 mg/kg bw/day group had total litter loss (5 missing and 5 dead offspring) due to the inadequate nourishing instinct of mother.
- Sex Distribution: There were no significant differences in the litter means of genders at any dose level with respect to the control on postnatal days 0 or 4.
- Clinical observations: Test item related clinical signs did not appear in offspring.
- Body weight: A test item influence was detected on the body weight development of offspring in 500 mg/kg bw/day group. The mean litter weight gain between postnatal days 0 and 4 and the offspring’s mean weight at the birth and on postnatal day 4, and mean offspring’s weight gain in 500 mg/kg bw/day group was less than in the control. The mean litter weight was slightly but statistically significantly higher with respect to control in 250 mg/kg bw/day group on postnatal days 0 and 4.
- Necropsy: No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The offspring’s body weight development was depressed at 500 mg/kg bw/day between postnatal days 0 and 4.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this repeated-dose toxicity study with reproduction/developmental toxicity screening test in rats (OECD 422, GLP), the NOAEL for general toxicity, fertility and developmental toxicity was determined to be 100, 500, and 250 mg/kg bw, respectively.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, four groups of 12 Wistar rats per sex were administered the test substance by gavage once a day at 0 (sunflower oil), 500, 250 and 100 mg/kg bw/day. Stability, homgeneity and correct concentration of the test substance in the vehicle were confirmed by analysis. All animals of the parent generation received test item or vehicle prior to mating (14 days) and throughout the mating period. Test item or vehicle was administered to male animals until day before the necropsy post mating (altogether for 44 days). For females, test item or vehicle was administered through the gestation period and up to lactation days 3 - 7, i.e. up to the day before the necropsy (altogether for 44 - 59 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. Five dams and males cohabited with were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on day 4 postpartum. Offspring were weighed and observed for possible abnormalities and were euthanized on postnatal day 4. Two male and two female animals were found dead during the study due to the toxic effect of 500 mg/kg bw/day dose of the test item. Clinical signs (decreased activity, diarrhea, dyspnea. hunched back, incoordination, narrow eye aperture, noisy breathing, piloerection, salivation, swollen abdomen), necropsy observations (atrophy of spleen and thymus, gas content in the intestines and stomach, dark red liver, reddish colored duodenum, undernourishment, yellowish gelatinous intestinal content) and histopathology findings (histiocytic infiltration in the mucous membrane of small intestines, bile duct hyperplasia in the liver, multifocal tubulonephrosis in the kidney, alveolar edema in the lungs, lymphoid atrophy in the spleen and thymus) referred to test item effect and explain the fatal death of these animals. In the surviving animals, test item related clinical signs (decreased body tone, diarrhea, salivation, noisy breathing, nuzzling up the bedding material, piloerection, prone position and sanguineous fur around nose) were observed in male or female animals of 500 mg/kg bw/day group. Salivation (male and female) and noisy breathing (male) were detected in 250 mg/kg bw/day group. At the detailed weekly observations, most of the above listed signs appeared in male (decreased body tone, diarrhea, noisy breathing, nuzzling up the bedding material, piloerection, prone position or sanguineous fur around nose) or female (diarrhea or piloerection) animals of 500 mg/kg bw/day group mostly during the first half of the treatment period. Noisy breathing was recorded also for two male rats of 250 mg/kg bw/day group at the weekly observations. Functional observations did not demonstrate any test item related changes in the behavior, physical condition and reactions to different type of stimuli of animals selected for examination (500, 250 or 100 mg/kg bw/day, control) at the end of the treatment period. The body weight gain was reduced in male animals of 500 and 250 mg/kg bw/day group during the entire treatment period and in some female animals of 500 mg/kg bw/day during the first week of the treatment. The mean daily food consumption was less in full correspondence with the body weight development of male animals in 500 and 250 mg/kg bw/day groups during the first two weeks of the treatment period and in female animals of 500 mg/kg bw/day group between Days 0 and 7. Test item related haematological changes were observed in a higher percentage of neutrophil granulocytes (NEU) and in a lower percentage of lymphocytes (LYM) at 500 mg/kg bw/day (male and female) and at 250 mg/kg bw/day (male). In females of the 250 mg/kg bw group, a statistically significant increase (~75%) in total number of white blood cells (WBC) were observed. In female rats, a statistically significant increase of ~75 and ~240% in total number of white blood cells (WBC) were observed in the 250 and 500 mg/kg bw dose group, respectively. Specific pathologic alterations related to the test item were not detected at the evaluation of red blood cell parameters, in clinical chemistry or blood coagulation parameters. Gross pathology examination did not reveal test item related macroscopic changes in the organs or tissues of surviving animals subjected to necropsy at any dose level (500, 250 and 100 mg/kg bw/day). The mean spleen weights relative to body and brain weights of female animals dosed with 500 mg/kg bw/day slightly exceeded the control value and although no related histopathological effect was noted, it cannot be excluded that these findings are related to a test item influence on the splenic function. Test item related lesions were observed in the intestines (histiocytic infiltration in the mucous membrane of small intestines; male and female) and liver (minimal or mild bile duct hyperplasia; male) of surviving animals of 500 mg/kg bw/day group. There were no differences between the control and test item treated groups in the reproductive ability of male and female animals and in the delivery data of dams. A test item effect on the offspring development was observed in the lower mean litter weight gain between postnatal days 0 and 4 and also on the offspring’s mean weight at the birth, on postnatal day 4 and mean offspring’s weight gain in 500 mg/kg bw/day group. Under the conditions of the present study, the test substance caused death, clinical signs, changes in body weight and body weight gain, food consumption, hematological parameters (white blood cells (WBC, NEU, LYM), accompanied with histological alterations in the small intestines (histiocytic infiltration) and in the liver (bile duct hyperplasia), in dead animals renal tubulonephrosis, pulmonary alveolar edema and lymphoid atrophy in the spleen and thymus after repeated dose oral administration to male or female rats at 500 mg/kg bw/day dose. At 250 mg/kg bw/day, clinical signs (male and female), slight changes in the body weight gain, food consumption and hematological parameters (NEU, LYM in male animals, WBC in female animals) were detected. At 100 mg/kg bw/day, there were no test item related effects. The male and female reproductive performance (gonad function, mating behavior, conception) was normal in parental male and female animals. Dam’s delivery data was not affected by the test item at any dose level. The offspring’s body weight development was depressed at 500 mg/kg bw/day between postnatal days 0 and 4. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined for general toxicity, fertility and developmental toxicity to be 100, 500, and 250 mg/kg bw, respectively.