Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- Under the conditions of the 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.

Link to relevant study records
Reference
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other: GLP-compliant guideline study available as unpublished report, no restrictions, fully adequate for assessment.
Reason / purpose:
reference to same study
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
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
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
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.
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.
- 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.
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.
- 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.
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.
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Species:
rat
Quality of whole database:
The study available is a GLP-compliant guideline study, fully adequate for assessment.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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 (TOXI-COOP Zrt, 2014). Stability, homogeneity 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 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.

Effects on developmental toxicity

Description of key information

Under the conditions of the developmental toxicity study test in rats (OECD 414, GLP), the NOAEL for maternal toxicity cannot be defined, the NOAEL for developmental toxicity was determined to be >= 200 mg/kg bw.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Feb 2018 to 09 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Toxicological Studies, ul. Doświadczalna 27, 43–200 Pszczyna, Poland
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Cmdb: Wi; outbred
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University in Białystok kept behind the breeding barrier.
- Weight at study initiation: Mean body weight 231.7 g (200 to 268 g)
- Housing: The animals were housed in plastic cages with metal wire lid and dimensions: 58 x 37 x 21 cm (length x width x height). Autoclaved, and additionally UV-irradiated wood chips were used as bedding. At mating one female was placed in one cage with one male. At pregnancy the females were housed individually. The environment of the animals was enriched by placing wooden blocks, tunnels and nesting materials for laboratory animals in the cages.
- Diet: “ALTROMIN” standard laboratory fodder, Altromin Spezialfutter GmbH & co.KG, Seelenkamp, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: at least 5 days.
- A general medical-veterinary examination was performed on the day of the introduction of the animals to the acclimatisation, whereas a detailed medical-veterinary examination was performed prior to the beginning of the experiment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 24.0
- Humidity (%): 33.5 to 58.5
- Air changes (per hr): about 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
28 Feb 2018 to 09 Jul 2018
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared every two days based on results of the stability test. To ensure the appropriate concentration for doses, the right quantity of test item was dissolved for each dose. The following establishments was fulfilled during preparation of doses:
Group 1: 1 g of solution contained 20 mg of test item at dose of 80 mg/kg bw
Group 4: 1 g of solution contained 31,25 mg of test item at dose of 125 mg/kg bw
Group 2: 1 g of solution contained 50 mg of test item at dose of 200 mg/kg bw
Group 3: 1 g of solution contained 125 mg of test item at dose of 500 mg/kg bw
The test item was given from the 5th to the 19th day of gestation using a stomach tube. The control group of pregnant females (group 0) was given sunflower oil at the same volume as groups treated with the test item. In order to maintain a constant dose level appropriate to the animals’ body weight, the doses were adjusted on days of body weight determination

VEHICLE
- Amount of vehicle: A constant volume of the solution, i.e. 0.4 mL/100 g bw was used at each dose level.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to confirm correct preparation of the test item in the study, the solutions of the test item in sunflower oil were chemically analysed. Samples of the test item solution were transferred for chemical analyses to the Laboratory of Analytical Chemistry, which is a part of the Department of Ecotoxicological Studies at the Institute of Industrial Organic Chemistry, Branch Pszczyna.
Before the beginning of the study analytical method validation was conducted. During the study the samples were transferred thrice for chemical analysis: at the beginning, in the middle and at the end of the study. The concentration of tes substance in sunflower oil was chemically determined with a High Performance Liquid Chromatography (HPLC), Shimadzu, Prominence LC-2030C with Diode Array Detector (DAD).
Details on mating procedure:
- Impregnation procedure: cohoused, not related males used for mating with nulliparous female rats came from the same husbandry and stock and had a similar age.
- M/F ratio per cage: 1/1, one male was used to mate with to females.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of gestation.
Duration of treatment / exposure:
The test item/medium were administered to the females from the 5th to the 19th day of gestation.
Frequency of treatment:
Once daily
Duration of test:
On the 20th day of gestation the animals were euthanised.
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Group 4 (new group after termination of treatment goup 3)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 3 (group terminated for humanitatian reasons)
No. of animals per sex per dose:
Group 0,1: 22 females per group
Group 2,4: 23 females per group
Group 3: 10 females per group (terminated)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 80, 200 and 500 mg/kg bw and also the vehicle were selected on the basis on the results of Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening test (OECD 422). The high dose group was terminated due to occurrence of clinical signs (such as respiratory murmurs, difficult respiration, porphyrin discharge around nostrils, porphyrin discharge around the eyes, bristled coat, rounded back, dejection, decrease of locomotor activity, diarrhoea, urination, moisture around the snout, tremors, hypothermia and more than 20% decrease of body weight) and high maternal mortality. A new dose group, 125 mg/kg bw/day, was introduced.
- Rationale for animal assignment: Random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during labour week and once a day on days off.
- Cage side observations included: general evaluation of their health state and observations of morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.
- Detailed clinical observations included: skin changes, coat changes, changes in eyes and mucous membranes, respiratory system, circulatory system, nervous system, somatic activity and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 0, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20. Also females which died before gestation day 20 were macroscopically examined for any structural abnormalities or pathological changes which could have influenced gestation.
- Organs examined: Lungs, stomach, liver, small intestine, duodenum, jejunum, cecum, whole intestines, spleen, ovaries and area around the anus.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: the left and right uteri horn were examined. The uteri of non-pregnant females and non-pregnant horns of the uteri were fixed between two glass plates and overexposed by electric lamp to confirm the non-pregnant status.
Fetal examinations:
- External examinations: Yes: all per litter, sex, body weight with and without placenta, placenta weight alone, body lenght with and without tail.
During gross evaluation attention was paid to: reactions to tactile impulses, formation of body coverings, formation of limbs, number of fingers and toes, shape of head, formation of auricular conchas, presence of nasal apertures, formation of oral cavity, presence of anus and tail.
- Soft tissue examinations: Yes: half per litter. Fetuses were cut in sagittal planes and the formation of particular cavities and presence of internal organs was evaluated.
- Skeletal examinations: Yes: half per litter. The stained fetuses were examined and formation of skull bones, back-bone, ribs, acromial and pelvic girdles with limbs was evaluated. Points of ossification in sternum, metacarpus of fore limbs and metatarsus of hind limbs were counted during evaluation
- Head examinations: Yes: half per litter, the skull bones were examined.
Statistics:
When discussing the results of the prenatal developmental toxicity study, all females used in the study were taken into consideration. The evaluation of results included the relationship, or lack thereof, between the exposure of the animals to the test item and the incidence and severity of all findings, and also, when appropriate, historical control data to enhance interpretation of study results.
Only females whose pregnancy status is confirmed were taken into consideration when conducting statistical analyses with the use of STATISTICA 10, with p ≤ 0.05 concerning the following elements: body weight of pregnant females, food consumption of pregnant females, number of corpora lutea, number of fetuses in litter, number of males and females in litter, weight of uteri, weight of fetuses, length of fetuses, number of ossification points in sternum and limbs. For each quantitative parameter mean and standard deviation were determined.
The results obtained in the treated groups (group 1, group 4, group 2) were compared with the control group (group 0).
The course of the statistical analysis was as follows:
- first of all, the normality of distribution with the Shapiro-Wilk test was examined and the homogeneity of variance with the Brown-Forsythe test.
- if the test results were characterized by normal distribution and homogeneous variances, a one-way analysis of variance was used, if necessary confirmed with Dunnett’s test.
- in the absence of normality of distribution or non-homogeneous variances, the nonparametric Kruskal-Wallis test was used, if necessary confirmed with Dunnett’s test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Respiratory murmurs were observed in all treatment groups and the incidence increased with higher doses, no similar changes were observed in the control group. In Group 2 (200 mg/kg bw/day), one female suffered from difficulty breathing, accelerated respiration and intensifying dyspnoea. Another female suffered from porphyrin discharge around the nostrils.
In the terminated group 3 (dose of 500 mg/kg bw) clinical signs consisted of: respiratory murmurs, difficult respiration, porphyrin discharge around nostrils, porphyrin discharge around the eyes, bristled coat, rounded back, dejection, decrease of locomotor activity, diarrhoea, urination, moisture around the snout, tremors, hypothermia and more than 20% decrease of body weight, and high maternal mortality.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no mortality in group 0 (0 mg/kg bw/day), group 1 (80 mg/kg bw/day) or group 4 (125 mg/kg bw/day).
Four animals died and one was euthanised in group 2 (200 mg/kg bw/day).
Four animals died and two were euthanised in group 3 (500 mg/kg bw/day; group was terminated).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decrease of average body weight of pregnant females from 8th to 20th day of experiment was observed in all treated groups and statistically significant decrease stated in group 2 (200 mg/kg bw) indicated influence of test item (Table 1 in 'Any other information on results incl. tables'). Individual body weight losses of pregnant females in treated groups also indicated influence of the test item because there were no similar body weight losses in control group. Mild body weight losses stated in two non-pregnant females of control groups were interpreted as accidental however in case of similar body weight losses in one non-pregnant female of group 2 (200 mg/kg bw) and one non-pregnant female of group 4 (125 mg/kg bw) influence of test item cannot be excluded.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease of average food consumption from 8th to 20th day of experiment and statistically significant decreases were stated in all treated groups compared to control group which indicated influence of test item (Table 2 in 'Any other information on results incl. tables'). On the basis of results, it can be seen that decrease of average food consumption increased with increasing dose.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In females, which were euthanised for humanitarian reasons or died in group 2 (200 mg/kg bw) and 3 (500 mg/kg bw), many gross lesion were stated (Table 4 in 'Any other information on results incl. tables'). Extension of intestines or stomach, bloating, ulcers and erosions of the mucosa of the stomach, congestion of intestines, intestinal gas, content of the intestines tinged with blood, area around the anus contaminated with faeces and also haemorrhagic inflammation of intestines, indicated influence of test item on the gastrointestinal track. The circulatory disorders (i.e. congestion of lungs, ovaries and also intestines) and foci of emphysema, enlargement of the lungs (as a results of emphysema) and fragile spleen most likely represent euthanasia-related agonal and/or death-related events.
The circulatory disorders (i.e. congestion of stomach and intestines) which appeared in females euthanised on the 20th day of pregnant from control and all treated groups, most likely represent euthanasia-related agonal events (Table 3 in 'Any other information on results incl. tables'). However, increase in the numbers of females with these lesions in group 1, 4 , 2 and 3 compared to control group can be observed.
Extension of intestines, bloating and gas in intestines, ulcers and erosions of the mucosa of the stomach and thickened wall with an inflammatory character observed in females euthanised on the 20th day of pregnant from group 1, 4, 2 and 3 indicated influence of test item on the gastrointestinal track.
Spread, bright lesion with a firm consistency in the caudate lobe of the liver (probably of a cancerous nature) and lesion in size 6x1,5 mm, red, pedunculated in adipose tissue around the left ovary observed in 2 females from group 0 and bright red-yellow lesion, size 3x1 mm in the right corner of the uterus at the height of the first fetus observed in female from group 1 (80 mg/kg bw) can be considered as a spontaneous lesions. The lack of association with the influence of the test item is evidenced by the presence of these changes in the control group.
Other lesions observed in the liver i.e. fragile lesion with a clear lobular structure and with bright foci and two bright lesion with granular consistency are probably associated with the administration of the euthanasia dose to the liver instead of to the peritoneal cavity and should not be combined with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Pre- and post-implantation losses were observed in control and all experimental groups (Table 6 in 'Any other information on results incl. tables'). The percentage of pre- and post-implantation losses was comparable in all groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no implantation sites in non pregnant females, indicating that there was no total litter loss by resorption (Table 6 in 'Any other information on results incl. tables').
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Resorptions were observed in control and all experimental groups (Table 6 in 'Any other information on results incl. tables'). The number of resorptions was comparable for all groups.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Intrauterine mortality was equal in control group 0, 1 and 2 . No intrauterine mortality was stated in group 4 (Table 7 in 'Any other information on results incl. tables').
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of non-pregnant females was equal in control group and group 4 (125 mg/kg bw) and 2 (200 mg/kg bw). In the remaining groups, no non-pregnant females were stated (Table 6 in 'Any other information on results incl. tables').
Other effects:
not examined
Details on maternal toxic effects:
An overview of the result of mating can be found in Table 6 in 'Any other information on results incl. tables'.
Key result
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
gross pathology
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase of weight of the fetuses without placenta and fetal membranes in group 4 (125 mg/kg bw) should be interpreted as accidental because there were no changes in group 2 (with higher dose - 200 mg/kg bw) (Table 8 in 'Any other information incl. results').
The weight of placenta was statistically significantly lower in group 2 (200 mg/kg bw) compared with the control group but weight of fetuses with and without placenta and fetal membranes were in the normal range so this change should not be use in isolation as the only criterion for assessment and should not be combined with the test item.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Intrauterine mortality was equal in control group 0, 1 and 2 . No intrauterine mortality was stated in group 4 (Table 7 in 'Any other information on results incl. tables').
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The average numbers of the males in litter were statistically significantly lower in group 4 compared with the control group but there were no changes in group 2 (with higher dose - 200 mg/kg bw) so it should be interpreted as accidental and should not be combined with the test item (Table 7 in 'Any other information on results incl. tables').
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No changes in litter size were observed (Table 7 in 'Any other information on results incl. tables').
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An overview of the external malformations can be found in Table 9 in 'Any other information on results incl. tables'.
No statistically significant difference in lengths of the fetuses with tail and lengths of the fetuses without tail in all treated groups in comparison with the control group were stated.
Subcutaneous haemorrhages in different parts of the fetal bodies which occurred in the treated groups and in the control group, should not be perceived as the effects of the test item. Subcutaneous haemorrhages in fetuses were often observed in other prenatal developmental toxicity studies performed at the Institute of Industrial Organic Chemistry Branch Pszczyna and should be considered as typical disturbances in fetuses. Similarly, three case of internal haemorrhage in fetuses were probably accidental and should not be considered as the test item related effect.
Omphalocele which occurred in one fetus of group 2 (200 mg/kg bw) is a serious defect in the abdominal wall at the umbilicus, through which the intestines protrude and is regarded as a serious malformation, but it occurred also in control group in one fetus and hence should not be connected with the influence of the test item. Significantly smaller fetuses occurred in all groups (control and treated) and because there were no statistically significant decrease of weight of fetuses and no statistically significant difference in lengths of the fetuses from treated groups, this lesion should not be combined with the test item.
In group 1 (80 mg/kg bw), occurrence of two smaller fetuses with deformed tail and 4-fingered forelimbs from the same litter, one fetus with diaphragmatic hernia and one, dead fetus with hypoplasia of the right ventricle should be treated as accidental due to the significantly smaller number of fetuses with malformation in groups with higher dose of the test item (groups 4 and 2). In groups with higher dose of the test item there were no increased number of resorptions and intrauterine mortality of fetuses, which confirms the randomness of malformation in group 1 (80 mg/kg bw). Additionally, based on historical data (previous studies conducted at Institute of Industrial Organic Chemistry Branch Pszczyna), occurrence of 4 fetuses with serious malformations per 294 fetuses in group are within the normal range.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The average number of ossification points in the sternum and metacarpus can be found in Table 10 and 11 in 'Any other information of results incl. tables'. An overview of these findings is represented in Table 12 in the same field.
Variations in sternum ossification centres were of unilateral, bipartite and misaligned character and occurred in all treated groups and in control group. The lack of one of ossification point occurred in all treated groups and in control group. Changes in skull, vertebra column and ribs were observed in one fetus from group 1 (80 mg/kg bw). In gross examination of the same fetus, the deformed tail and 4-fingered forelimbs were observed and, as mentioned, this changes should be treated as accidental.
Analysis of percentage of fetuses with particular number of ossification points of sternum showed no difference between control group and treated groups.
Statistically significantly greater number of ossification points in metacarpus was stated in group 1 (80 mg/kg bw), group 4 (125 mg/kg bw) and group 2 (200 mg/kg bw) compared with the control group. It is possible that the average number of ossification points in metacarpus in control group was accidentally and exceptionally low because the highest average number of ossification points in metacarpus was reported in group 1 (80 mg/kg bw) and the averages were lower in groups 4 (125 mg/kg bw) and 2 (200 mg/kg bw) which may indicate a lack of connection between the dose level and the average number of ossification points in metacarpus. No statistically significant difference of ossification points in sternum and in metatarsus in treated groups compared with the control group indicates the randomness of statistical significant changes in the average number of ossification point in metacarpus in treated groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Evaluation of sagittal sections of fetuses from control group confirmed findings from gross evaluation.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Table 1. Body weight of pregnant females [g] (mean ± SD).

 

Day of gestation

Group 0

0 mg/kg bw

n=20

Group 1

80 mg/kg bw

n=22

Group 4

125 mg/kg bw

n=21

Group 2

200 mg/kg bw

n=16

Group3 500 mg/kg bw

n=4 #

0

227.5 ± 14.57

230.7 ± 16.70

228.3 ± 13.06

231.6 ± 16.12

234.3 ± 17.35

5

247.0 ± 15.06

252.5 ± 17.91

247.7 ± 15.46

252.3 ± 18.30

256.8 ± 16.82

8

254.8 ± 15.39

252.2 ± 22.49

243.5 ± 16.01

247.2 ± 19.46

238.8 ± 16.58

11

266.7 ± 17.19

264.3 ± 21.33

254.8 ± 18.60

255.9 ± 21.78

240.3 ± 29.15

14

280.0 ± 17.54

277.0 ± 20.80

270.4 ± 15.26

261.0 ± 19.20 *

245.3 ± 23.91

17

304.3 ± 17.41

302.8 ± 22.89

290.8 ± 16.82

279.3 ± 25.52 *

267.0 ± 21.37

20

339.3 ± 24.63

336.5 ± 26.64

323.0 ± 19.93

308.0 ± 25.92 *

300.8 ± 23.47

n – number of animals in group

* - statistically significant difference with p ≤ 0.05

# - no statistical analysis

 

Table 2. Food consumption [g/100g of bw/day] (mean ± SD).

Day of gestation

Group 0

0 mg/kg bw

n=20

Group 1

80 mg/kg bw

n=22

Group 4

125 mg/kg bw n=21

Group 2

200 mg/kg bw n=16

Group3 500 mg/kg bw

n=4 #

5

8.51 ± 0.73

8.07 ± 0.94

7.93 ± 0.72

8.21 ± 0.80

8.38 ± 0.54

8

7.05 ± 0.76

6.52 ± 0.82

5.80 ± 0.92 *

5.67 ± 1.19 *

3.45 ± 1.57

11

7.27 ± 0.60

6.32 ± 0.59 *

5.95 ± 0.60 *

5.50 ± 0.71 *

3.63 ± 1.28

14

7.31 ± 0.54

6.57 ± 0.76 *

6.33 ± 0.65 *

5.46 ± 1.06 *

4.83 ± 1.73

17

7.05 ± 0.75

6.45 ± 0.55

6.45 ± 0.56

5.42 ± 1.39 *

6.63 ± 0.61

20

6.22 ± 0.59

5.90 ± 0.53

5.67 ± 0.59

5.55 ± 1.16 *

6.63 ± 0.38

n – number of animals in group

* - statistically significant difference with p ≤ 0.05

# - no statistical analysis

 

Table 3. Gross lesions – euthanised animals on the 20th day of experiment.

 

Examined organ

 

Type of change

Group

0

0 mg/kg bw n=22

1

80 mg/kg bw n=22

4

125 mg/kg bw n=23

2

200 mg/kg bw n=18

3

500 mg/kg bw n=4

 

Stomach

Congestion

2

[81 ,9]

2

[9,19]

-

4

[7,81,9,18]

1

[9]

Erosions of the mucosa

-

4

[6,9,10,12]

4

[7,9,181,21]

5

[1,7,9,16,21]

1

[3]

Ulcers

-

-

-

2

[4,14]

3

[3,6,9]

 

Jejunum

Extension

-

-

-

2

[10,20]

-

Thickened wall with an inflammatory

character

 

-

 

-

 

-

1

[20]

 

-

(Intestinal) gas

-

-

-

1

[10]

-

 

Cecum

Slight congestion

2

[15,21]

6

[1,2,5,6,11,21]

2

[15,21]

2

[1,10]

1

[6]

 

Congestion

2

[17,181]

4

[9,10,12,19]

3

[9,161,181]

7

[2,6,7,81,9,131

,18]

2

[1,3]

Extension

-

-

1

[181]

2

[10,131]

1

[3]

Small amount of (intestinal) gas

-

-

-

-

1

[6]

(intestinal) gas /

bloating

-

1

[17]

6

[1,4,6,7,8,181]

2

[10,131]

2

[3,9]

 

Colon

Slight congestion

-

1

[11]

-

-

-

Congestion

-

-

-

1

[6]

-

Extension

-

-

-

-

1

[9]

Bloating

-

-

1

[6]

-

-

Whole intestines

Congestion

2

[81,9]

-

-

-

-

 

Liver

Fragile liver with a

clear lobular structure and with bright foci

 

-

1

[10]

 

-

 

-

 

-

Bright lesion, size 5x5 mm with a granular consistency in the left

lateral lobe

 

-

 

1

[8]

 

-

 

-

 

-

Lesion spread, bright with a firm consistency in the caudate lobe,

probably of a cancerous nature

 

1

[14]

 

-

 

-

 

-

 

-

Bright lesion with granular consistency (10x 6 mm) in

caudate lobe

 

-

 

-

 

1

[5]

 

-

 

-

Lesion in size 6x1,5 mm, red, pedunculated in adipose tissue

around the left ovary

1

[81]

 

-

 

-

 

-

 

-

Bright red-yellow lesion, size 3x1 mm in the right corner of the uterus

at the height of the first fetus

 

-

1

[10]

 

-

 

-

 

-

[ ] – computer numbers of animals with gross lesions

n – number of tested animals

X1 - females not pregnant

 

 

Table 4. Gross lesions – dead and euthanised for humanitarian reason animals.

Examined organ

 

Type of change

Group

0

0 mg/kg bw n=0

1

80 mg/kg bw n=0

4

125 mg/kg bw n=0

2

200 mg/kg bw n=5

3

500 mg/kg bw n=6

 

Lungs

Strong congestion

-

-

-

2

[11,12]

1

[7]

Congestion

-

-

-

3

[15,221,23]

-

Enlargement

-

-

-

2

[11,12]

4

[2,5,7,10]

Foci of emphysema

-

-

-

3

[11,12,15]

4

[2,5,7,10]

Foci of atelectasis

-

-

-

1

[23]

-

 

Stomach

Strong extension

-

-

-

3

[11,12,15]

2

[7,10]

Extension

-

-

-

1

[221]

4

[2,41,5,81]

 

Gas (bloating)

 

-

 

-

 

-

3

[12,15,221]

6

[2,41,5,7,81,10

]

Erosions of the mucosa

-

-

-

3

[12,15,23]

4

[41,5,7,10]

Ulcers

-

-

-

1

[15]

2

[7,10]

 

Small intestine

Congestion

-

-

-

1

[12]

-

Extension

-

-

-

-

3

[2,5,81]

Content tinged with blood

-

-

-

1

[12]

-

Duodenum

Haemorrhagic

inflammation

-

-

-

1

[23]

-

Jejunum

Haemorrhagic inflammation

-

-

-

1

[23]

-

 

Cecum

Congestion

-

-

-

-

3

[2,41,81]

Extension

-

-

-

-

5

[2,41,5, 81,10]

(Intestinal) gas/bloating

-

-

-

1

[15]

5

[2,41,5,81,10]

 

Whole intestines

Extension

-

-

-

2

[11,12]

3

[41,7,10]

(Intestinal) gas

-

-

-

2

[11,12]

1

[7]

Content slightly tinged with blood

-

-

-

1

[11]

-

Haemorrhagic inflammation

-

-

-

1

[12]

-

Spleen

Fragile

-

-

-

1

[221]

-

Ovaries

Congestion

-

-

-

1

[11]

-

Area around the anus contaminated with light / yellow and rare / liquid faeces

 

-

 

-

 

-

 

-

3

[2,41,81]

[ ] – computer numbers of animals with gross lesions

n – number of tested animals

X1 - euthanized for humanitarian reason animal

 

 

Table 5. Number of corpora lutea, implantations, resorptions, pre- and post-implantation losses (mean ± SD)

 

Group

Number of  pregnant females

Average number of corpora lutea per one female

Number of implantations in not pregnant

females

Number of resorptions

Preimplantation losses

Postimplantation losses

total

in females min - max

average in one female

number

percent

number

percent

0

20

16.100 ± 2.198

0

24

0 – 5

1.200 ± 2.198

38

11.801

25

8.803

1

22

15.591 ± 2.062

0

16

0 – 5

0.727 ± 1.202

33

9.621

17

5.484

4

21

15.238 ± 3.015

0

29

0 – 8

1.381 ± 2.085

34

10.625

29

10.140

2

16

14.313 ± 1.352

0

13

0 – 3

0.813 ± 0.911

21

9.170

14

6.731

 

Table 6. Result of mating.

 

Parameter

Number of females

Group 0

0 mg/kg bw

Group 1

80 mg/kg bw

Group 4

125 mg/kg bw

Group 2

200 mg/kg bw

Group 3

500 mg/kg bw

Number of females per group

22

22

23

23

10

Number of pregnant females

20

22

21

21

10

Number of not pregnant females

2

[8,18]

0

2

[16,18]

2

[8, 13]

0

Number of died females

0

0

0

4

[11, 12, 15, 23]

4

[2, 5, 7, 10]

Number of euthanized females

0

0

0

1

[22]

2

[4, 8]

 

Females with resorptions

11

[2(2),4(1),5(2),

6(3),9(1),10(1),

11(2),13(2),15(5),

17(4),21(1)]

10

[1(5),3(1),9(1),

12(1),13(1),17(1),

18(1),19(3),

21(1)22(1)]

11

[2(4),4(1),5(1),

7(8),8(1),9(1),

12(2),13(2),17(5),

20(1),23(3)]

9

[1(1),2(1),3(3),

4(2),7(2),9(1),

14(1),19(1),20(1)]

 

no calculations were performed due to too small group size

Number of females for statistical analysis

20

22

21

16

0

[ ] computer number of female

() number of resorptions

 

Table 7. Number of fetuses (mean ± SD).

 

Group

Number of  pregnant females

Number of fetuses

 

Number of resorptions

 

total

 

dead

in litters min.max.

average in litter in 1 female

 

females

 

males

0

20

260

1

5 - 18

12.950 ± 3.517

5.700 ± 2.179

7.250 ± 2.573

24

1

22

294

1

7 - 19

13.318 ± 2.784

6.636 ± 1.840

6.682 ± 2.418

16

4

21

257

0

7 - 16

12.238 ± 2.548

7.000 ± 2.258

5.238 ± 1.841 *

29

2

16

195

1

8 - 16

12.125 ± 2.391

5.688 ± 2.056

6.438 ±1.931

13

* statistically significant difference with p ≤ 0.05

 

Table 8. Average weight of fetuses and placenta [g] (mean ± SD).

 

Group

 

Number of examined fetuses

Weight of fetuses

 

Weight of placenta

with placenta and fetal membranes

without placenta and fetal membranes

0

259

4.774 ± 0.338

3.386 ± 0.304

0.542 ± 0.077

1

293

4.745 ± 0.474

3.450 ± 0.326

0.531 ± 0.086

4

257

4.793 ± 0.420

3.491 ± 0.352*

0.543 ± 0.099

2

194

4.664 ± 0.507

3.449 ± 0.391

0.515 ± 0.096*

* statistically significant difference with p ≤ 0.05

 

Table 9. Gross lesions in fetuses.

 

Pathological changes

Number of fetuses

Group 0

0 mg/kg b.w.

Group 1

80 mg/kg b.w.

Group 4

125 mg/kg b.w.

Group 2

200 mg/kg b.w.

Total number of fetuses

260

294

257

195

Number of alive fetuses

259

293

257

194

Number of dead fetuses

1[17]

1[8]

0

1[5]

Number of fetuses with haemorrhages

12

12

7

4

Haemorrhage on the lateral, left elbow region

1

[2]

-

-

-

Haemorrhage on left metatarsus

1

[3]

-

1

[3]

-

Haemorrhage on right metatarsus

-

1

[13]

-

-

Haemorrhage on interscapular region

3

[3,13,19]

3 [3x3]

-

2

[16,17]

Haemorrhage on the right side of the thoracic

1

[3]

-

-

-

Haemorrhage on the left glenohumeral joint

1

[12]

-

-

-

Haemorrhage on the right, dorsal side of the neck

1

[15]

-

-

1

[5]

Haemorrhage on the right, ventral side of the neck

1

[16]

-

1

[20]

-

Haemorrhage on the ventral side of the neck

1

[19]

-

-

1

[2]

Haemorrhage at the base of the tail

-

3  [2x2,4]

-

-

Haemorrhage on the tail

1

[16]

3

[6,21,22]

3

[3,13,17]

-

Haemorrhage on temporomandibular joint region

-

1

[3]

-

-

Haemorrhage on the right forearm

-

-

1

[9]

-

Haemorrhage on the skin of the abdomen - abdominal cavity strongly enlarged, probably part of the euthanasia dose for the mother was

given to the fetus

 

1

[14]

 

-

 

-

 

-

Haemorrhage on the entire abdominal wall - abdominal cavity enlarged

-

1

[15]

-

-

Haemorrhage on the entire torso and

abdomen - abdominal cavity enlarged

 

-

 

-

1

[3]

 

-

Number of fetuses with other changes

2

9

3

3

Omphalocele

1

[13]

-

-

1

[10]

Significantly smaller

1 [17*]

4 [8,8*,2x17]

3 [3x7]

1

[2]

Deformed (shortened) tail

-

2 [2x17]

-

-

4-fingered forelimbs

-

2 [2x17]

-

-

Anasarca and amniotic fluid tinged with blood

-

1 [8*]

-

1 [5*]

[ ] computer numbers of females

2x, 3x etc.- number of fetuses from one female, with the same lesion

* dead fetus

 

Table 10. Percentage of fetuses with a given number of ossification points of sternum (%).

 

Group

Number of   examined fetuses

Number of ossification points

0

1

2

3

4

5

6

0

134

-

-

-

0.75

6.72

16.42

76.12

1

152

0.66

0.66

-

-

4.61

9.21

84.87

4

132

-

-

0.76

-

6.82

15.15

77.27

2

100

-

-

-

-

2.00

15.00

83.00

 

Table 11. Percentage of fetuses with a given number of ossification points of limbs (%).

 

Group

Number of   examined fetuses

Metacarpus of forelimbs Number of ossification points

Metatarsus of hindlimbs Number of ossification points

3

2.5*

3.5*

4

3

4

0

134

29.10

-

5.22

65.67

-

100.00

1

152

9.21

0.66

4.61

85.53

0.66

99.34

4

132

16.67

-

5.30

78.03

-

100.00

2

100

13.00

-

-

87.00

-

100.00

*value 3.5 means 3 ossification points in one limb and 4 in second limb; value 2.5 means 2 ossification points in one limb and 3 in second limb

 

Table 12. Average number of ossification points of sternum and limbs (mean ± SD).

 

Group

Number of

examined fetuses

 

Sternum

Metacarpus of forelimbs

Metatarsus of hindlimbs

0

134

5.679 ± 0.632

3.683 ± 0.453

4.000 ± 0.000

1

152

5.743 ± 0.785

3.875 ± 0.322*

3.993 ± 0.081

4

132

5.682 ± 0.669

3.807 ± 0.379*

4.000 ± 0.000

2

100

5.810 ± 0.443

3.870 ± 0.338*

4.000 ± 0.000

* statistically significant difference with p ≤ 0.05

Conclusions:
In this GLP compliant OECD 414 study, the possible toxic effects of Rokopol RF-151 to pregnant Wistar rats (Cmdb: WI; outbred) and their fetuses was evaluated. On the basis of the clinical examinations and gross examination the NOAEL for maternal toxicity cannot be defined. On the basis of the results of the evaluation of fetuses the NOAEL for developmental toxicity can be defined as 200 mg/kg bw.
Executive summary:

In this GLP compliant OECD 414 study, the possible toxic effects of Rokopol RF-151 to pregnant Wistar rats (Cmdb: WI; outbred) and their fetuses was evaluated. The study was conducted on 100 females divided into five experimental groups: four treated groups and one control group. From day 5 to 19 of gestation the test item, dispersed in sunflower oil (solution), was administered via oral gavage at doses of 80 mg/kg bw (group 1), 125 mg/kg bw (group 4), in 200 mg/kg bw (group 2) and 500 mg/kg bw (group 3). Due to severe toxicity in the high dose group, this group was terminated for humanitarian reasons and a dose group of 125 mg/kg bw (group 4) was introduced. Group 0 served as control and was treated with sunflower oil.

During the entire study clinical observations for mortality and signs of toxic influence of the test item were performed in females, their body weight and food consumption were controlled. One day before an expected delivery, on day 20 of gestation, all females were killed with the use of Euthasol and subjected to caesarean section with gross examination. Moreover gross examinations were performed in all females which died or were euthanised for humane reason before the end of the study.

The following endpoints were determined in each pregnant female: number of alive and dead fetuses and number of resorptions. After the removing of fetuses each uterus was weighed. The non-gravid uteri and non-gravid horns were overexposed by electric lamp and were stained using ammonium sulphide in order to confirm the non-pregnant status. The number of corpora lutea was determined in fixed ovaries. The following endpoints were determined in the study: sex, body weight with and without placenta, weight of placenta, body length with and without tail. Each fetus was subjected to gross examination. Half of fetuses from each litter were stained with alizarin and subjected to evaluation of skeleton. The rest of them was fixed in 10% solution of formalin and evaluated for formation of body cavities and internal organs.

For the maternal generation, there was no mortality in group 0, 1 and 4. Four females died and one female was euthanised during the experiment in group 2. Four females died and two females were euthanised during the experiment in group 3. During the entire experiment, no clinical signs were observed in the control group. Respiratory murmurs were observed in all treatment groups and the incidence increased with higher doses. Average body weight of the pregnant females of group 1 and 4 did not differ statistically significantly from average body weight of the control group. In group 2, the average body weight was statistically significantly lower in comparison with the pregnant females of control group on 14th ,17th and 20th day of gestation. Decrease of average food consumption from 8th to 20th day of experiment and statistically significant decreases were stated in all treated groups compared to control group which indicated influence of test item. At necropsy, multiple effects were observed in females euthanised on the 20th day of pregnant from group 1, 4, 2 and 3 on the gastrointestinal track: congestion, extension of intestines, bloating and gas in intestines, ulcers and erosions of the mucosa of the stomach and thickened wall with an inflammatory character. These effects intensified with increasing concentrations.

Six females were non pregnant in the study (two in control group, two in group 4 and two in group 2). In the remaining groups, no non-pregnant females were stated. Resorptions and pre- and post-implantation losses were observed in control and all experimental groups. The number of resorption and number and percent of pre- and post-implantation losses in all groups was comparable. Intrauterine mortality was equal in control group and group 1 (80 mg/kg bw) and 2 (200 mg/kg bw). No intrauterine mortality was stated in group 4 (125 mg/kg bw). The average number of corpora lutea in all treated groups were comparable with the number of corpora lutea in the control group.

Due to too small number of females in group 3, evaluation of fetuses was not performed. Subcutaneous haemorrhages in different parts of the fetal bodies which occurred in the treated groups and in the control group, should not be perceived as the effects of the test item. Other external observations were incidental and not treatment- related. Variations in sternum ossification centres were of unilateral, bipartite and misaligned character and occurred in all treated groups and in control group. The lack of one of ossification point occurred in all treated groups and in control group. Changes in skull, vertebra column and ribs were observed in one fetus from group 1 (80 mg/kg bw). In gross examination of the same fetus, the deformed tail and 4-fingered forelimbs were observed and, as mentioned, this changes should be treated as accidental. Analysis of percentage of fetuses with particular number of ossification points of sternum showed no difference between control group and treated groups. Statistically significantly greater number of ossification points in metacarpus was stated in group 1, 4 and 2 compared with the control group. It is possible that the average number of ossification points in metacarpus in control group was accidentally and exceptionally low because the highest average number of ossification points in metacarpus was reported in group 1 and the averages were lower in groups 4 and 2, which may indicate a lack of connection between the dose level and the average number of ossification points in metacarpus. No statistically significant difference of ossification points in sternum and in metatarsus in treated groups compared with the control group indicates the randomness of statistical significant changes in the average number of ossification point in metacarpus in treated groups.

Based on the study and analysis of the results it can be stated that the test item Rokopol RF-151 affected results of the clinical examinations and gross examination of pregnant females in all treated groups which indicated that the NOAEL for maternal toxicity can not be defined. On the basis of the results of the evaluation of fetuses the NOAEL for developmental toxicity can be defined as >= 200 mg/kg bw.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study available is a GLP-compliant guideline study, fully adequate for assessment.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 - rat

In a GLP compliant OECD 414 study, the possible toxic effects of Rokopol RF-151 to pregnant Wistar rats (Cmdb: WI; outbred) and their fetuses was evaluated. The study was conducted on 100 females divided into five experimental groups: four treated groups and one control group. From day 5 to 19 of gestation the test item, dispersed in sunflower oil (solution), was administered via oral gavage at doses of 80 mg/kg bw (group 1), 125 mg/kg bw (group 4), in 200 mg/kg bw (group 2) and 500 mg/kg bw (group 3). Due to severe toxicity in the high dose group, this group was terminated for humanitarian reasons and a dose group of 125 mg/kg bw (group 4) was introduced. Group 0 served as control and was treated with sunflower oil.

During the entire study clinical observations for mortality and signs of toxic influence of the test item were performed in females, their body weight and food consumption were controlled. One day before an expected delivery, on day 20 of gestation, all females were killed with the use of Euthasol and subjected to caesarean section with gross examination. Moreover gross examinations were performed in all females which died or were euthanised for humane reason before the end of the study.

The following endpoints were determined in each pregnant female: number of alive and dead fetuses and number of resorptions. After the removing of fetuses each uterus was weighed. The non-gravid uteri and non-gravid horns were overexposed by electric lamp and were stained using ammonium sulphide in order to confirm the non-pregnant status. The number of corpora lutea was determined in fixed ovaries.The following endpoints were determined in the study: sex, body weight with and without placenta, weight of placenta, body length with and without tail. Each fetus was subjected to gross examination. Half of fetuses from each litter were stained with alizarin and subjected to evaluation of skeleton. The rest of them was fixed in 10% solution of formalin and evaluated for formation of body cavities and internal organs.

For the maternal generation, there was no mortality in group 0, 1 and 4. Four females died and one female was euthanised during the experiment in group 2. Four females died and two females were euthanised during the experiment in group 3. During the entire experiment, no clinical signs were observed in the control group. Respiratory murmurs were observed in all treatment groups and the incidence increased with higher doses. Average body weight of the pregnant females of group 1 and 4 did not differ statistically significantly from average body weight of the control group. In group 2, the average body weight was statistically significantly lower in comparison with the pregnant females of control group on 14th ,17th and 20th day of gestation. Decrease of average food consumption from 8th to 20th day of experiment and statistically significant decreases were stated in all treated groups compared to control group which indicated influence of test item. At necropsy, multiple effects were observed in females euthanised on the 20th day of pregnancy from group 1, 4, 2 and 3 on the gastrointestinal track: congestion, extension of intestines, bloating and gas in intestines, ulcers and erosions of the mucosa of the stomach and thickened wall with an inflammatory character. These effects intensified with increasing concentrations.

Six females were non pregnant in the study (two in control group, two in group 4 and two in group 2). In the remaining groups, no non-pregnant females were stated. Resorptions and pre- and post-implantation losses were observed in control and all experimental groups. The number of resorption and number and percent of pre- and post-implantation losses in all groups was comparable. Intrauterine mortality was equal in control group and group 1 (80 mg/kg bw) and 2 (200 mg/kg bw). No intrauterine mortality was stated in group 4 (125 mg/kg bw). The average number of corpora lutea in all treated groups were comparable with the number of corpora lutea in the control group.

Due to too small number of females in group 3, evaluation of fetuses was not performed. Subcutaneous haemorrhages in different parts of the fetal bodies which occurred in the treated groups and in the control group, should not be perceived as the effects of the test item. Other external observations were incidental and not treatment- related. Variations in sternum ossification centres were of unilateral, bipartite and misaligned character and occurred in all treated groups and in control group. The lack of one of ossification point occurred in all treated groups and in control group. Changes in skull, vertebra column and ribs were observed in one fetus from group 1 (80 mg/kg bw). In gross examination of the same fetus, the deformed tail and 4-fingered forelimbs were observed and, as mentioned, this changes should be treated as accidental. Analysis of percentage of fetuses with particular number of ossification points of sternum showed no difference between control group and treated groups. Statistically significantly greater number of ossification points in metacarpus was stated in group 1, 4 and 2 compared with the control group. It is possible that the average number of ossification points in metacarpus in control group was accidentally and exceptionally low because the highest average number of ossification points in metacarpus was reported in group 1 and the averages were lower in groups 4 and 2, which may indicate a lack of connection between the dose level and the average number of ossification points in metacarpus. No statistically significant difference of ossification points in sternum and in metatarsus in treated groups compared with the control group indicates the randomness of statistical significant changes in the average number of ossification point in metacarpus in treated groups.

Based on the study and analysis of the results it can be stated that the test item Rokopol RF-151 affected results of the clinical examinations and gross examination of pregnant females in all treated groups which indicated that the NOAEL for maternal toxicity cannot be defined. On the basis of the results of the evaluation of fetuses the NOAEL for developmental toxicity can be defined as >= 200 mg/kg bw.

OECD 422 - rat

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, four groups of 12 female Wistar rats were administered the test substance by gavage once a day at 0 (sunflower oil), 500, 250 and 100 mg/kg bw/day. Stability, homogeneity 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 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 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 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, noisy breathing, piloerection, salivation, swollen abdomen), necropsy observations (enlarged adrenal glands, white knots on the wall of the stomach, gas content in the intestines and stomach, reddish colored duodenum, 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 (diarrhea, salivation, piloerection) were observed in female animals of 500 mg/kg bw/day group. Salivation was detected in 250 mg/kg bw/day group. At the detailed weekly observations, most of the above listed signs appeared in female (diarrhea or piloerection) animals of 500 mg/kg bw/day group mostly during the first half of the treatment period. 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 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 female animals of 500 mg/kg bw/day group between Days 0 and 7. Test item related statistically significant haematological changes consisted of a higher percentage of neutrophil granulocytes (NEU), a lower percentage of lymphocytes (LYM) and an increase (~2.4 fold) in total number of white blood cells (WBC) in the 500 mg/kg bw dose group. In the 250 mg/kg bw group, a statistically significant increase (~75%) in total number of white blood cells (WBC) was observed. 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) 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 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. Based on these observations the No Observed Adverse Effect Levels (NOAEL) for maternal and developmental toxicity were determined to be 100, and 250 mg/kg bw, respectively.

Justification for classification or non-classification

Based on the absence of adverse effects on reproductive organs or tissues in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, classification is not necessary for toxicity to fertility in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Based on the absence of developmental effects, in the absence of parental toxicity, in the available study (OECD422), classification is not necessary for developmental toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.