Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: About 10 - 11 weeks (male animals), About 9 weeks (female animals)
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually in polycarbonate cages type III; during pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany; during pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 4 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.

VEHICLE
- Concentration in vehicle: 2.5, 7.5, 25 g/100ml
- Amount of vehicle (if gavage): 4 ml/kg bw
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in corn oil over a period of 7 days was proven. At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, one sample from the mid concentration was additionally taken for concentration control analysis.
The samples collected at the beginning of the administration period and during the lactation period were analyzed.
Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test article was distributed homogeneously in corn oil. The concentrations of the test item in corn oil were found to be in the range of 90-110% of the nominal concentration. The results demonstrated the correctness of the concentrations of the test substance in corn oil.
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), ssessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the premating phase, body weight was determined twice a week, i.e. on study days 3, 7, 10 and 14.
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
• Females without litter and after weaning (PND 13) were weighed once a week.

FOOD CONSUMPTION: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA),

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
• Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings
• Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:behavior on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait abnormalities, activity/arousal level, urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
• Motor activity assessment: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.
Oestrous cyclicity (parental animals):
Estrous cycle determination prior to treatment was performed in a pool of up to 50 non-randomized female animals. Only animals with regular estrous cycle were selected for randomization before the start of the treatment period. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups or 2 preferably female pups per litter were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for possible determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

PUP NUMBER AND STATUS AT DELIVERY
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations”.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

PUP CLINICAL OBERVATIONS
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

PUP BODY WEIGHT
The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

NIPPLE/AREOLA ANLAGEN
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

ANOGENITAL DISTANCE
Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights will be determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed), Uterus. The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes
Organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution. See table below for details on examinations.
Postmortem examinations (offspring):
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations (see 3.9.). After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for further processing.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
DUNNETT-test (two-sided): Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index
FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index
WILCOXON test (two-sided): % live male day x, %live female day x
KRUSKAL-WALLIS test (two-sided); If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided): Number of cycles and Cycle Length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, Blood parameters, Weight parameters
Reproductive indices:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Postimplantation loss
Offspring viability indices:
Viability index, Survival index, Sex Ratio, Anogenital index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Protruding left eyeball was observed in female animal No. 114 of test group 1 (100 mg/kg bw/d) during the entire study period with beginning on pre-mating day 10. This finding was assessed to be incidental. Piloerection was observed in female animal No. 137 of test group 3 (1000 mg/kg bw/d) from gestation day 23 onwards. The finding was assessed to be related to treatment.
All pups of female animal Nos. 131 and 137 of test group 3 (1000 mg/kg bw/d) were found stillborn on PND 0. In addition, blood in bedding was observed in animal No. 137 (on PND 0 only). Female animal No. 140 of test group 3 (1000 mg/kg bw/d) lost its complete litter on PND 0. Note: On 07 May 2017 parts of pup bodies were found in the cage. It was not possible to determine number and sex of born pups. The finding “complete litter loss” was chosen.
Pale skin was observed in female animal Nos. 131 (on lactation day 0), 137 (between lactation days 0 to 1), and No. 138 (between lactation day 0 to 2). All individuals belonged to test group 3 (1000 mg/kg bw/d). In addition, piloerection was observed in female animal Nos. 137 (between lactation days 0 to 3), No. 138 (between lactation days 0 to 2), and No. 136 (on lactation day 3). All findings were assessed to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in mean body weights and mean body weight change values were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in male animals during pre-mating and mating periods.
Food consumption was significantly increased in female animals of test group 1 (100 mg/kg bw/d) between pre-mating days 7-13 and gestation days 0-7. The changes were assessed to be incidental.
In female animals of test group 3 (1000 mg/kg bw/d) food consumption was significantly increased during gestation days 0-7 and significantly decreased between lactation days 4-7, 7-10, 10-13 and 1-13. The changes were assessed to be secondary to the litter losses, but not a direct consequence of the test substance on the dams’ organisms.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
In females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) red blood cell (RBC) counts were significantly decreased and mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly increased. RCB count medians were not dose-dependently changed. MCH and MCV are calculated red blood cell indices which were affected only by the decreased RBC counts. The measured parameters, hemoglobin and hematocrit were not changed. Therefore, the altered RBC counts, MCV and MCH indices were regarded as incidental and not treatment-related.
In males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) absolute reticulocyte counts were significantly increased. However, the alteration was not dose-dependent. Therefore, this change was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed. In males of test group 2 (300 mg/kg bw/d) total bilirubin levels were significantly increased, but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed. Exophthalmos was observed in female animal No. 114 of test group 1 (100 mg/kg bw/d).
Sensorimotor tests/reflexes: No test substance-related effects were observed. Female animal No. 114 of test group 1 (100 mg/kg bw/d) showed retarded adaptation of the pupil to light. The finding was assessed to be incidental.
Quantitative parameters: No test substance-related effects were observed.
Motor activity measurement: Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals. Comparing the single intervals with the control groups, one significantly decreased value was measured for female animals of test group 3 (1000 mg/kg bw/d) at interval 1. The change was regarded to be incidental and not related to treatment as neither other single intervals nor the overall motor activity was affected.
No deviations to control values were observed for female animals in test groups 1 to 3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormones: In parental males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) T4 levels were significantly higher compared to controls. The change was not dose-dependent and the means were within the historical control range (T4 males 44.87-88.29 nmol/L). TSH levels of these individuals were not changed. Therefore, the T4 increase was regarded as incidental and not treatment-related. In male and female pups at PND 13 (test groups 11, 12 and 13; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was 4 days.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- Male mating index
The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups.

- Male fertility index
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
Male animal No. 10 of test group 0 (control), which was mated with female No. 110, male animal No. 13 of test group 1 (100 mg/kg bw/d), which was mated with female animal No. 113, male animal No. 24 of test group 2 (300 mg/kg bw/d), which was mated with female animal No. 124, and male animal No. 32 of test group 3 (1000 mg/kg bw/d), which was mated with female animal No. 132, did not generate F1 pups. Thus, the male fertility index was 90% in all test groups 0-3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

-Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 3.3 days for test group 0, 4.0 days for test group 1, 4.1 days for test group 2 and 2.2 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

- Female fertility index
Most sperm positive rats delivered pups with the exception of female animal No. 110 of test group 0 (control), which was mated with male No. 10, female animal No. 113 of test group 1 (100 mg/kg bw/d), which was mated with male animal No. 13, female animal No. 124 of test group 2 (300 mg/kg bw/d), which was mated with male animal No. 24, and female animal No. 132 of test group 3 (1000 mg/kg bw/d), which was mated with female animal No. 32. These female animals had sperm in vaginal smear but did not deliver pups and did not show any implants.
Thus, the female fertility index was 90% in all test groups 0-3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data
The mean duration of gestation was 22.1 days in control group 0, 22.4 days in test group 1 and 22.6 days in test group 2. In test group 3, the mean duration of gestation was 23.3 days and significantly increased when compared to the controls. The prolonged mean gestation period was considered to be related to treatment.

- Gestation index
The gestation index was 100% in test groups 0-2 (0, 100 and 300 mg/kg bw/d), but reduced to 66.7% in test group 3 (1000 mg/kg bw/d) due to the reduced number of females with live pups on the day of birth. The decreased value was assessed to be related to treatment.

- Live birth indices
The rate live birth indices were 100% in test group 0 (control group), 98.2% in test group 1 (100 mg/kg bw/d), and 95.9% in test group 2 (300 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. The index was reduced to 58.1% in test group 3 (1000 mg/kg bw/d). The decreased value was assessed to be related to treatment

-Postimplantation loss
The postimplantation loss was 7.6% in control group 0, 5.0% in test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d), but 18.5% in test group 3 (1000 mg/kg bw/d). The values in test groups 0, 1 and 2 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. The postimplantation loss in test group 3 was clearly outside the range when taking female animal No. 140 into account. However, postimplantation loss in high dose animals was well within the historical control data if animal No. 140 is taken out of the calculation. Animal 140 showed 100% postimplantation loss but had only 2 implantation sites and could be regarded as an outlier.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
other: increased gestation length
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Three male pups of test group 1 (100 mg/kg bw/d), 5 male and 3 female pups of test group 2 (300 mg/kg bw/d), 24 male and 19 female pups of test group 3 (1000 mg/kg bw/d) died prematurely.
All other F1 pups in all test groups (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 and PND 13.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- Pup number and status at delivery
The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred, but the number of liveborn pups was clearly decreased for test group 3 (1000 mg/kg bw/d).

- Pup viability index/mortality
The viability index indicating pup mortality between PND 0 and 4 was 100.0% in test group 0 (control), 99.2% in test group 1 (100 mg/kg bw/d), 95.8% in test group 2 (300 mg/kg bw/d) and 94.6% in test group 3 (1000 mg/kg bw/d). The rate live birth indices were 100% in test group 0, 98.2% in test group 1, and 95.9% in test group 2, but reduced to 58.1% in test group 3. The number of dams with stillborn pups was significantly increased in test group 3, i.e. 5 female animals with individual Nos. 131, 133, 134, 137 and 138. In 2 litters, i.e. Nos. 131 and 137, all pups were stillborn. The mean number of stillborn pups/litter was also significantly increased with a value of 4.9. Mean value of perinatal loss was significantly increased (47.2%) in test group 3 (1000 mg/kg bw/d).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group with the following exceptions.
Mean body weight of male pups in test group 3 (1000 mg/kg bw/d) was significantly higher on lactation day 13 (+12.9%) when compared to control. In addition, the body weight change values of male pups were also higher between lactation days 7-13 and 1-13. These deviations to the control were assessed to be incidental and not related to the test item.
One male runt was found in one litter of test group 0 (0 mg/kg bw/d), one female runt was found in one litter of test group 1 (100 mg/kg bw/d) and 2 male runts were found in each one litter of test group 2 (300 mg/kg bw/d). A relation to dosing was not observed, test substance-related effects did not occur.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Three male and 2 female pups of test group 2 (300 mg/kg bw/d), 22 male and 17 female pups of test group 3 (1000 mg/kg bw/d) showed post mortem autolysis. These findings were observed as the pups were found dead and the autolysis was in progress before the daily clinical observations started.
Dilated renal pelvis was observed in each one female pup of test groups 1 (control group) and 3 (1000 mg/kg bw/d).
Hydronephrosis was observed in one female pup of test group 3 (1000 mg/kg bw/d). These findings were assessed to be incidental and not related to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

- Anogenital distance
Anogenital distance and anogenital distance index of female pups were significantly higher in test group 3 (1000 mg/kg bw/d). However, these changes were considered to be incidental and not treatment-related.

- Nipple/areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: perinatal loss was significantly increased, gestation index as well as the live birth index significantly reduced

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: viability
Treatment related:
yes
Dose response relationship:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

MATING & DELIVERY DATA

0

100 mg/kg

300 mg/kg

1000 mg/kg

Females mated

10

10

10

10

Mating Index

100%

100%

100%

100%

Females pregnant

9

9

9

9

Fertility Index

90%

90%

90%

90%

With liveborn pups

9

9

9

6

With stillborn

0

2

1

5*

With all stillborn

0

0

0

2

Gestation Index

100%

100%

100%

67%

Gestation Days

22.1

22.4

22.6

23.3**

LITTER DATA

0 mg/kg

100 mg/kg

300 mg/kg

1000 mg/kg

Implantation Sites

113

116

117

103

Pups delivered

104

110

111

93

Mean pups delivered

11.6

12.2

12.3

11.6

PostimplantationLoss

7.6%

5.0%

5.0%

18.5%

Liveborn Pups

104

108

106

54

Mean

11.6

12.0

11.8

6.8

Livebirth Index

100%

98.2%

95.5%

58.1%

Pups stillborn

0

2

5

39

Perinatal Loss

0%

2.2%

4.3%

47.2%

Pups died day 0-4

0

1

3

4

Viability Index

100%

99.2%

95.8%

94.6%

INDIVIDUAL FEMALE ANIMAL DATA HIGH DOSE

1000 mg/kg

Status

Gestation Days

Dead

Alive

Total

ImplanatationSites

Post implantation Loss

Live Birth Index

Perinatal Loss

Female 131

Pregnant

24

9

0

9

11

18.18%

0%

100%

Female 132

No implants

-

-

-

-

0

-

-

-

Female 133

Pregnant

24

8

4

12

13

7.69%

33.3%

66.7%

Female 134

Pregnant

23

3

12

15

16

6.25%

80%

20%

Female 135

Pregnant

22

0

12

12

13

7.69%

100%

0%

Female 136

Pregnant

23

0

14

14

14

0%

100%

0%

Female 137

Pregnant

24

9

0

9

11

18.18%

0%

100%

Female 138

Pregnant

23

10

1

11

12

8.33%

9.9%

90.9%

Female 139

Pregnant

23

0

11

11

11

0%

100%

0%

Female 140

Pregnant

24

0

0

0

2

100%

-

-

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test item to Wistar rats revealed adverse signs of toxicity in female animals at a dose level of 1000 mg/kg bw/d. No effects were observed in male animals at any dose level. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and 300 mg/kg bw/d for female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and 300 mg/kg bw/d for female Wistar rats because of the increased perinatal loss of pups. Thus, the NOAEL for developmental toxicity was 300 mg/kg bw/d.
Executive summary:

The test item was given daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Corn oil served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7,14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted. At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental animals per sex and test group. Clinicochemical and hematological examinations were performed in 5 parental animals per sex and group towards the end of the administration period. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The following test substance-related, relevant findings were noted in test group 3 (1000 mg/kg bw/d)

•       Three female animals of test group 3 (1000 mg/kg bw/d) showed piloerection and pale skin during end of gestation and early lactation, each one additional individual showed either one finding, only. The observed effects were most likely a consequence of dystocia, the labored birthing process.

•       The gestation index was reduced to 66.7%.

•       The live birth index was reduced to 58.1% due to the reduced number of females with live pups on the day of birth.

•       The mean duration of gestation was 23.3 days and significantly increased when compared to the controls.

•       Postimplantation loss was clearly increased (18.5%) when taking female animal No. 140 into account but well within the historical control data if animal No. 140 would has been taken out of mean calculation.

•       The number of dams with stillborn pups was significantly increased and in 2 litters all pups were stillborn. The mean number of stillborn pups/litter was also significantly increased.

•       Mean value of perinatal loss was significantly increased (47.2%).

•       Yellow discoloration of the adipose tissue was noted in male and female animals. The yellow discoloration of the adipose tissue was also noted in males and females of test group 2 (300 mg/kg bw) and in females of test group 3 (100 mg/kg bw).

•       24 male and 19 female pups died prematurely before lactation day 13.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test item to Wistar rats revealed adverse signs of toxicity in female animals at a dose level of 1000 mg/kg bw/d. No effects were observed in male animals at any dose level. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and 300 mg/kg bw/d for female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and 300 mg/kg bw/d for female Wistar rats because of the increased perinatal loss of pups. Thus, the NOAEL for developmental toxicity was 300 mg/kg bw/d.