Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Toxicity to reproduction

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 for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

EC Number:

255-527-0

EC Name:

Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Cas Number:

41741-86-0

Molecular formula:

C34 H24 Cr N8 O6 .Na

IUPAC Name:

Sodium;chromium(3+);2-[(3-methyl-5-oxido-1-phenylpyrazol-4-yl)diazenyl]benzoate

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 or test system 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:

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:

other: 0.5% Sodium carboxymethyl cellulose suspension in drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5% sodium carboxymethyl cellulose in drinking water 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: 1, 3, 10 g/100ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Details on mating procedure:

Two weeks after the beginning of treatment, the surviving males and females from the same test group were mated overnight in a ratio of 1:1. 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 0.5% sodium carboxymethyl cellulose in drinking water 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 item was distributed homogeneously in 0.5% sodium carboxymethyl cellulose in drinking water. The concentrations of the test item in 0.5% sodium carboxymethyl cellulose in drinking water were found to be in the range of 95-117% of the nominal concentration. These results demonstrated the correctness of the concentrations of Oracet Yellow 160 FA in 0.5% sodium carboxymethyl cellulose in drinking water.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a range finder study

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 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 (for the calculation of the administration volume)
• Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume)

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.

OPHTHALMOSCOPIC EXAMINATION: No

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),

URINALYSIS: No

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.

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. 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: 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 or less.
On post-natal day (PND) 4, as a result of standardization, surplus pups, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, these pups were examined externally, eviscerated and their 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 possible further processing. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically.
All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.


EXAMINATIONS
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.

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 “Necropsy observations”.

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 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.

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.

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

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). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females.
“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.

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.

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.

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):

All pups with scheduled sacrifice on PND 4, resp. PND 13 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their 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 transferred to the Pathology Laboratory.

All stillborn pups and all pups that died before day 13 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

Offspring viability indices:

Live birth index, Postimplantation loss, Viability index, Survival index, Sex ratio, Anogenital index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse signs of toxicity were observed. Female animal No. 102 of test group 0 (control) showed piloerection and reddish discolored vaginal discharge on gestation day (GD) 23. Blood in bedding was found on the same day. The animal was not treated, a relation to the test substance was excluded. No other female animal of test groups 0 to 3 (control, 100, 300 and 1000 mg/kg bw/d) showed clinical signs. In the litter of female animal No. 102 of test group 0 (control) all pups were stillborn, the dam lost its complete litter. On the day of delivery, this animal showed piloerection. From postnatal day (PND) 1 until sacrifice, the animal did not show any clinical finding. The animal was not treated, a relation to the test substance could be excluded.

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):

During the entire administration period, no test substance-related 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 relevant changes with regard to food consumption was observed in male and female animals on any day of the administration period. Food consumption values in female animals of test group 1 (100 mg/kg bw/d) were significantly lower between pre-mating days 0 to 7 as well as between GD 0 to 7 and GD 0 to 20. These values were still within a normal range typical for this strain of rats and a dose-response relationship did not occur. Therefore, the deviations to the control were assessed to be without toxicological relevance.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test substance-related changes in water consumption were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. In males of test group 1 (100 mg/kg bw/d) platelet counts were significantly increased, but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. 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:
Quantitative Parameters: No test substance-related effects were observed. Grip strength of hindlimbs was significantly increased in male animals of test group 1 (100 mg/kg bw/d). As no dose-response relationship occurred, the change was assessed as being spontaneous in nature and not related to treatment.
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: 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, a significantly decreased value was measured for male animals of test group 3 (1000 mg/kg bw/d) at interval 4. The difference was regarded to be incidental and not related to treatment as only a single interval was changed and the overall motor activity was not affected. No changes were observed for female animals in test groups 1, 2 and 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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item related microscopic observations. Widespread fine, gritty, foreign alveolar content, foamy alveolar macrophages and multiple cholesterol granuloma (with foreign body-type multinucleated macrophages) were observed in the lung of female 122 of the group treated with 300 mg/kg bw/d. Additionally, signs of alveolar emphysema were noted in this animal. Few macrophage aggregates were observed within the mesenteric lymph node of a single male (male 33) and 2/5 females (female 136, 139) of the group treated with 1000 mg/kg bw/d. Thorough microscopic examination of the male and female reproductive organs did not reveal any relevant histopathologic change. The morphology and distribution of the different successive stages during spermatogenesis (Russell et al., 1990) was normal for all males examined. Other microscopic findings reported in the individual animal table are considered spontaneous, incidental or related to technical procedures and consistent with the usual pattern of findings in animals of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In parental males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) and in male and female pups of test groups 11, 12 and 13 (100, 300 and 1000 mg/kg bw/d) at PND 13, no treatment-related alterations of T4 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 was 3.9 days in all test groups including the control.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The male mating index calculated after the mating period for F1 litter was 100% in all test groups including the control. Fertility was proven for all of the F0 parental males within the scheduled mating interval to produce F1 litter. Thus, the male fertility index was 100% in all test groups including the control.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups including the control. The mean duration until sperm was detected (GD 0) was 2.5 days for test group 0, 3.7 days for test group 1, 2.7 days for test group 2 and 2.9 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. All sperm positive rats delivered living pups with the exception of female animal No. 102, which was mated with male animal No. 2 of test group 0 (control), which delivered only stillborn pups. Thus, the female fertility index was 100% in all test groups including the control. The mean duration of gestation was similar in all test groups, i.e. 22.3 days in test groups 0 and 1, and 22.1 days in test groups 2 and 3. These findings 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 gestation index was 90% in test group 0 (control) and 100% in test groups 1-3 (100, 300 and 1000 mg/kg bw/d).
The rate live birth indices were 90% in test group 0 (control) and 100% in test groups 1-3 (100, 300 and 1000 mg/kg bw/d).
The postimplantation loss was 11.5% in test group 0 (control), 4.5% in test group 1 (100 mg/kg bw/d), 6.2% in test group 2 (300 mg/kg bw/d) and 4.3% in test group 3 (1000 mg/kg bw/d). These findings 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. A treatment-related increase in postimplantation loss was not observed in any test group.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects reported

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

All F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 or PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during PND 0-4 was 100.0% in test group 0 (control), 99.1 in test group 1 (100 mg/kg bw/d), 95.9% in test group 2 (300 mg/kg bw/d) and 99.3% in test group 3 (1000 mg/kg bw/d). The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100% in test groups 1-3.

Body weight and weight changes:

no effects observed

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. All values were within the range of the biological variation inherent in the strain of rats used for this study.

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed. The enlarged heart which was observed in a single male pup in test group 3 (1000 mg/kg bw/d) was assessed to be spontaneous in nature.

Description (incidence and severity):

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 including the significant deviation in test group 3 were regarded to be spontaneous in nature. Anogenital distance and anogenital index were not affected in all F1 pups. 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:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

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 material to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus, the NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Executive summary:

The test article was administered by gavage to groups of 10 male and 10 female Wistar rats (F0 generation) 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). Drinking water containing 0.5% sodium carboxymethyl cellulose 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 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. 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 Pathology Laboratory 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.

No treatment-related, adverse effects were observed for F0 PARENTAL ANIMALS in any dose group during Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology. No treatment-related, adverse effects were observed for F1 PUPS of all dose groups during Clinical Examinations/ Gross examinations. 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 material to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.