4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

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

Study period:

24 Jul 2014 - 24 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 11 weeks (males/females)
- Weight at study initiation: mean weights male: 310 g; mean weight females: 205 g
- Housing: During the study period, the rats were housed individually in polycarbonate cages type III (floor area of about 800 cm²), with the following exceptions: During overnight matings, male and female mating partners were housed together in polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 4 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm²) and small amounts of bedding material.
- Diet: ground Kliba maintenance diet mouse-rat "GLP", ad libitum
- Water: ad libitum from water bottles
- Acclimation period: 6 days

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

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% and 0.5%

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, drinking water with 0.5% or 1% Carboxymethylcellulose was filled up to the desired volume, subsequently mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
The concentration of Carboxymethylcellulose had to be changed during the study because the supplier changed the specification of Carboxymethylcellulose without communicating to the customer beforehand. Unexpectedly, the viscosity of its suspensions increased. It was not suitable for gavage application anymore. The concentration in suspensions had to be reduced to be able to gavage the test substance preparations.
Test substance preparations in drinking water with 1% Carboxymethylcellulose were used in test group 3 on study day 0 and 1 and test group 1
and 2 on study day 0 to 7; test substance preparations in drinking water with 0.5% Carboxymethylcellulose were used in test group 3 from study day 2 onwards and test group 1 and 3 from study day 8 onwards. For the control group, drinking water containing 1% Carboxymethylcellulose was used on study day 0 to 7; from study day 8 onwards drinking water containing 0.5% Carboxymethylcellulose was used.

VEHICLE
- Concentration in vehicle: 0, 1, 3, 10 g/100 ml
- Amount of vehicle (if gavage): 10 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 stability of the test substance in drinking water over a period of 7 days at room temperature was proven before the start of the study. Samples of the test substance preparations were sent to the analytical laboratory for verification of the concentrations. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and highconcentrations (100 and 1000 mg/kg bw/d).
Homogeneity of the test substance preparations was demonstrated. Because of technical challenges during analyses the reserve samples of test substance
preparation were analysed. However, the concentrations of test material in drinking water containing 1% Carboxymethylcellulose used in the first days of
administration were found below the nominal concentration. The analyses demonstrated a recovery of 83-88% in the low dose (100 mg/kg bw/d), 76% in the mid dose (300 mg/kg bw/d) and 93-98% in the high dose (1000 mg/kg bw/d). The concentrations of test material in drinking water containing 0.5% Carboxymethylcellulose used starting from study day 2 onwards in the test group 3 and starting from study day 8 in test group 0, 1 and 2 were found below the nominal concentration. The analyses demonstrated a recovery of 58-61% in the low dose (100 mg/kg bw/d), 63% in the mid dose (300 mg/kg bw/d) and 75-76% in the high dose (1000 mg/kg bw/d). In a conservative assumption the nominal dose levels tested were interpreted as lower dose on the basis of the lowest determined concentration in the test substance preparations. As a result of this the doses were adapted as follows:
100 mg/kg bw/d correspond to 58 mg/kg bw/d
300 mg/kg bw/d correspond to 189 mg/kg bw/d
1000 mg/kg bw/d correspond to 750 mg/kg bw/d

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 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100 mg/kg bw/d, 300 mg/kg bw/d, 1000 mg/kg bw/d
Basis:
other: nominal by gavage

Remarks:

Doses / Concentrations:
58 mg/kg bw/d, 189 mg/kg bw/d, 750 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.

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), assessment 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 parental 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) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear or waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
- Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 4.
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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION:
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: the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
- 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 (RET), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
- 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: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.) [g/L], Sediment, Color, turbidity (COL, TURB), Volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery was performed in the first five surviving 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.
- Battery of functions tested: Home cage observations, Open field observations, Sensory motor tests/ reflexes, Motor activity assessment

Sperm parameters (parental animals):

Special attention was given on the stages of spermatogenesis in the testes.

Litter observations:

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
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. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying
between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

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. The sex of the pups was finally confirmed at necropsy.

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

BODY WEIGHTS
The pups were weighed on the day after birth (PND 1) and on PND 4. 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). “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.

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 were determined in all animals sacrificed on schedule:
Anesthetized animals, Epididymides, Testes
The following weights were determined in 5 animals/sex and test group, sacrificed on schedule (females with litters only, same animals as used for clinical pathology examinations):
Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes
Organs or tissues of all parental animals were fixed in in 4% neutralbuffered formaldehyde or in modified Davidson’s solution. The testes and epididymides of the animal that died were fixed in 4% neutral-buffered formaldehyde solution. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:

Postmortem examinations (offspring):

All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 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-bycase basis, depending on the type of finding noted.

Statistics:

- DUNNETT test (two-sided): Food consumption (parental animals), Water consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days
- 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 (onesided+) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss,
- WILCOXON test (onesided-) with BONFERRONI-HOLM adjustment: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index
- WILCOXON test (twosided): % live male day x, %live female day x
- KRUSKAL-WALLIS test (two-sided): Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
- WILCOXON-test (one-sided): Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)
- KRUSKAL-WALLIS test (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 (twosided): Blood parameters, Urine pH, volume, specific gravity, color and turbidity, Weight parameters

Reproductive indices:

Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index, Post implantation loss

Offspring viability indices:

Viability index, Sex ratio

CLINICAL SIGNS AND MORTALITY
No parental animal died or was sacrificed moribund during the study period.
During premating, mating and post-mating yellow discolored feces were seen in all animals of the test groups 2 and 3 (300 and 1000 mg/kg bw/d). The sign came up first on study day 2 in test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d). This yellow discoloration was regarded to be caused by the test substance. The finding was considered to be related to treatment but assessed as being non-adverse.
Clinical observations for females during gestation: During gestation yellow discolored feces were seen in all animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d). This yellow discoloration was regarded to be caused by the test substance. The finding was considered to be related to treatment but assessed as being non-adverse. One female animal (No. 116) of test group 1 (100 mg/kg bw/d) was not able to deliver. In further consequence observations were vaginal discharge (GD 23 and 27), paleness (GD 23 – 26), piloerection (GD 26) and a mass was palpable in abdomen from GD 23 on to sacrifice. All these isolated findings were considered to be incidental and not treatment related. In one female animal (No. 127) of test group 2 (300 mg/kg bw/d), and in two females (No. 135 and 137) in test group 3 (1000 mg/kg bw/d) piloerection was observed on GD 22. It could not be excluded that these findings were related to treatment. However, these findings were assessed as being non-adverse.
Clinical observations for females during lactation: During lactation yellow discolored feces were seen in all animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d). This yellow discoloration was regarded to be caused by the test substance. The finding was considered to be related to treatment but assessed as being non-adverse. One female animal of test group 1 (100 mg/kg bw/d; animal No. 112) had a skin lesion in the neck region from lactation day 20 on to sacrifice (lactation day 22). One female (No. 133) of test group 3 (1000 mg/kg bw/d) showed hairless spots (alopecia) at its abdominal region (from lactation day 14 on to sacrifice) and its forelimbs (from lactation day 19 on to sacrifice). These isolated findings were considered to be incidental and not treatment related. Furthermore in one female animal of test group 2 (300 mg/kg bw/d; animal No. 127) and in two females in test group 3 (1000 mg/kg bw/d; animals No. 135 and 137) piloerection was observed on one to six days at the beginning of the lactation period. It could not be excluded that these findings were related to treatment. However, these isolated findings were assessed as being non-adverse.

DETAILED CLINICAL OBSERVATION
The detailed clinical observations on study days 0, 7, 14, 21, 28 in males and females and, additionally, on study days 35, 42, 49 and 56 in female animals did not reveal any additional abnormalities to yellow colored feces, which were seen from study day 7 in test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d). Only individual females showed additional signs, like skin lesion in neck region (test group 1, animal no. 112), pale skin, palpable mass in abdomen, unable to deliver (test group 1, animal no. 116), alopecia (test group 3, animal no. 133), and piloerection (test group 3, animal no. 137) mentioned in 4.2.1.2.. These isolated findings were assessed as spontaneous in nature and not treatment related.

BODY WEIGHT AND WEIGHT GAIN
Body weights were not statistically significantly affected in male and female animals during the study period by the test compound. At the end of gestation the body weight of a pregnant dam consists not only of the carcass weight of the dam but also of the fetuses growing inside of the uterus. The significant decreased body weight in test group 1 (100 mg/kg bw/d; 21%) on gestational day (GD) 20 was considered to be caused by the lower number of implantation sites in this test group (73) in comparison to control (120). This interpretation was supported by the body weight determined after giving birth being comparable in test group 1 and control (day 0 of lactation: test group 1 254.1 g and control 252.3 g). In correlation to that, the body weight changes were statistically significantly affected in test group 1 (100 mg/kg bw/d) from GD 7 on to the end of gestation period (-25.7% GD 7-14 and -29.7% GD 14-20). In consequence the overall body weight change in test group 1 (100 mg/kg bw/d) was affected during gestation period (-20.7% GD 0-20). Thereby, the observed significant effects on body weight and body weight gain during gestation were assessed as physiological adaptation to the lower number of implantation sites or fetuses in this test group and not as treatment related. Individual body weight loss during lactation period occurred occasionally across all dose groups and control group, because of this consistent distribution not considered to be substance related.

FOOD CONSUMPTION
No significant deviations from control in male and female animals were observed during the study period with one exception. In the lactation period the food consumption was significantly decreased in test group 1 (100 mg/kg bw/d). These findings were considered to be caused by the lower nutrition demand of the dams during lactation based on the lower number of pups in this test group (pups delivered 68) in comparison to control (pubs delivered 111). Thereby, the observed significant decreased food consumption during lactation was assessed as physiological adaptation to the lower number of pups delivered in this test group and not as treatment related.

WATER CONSUMPTION
No test substance-related changes were observed.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed. At the end of the administration period in females of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) large unstained cell (LUC) counts were higher compared to controls (not statistically significant in test group 3). The means of all three test groups were within the historical control range whereas in the study control the counts were lower (LUC 0.01-0.02 Giga/L, PART III, Supplement) and therefore, the alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period in females of test group 1 (100 mg/kg bw/d), alanine aminotransferase (ALT) activities were higher compared to controls, but the change was not dose-dependent and therefore it was regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
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 rats only, these observations were considered as incidental. No test substance-related effects were observed during home cage observations, ppen field observations, sensorimotor tests/reflexes and in quantitative parameters. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male animals of all test groups in comparison to the concurrent control group. The overall motor activity in female animals of test group 1 (100 mg/kg bw/d) and test group 2 (300 mg/kg bw/d) were statistically significant increased in comparison to control group, but this was not the case in any of the individual intervals 1 to 12. Without a dose response, this observation was assessed as being incidental and not treatment related. Regarding single intervals in males and females no significant deviations were detected with one exception. The statistically significant decreased number of interrupted beams in interval 9 of the test group 3 (1000 mg/kg bw/d) in females was considered as incidental and not treatment related.

MALE REPRODUCTION DATA
The male mating index was 100% in all test groups. Fertility was proven for all of the F0 parental males within the scheduled mating interval to produce F1 litter. Two males of test group 1 (No. 16 mated with female No. 116 and No. 18 mated with female No. 118) did not generate F1 pups. The male fertility index was 100% in all test groups.

FEMALE REPRODUCTION DATA
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 2.5, 2.2, 2.5, 2.1 days in test groups 0 - 3. This finding 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 pups with two exceptions in test group 1. Female animal No. 116 which was mated with male No. 16 and female No. 118 which was mated with male No. 18 did get sperm in vaginal smear and showed implants but delivered no pups. The female fertility index was 100% in all test groups.
The mean duration of gestation was similar in all test groups (22.1 days in test groups 0, 1 and 3; 22.0 days in test group 2). 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 100% in test group 0, 2 and 3 and 80% in test group 1 (100 mg/kg bw/d). The latter represents 8 females with live pups on the day of birth out of 10 females pregnant. Also in the historical control data not always 100% of the pregnant females had live pups. Consequently, the historical control data ranges from 89% to 100%. However, the incidence in test group 1 is below the lower limit of the historical control data, it was an isolated finding without any dose dependency. Therefore, the finding was considered incidental and not treatment related.
The rate live birth indices were 99.1% in test group 0, 100% in test group 1 (100 mg/kg bw/d), 99.2% in test group 2 (300 mg/kg bw/d) and 99.0% in test group 3 (1000 mg/kg bw/d). The values of test group 1, 2 and 3 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 was 8.4% in test group 0, 24.3% in test group 1 (100 mg/kg bw/d), 2.9% in test group 2 (300 mg/kg bw/d) and 4.3% in test group 3 (1000 mg/kg bw/d). With the exception of test group 1, the 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 (0.7% - 18.3%) of the historical control data. In test group 1 two females had only one implantation site and did not delivered F1 pups. Therefore, the loss of one fetus in each dam caused a postimplantation loss of 100% in 2 out of 10 dams. This was leading to an unusually high postimplantation loss of 24.3% in test group 1 as the mean of incidences in each dam. This finding was considered as incidental and not treatment related.

ORGAN WEIGHTS
All mean absolute weight parameters did not show significant differences when compared to the control group 0. In test group 3, the mean relative spleen weight was significanty increased (124%). The mean relative spleen weight (0.193%) in test group 3 (1000 mg/kg bw/d) was below the maximal historical control value (0.196%). Furthermore, this increase had no histopathological correlate and therefore, it was regarded as incidental and not treatmentrelated. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
Yellow discolorations of the contents were observed in the glandular stomach, jejunum, cecum and colon of males and females of test group 3 (1000 mg/kg bw/d). This finding was also observable in the glandular stomach of males and females of test group 2 and 1 (300 and 100 mg/kg bw/d). The discoloration was due to the color of the test substance and was not considered to be adverse. A thickened margo plicatus observed in 3 out of 10 males of test group 3 (1000 mg/kg bw/d) could not be correlated by histopathology and was therefore regarded as not treatment-related. All other 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.

HISTOPATHOLOGY: NON-NEOPLASTIC
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.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw (total dose)

Sex:

male/female

Basis for effect level:

other: No test substance-related adverse findings

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

750 mg/kg bw (total dose)

Sex:

male/female

Basis for effect level:

other: No test substance-related adverse findings

VIABILITY (OFFSPRING)
In test groups 0, 2 and 3 (0 mg/kg bw/d, 300 mg/kg bw/d and 1000 mg/kg bw/d; females No. 103, 126 and 135) each 1 pup was stillborn. The mean number of delivered F1 pups per dam was evenly distributed about test groups 0, 2 and 3. The mean number of delivered F1 pups is reduced in test group 1 (100 mg/kg bw/d, 8.5 pups/dam). This finding is slightly below the range (9.3 - 14.1 pups/dam) of the historical control data (PART III, Supplement) but there was no dose dependent alteration of the pup number. Therefore, it was considered to be incidental and not treatment related.
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% (test group 0 and 2), 99.0% (test group 1, 100 mg/kg bw/d) and 99.1% (test group 3) based on one pup of test group 1 (100 mg/kg bw/d) and test group 3 (1000mg/kg bw/d) were missing (cannibalized). 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. One pup in group 0 (0 mg/kg bw/d, animal no. 103-07), test group 2 (300 mg/kg bw/d, animal no. 126-05) and test group 3 (1000 mg/kg bw/d, 135-12) were found stillborn. 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.

SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in test groups 1, 2, and 3 (100 mg/kg bw/d, 300 mg/kg bw/d, and 1000 mg/kg bw/d) were comparable to the control group. Three male and one female runts were seen in test group 2 (300 mg/kg bw/d). Three female runts were observed in test group 3 (1000 mg/kg bw/d).

GROSS PATHOLOGY (OFFSPRING)
In test group 1 (100 mg/kg bw/d) one male pup showed a bilobed urinary bladder. In test group 3 (1000 mg/kg bw/d) one pup was not assessed because it was missing (cannibalized). Actually, also in test group 2 (100 mg/kg bw/d) one pup (animal no. 119-07) could not be assessed because it was also missing (cannibalized). This pup is not presented in the table IA-57 (Summary – Pup Necropsy Observation), because only its pup status was documented. It was not noted in pup necropsy why no data could be collected. This minor data gap had not influence on the validity of the study. Two female pups (each one pup of test group 2 (300 mg/kg bw/d) and test group 3 (1000 mg/kg bw/d)) showed a dilated renal pelvis. These findings were assessed as being spontaneous in nature and without toxicological relevance.

Dose descriptor:

NOAEL

Remarks:

Development

Generation:

F1

Effect level:

750 mg/kg bw (total dose)

Sex:

male/female

Basis for effect level:

other: No test substance-related adverse findings

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the present Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats the NOAEL (no observed adverse effect level) for general, systemic toxicity was at least 750 mg/kg bw/d. The NOEL (no observe effect level) for reproductive performance and fertility was at least 750 mg/kg bw/d in male and female Wistar rats. The NOEL for developmental toxicity was at least 750 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 in Wistar Rats the test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at nominal dose levels of 100, 300 and 1000 mg/kg bw/d which were interpreted as 58, 189, and 750 mg/kg bw/d based on the analyses of test substance preparations (BASF, 2015). Regarding clinical examinations, signs of general systemic toxicity were not observed in male and female parental animals of test groups 1-3 (58, 189, and 750 mg/kg bw/d) during the entire study period. During all phases of the study yellow discolored feces were seen in all animals of test groups 2 and 3 (189 and 750 mg/kg bw/d). This yellow discoloration was regarded to be caused by the test substance. The finding was considered to be related to treatment but assessed as being not adverse. One female animal (No. 116) of test group 1 (58 mg/kg bw/d) was not able to deliver. In further consequence observations were vaginal discharge (gestational day 23 and 27), paleness (GD 23 – 26), piloerection (GD 26) and a mass was palpable in abdomen from GD 23 on to sacrifice. During necropsy a thickening of the uterus wall colored beige brown was determined. This finding was correlated with a purulent inflammation with placental autolysis observed during histopathological evaluation. These findings were assessed as pyometra, a uterine infection. The mentioned finding in the single animal of the test group 1 was assessed as being incidental and not related to treatment. Fertility indices for male and female animals were not impaired by test-substance administration up to the dose level of 750 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not impaired by test-substance administration even at a dose level of 750 mg/kg bw/d. Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the compound of 750 mg/kg bw/d. Regarding pathology, there were neither treatment-related organ weight deviations nor gross lesions. Furthermore, all histopathological 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.

In conclusion, under the conditions of the present Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats the NOAEL (no observed adverse effect level) for general, systemic toxicity was at least 750 mg/kg bw/d. The NOEL (no observe effect level) for reproductive performance and fertility was at least 750 mg/kg bw/d in male and female Wistar rats. The NOEL for developmental toxicity was at least 750 mg/kg bw/d.

Short description of key information:
In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats no adverse effects were reported. The NOAEL for general, systemic toxicity was at least 750 mg/kg bw/d. The NOEL for reproductive performance and fertility was at least 750 mg/kg bw/d in male and female Wistar rats. The NOEL for developmental toxicity was at least 750 mg/kg bw/d.

Justification for selection of Effect on fertility via oral route:
GLP-compliant guideline study

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available