Benzonitrile, 4-[(1,6-dihydro-4-hydroxy-6-oxo-2-pyrimidinyl)amino]-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-18 to 2015-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2016

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD guideline 407

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated dose (28 days) toxicity (oral))

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050

Deviations:

no

Principles of method if other than guideline:

No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[(4,6-dihydroxypyrimidin-2-yl)amino]benzonitrile

EC Number:

609-425-1

Cas Number:

374067-80-8

Molecular formula:

C11H8N4O2

IUPAC Name:

4-[(4,6-dihydroxypyrimidin-2-yl)amino]benzonitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-4370561-AAA (T002326)
- Physical state: solid (powder)
- Appearance: slight beige powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15CB0987
- Expiration date of the lot/batch: 2016-07-29 (pilot phase); 2015-12-05 (main phase)
- Purity test date: 2016-05-19 (pilot phase); 2015-08-17 (main phase)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Test Facility Study no. 509773).

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension (Groups 2, 3 and 4).

OTHER SPECIFICS: correction factor is 1.00

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat was recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch had general and reproduction/developmental historical data in this species from the same strain and source. This animal model had been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 10-12 weeks (females, at start pretest); approx 10-12 weeks (males, at start treatment)
- Weight at study initiation: 200-243 g (females); 294-368 g (males)
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-onebasis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Use of restrainers for preventing ingestion (if dermal): no (not applicable)
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2015-09-18 (females, start pretest); 2015-10-02 (males, start treatment), 2015-11-08/09/10/11/12/14 (pups, delivery of litters)
To: 2015-11-02 (males, necropsy); 2015-11-20/23/24/26 (pups, necropsy on PND 13-15); 2015-11-13/16/23/24/25/27 (females, necropsy on PND 14-16)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036). A correction was made for the purity/composition of the test item. A correction factor of 1 was used.
- Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Concentration in vehicle: 0 mg/mL (Group 1; control), 20 mg/mL (Group 2), 60 mg/mL (Group 3), 200 mg/mL (Group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Lot/batch no. (if required): no data
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1/animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, until detection of mating was confirmed
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated as day 0 post-coitum. Once mating was confirmed, the males and females were seperated. Female no. 56 (100 mg/kg) was examined by palpation on 28 October 2015 and confirmed to be pregnant (i.e. mating was overlooked). The mating date was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (2015-10-14; Day 13 of treatment), according to a validated method (Test Facility Study no. 509773). Sextuplicate samples (i.e. 3 sets of duplicate samples) were collected. Two sets of duplicate samples were stored as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of the test item under test conditions was demonstrated in the method validation study (Test Facility Study no. 509773).

Duration of treatment / exposure:

31 days (males); 52-56 days (females that delivered); 42-45 days (females which failed to deliver)
Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on the results of a 10-day range finding study with T002326 (Test Facility Study no. 508974). At the administered dose levels of 500 and 1000 mg/kg/day, no mortality occurred. No toxicologically relevant clinical signs were noted. Body weight and food intake and organ weights were considered unaffected by treatment, and no necropsy findings were noted. Dose levels of 100, 300 and 1000 mg/kg/day were selected in consultation with the Sponsor.
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):no
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in P0 male parental generations: testis weight, additional slides of the testes (to examine staging of spermatogenesis)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on postnatal day 1 (=day the litter was found completed). When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
- maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND4; blood samples were collected from twe of the surplus pups; Excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring (if practically possible):
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 4.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead (if possible).
Pups found dead during the weekend were fixed in an identified container containing 70% ethanol, because they were not necrospied on the same day.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated.
- Male animals: following completion of the mating period (a minimum of 28 days of dose administration)
- Female animals: on PND 14-16 (females which deliver); on post-coitum day 25 (females which failed to deliver; nos. 62 and 69)

GROSS NECROPSY
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The number of implantation sites were recorded for all paired females.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of 5 selected animals/sex/group were collected and fixed in 10% buffered formalin: Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (males and females) (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes 4 (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin: Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides 4 (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (males and females) (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes 4 (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/group on the scheduled day of necropsy:
Adrenal glands, brain, Cowper's glands, epididymides, glans penis, heart, kidneys, levator ani plus bulbocavernosus muscle complex (LABC), liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid, uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals:
Cowper's glands, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex (LABC), testes, thyroid

HISTOPATHOLOGY
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire.
- All gross lesions of all animals (all dose groups).
- Thyroid gland of all selected males and females of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4.
- The reproductive organs of two Group 3 pairs that failed to sire or deliver healthy pups: male no. 22 / female no. 62 (not pregnant) and male no. 29 / female no. 69 (implantation sites only). For female no. 56 / male no. 16, reproductive organs were also examined histopathologically. However, mating was overlooked for this pair.
- All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age (PND 4, at culling) by decapitation between 7.00 and 10.30 a.m.; at 13-15 days of age (PND 13-15; 2 pups per litter) by aorta puncture under anaesthesia using isoflurane between 7.00 and 10.30 a.m.; and at 7-15 days of age (PND 7-15; all remaining pups) using Euthasol 20% by intraperitoneal injection.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling (see also sections 7.8. and 7.10.1), was preserved in 10% buffered formalin.
- The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated/Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females/ Number of females paired) x 100
- Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine impl antation sites) x 100
- Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
- Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
- Group mean values were calculated from individual litter values.
- Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg, pale faeces was noted among all females from Week 2 of the premating phase onwards, and for all males on the last day before necropsy.
Salivation seen after dosing among animals at 1000 mg/kg during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to the taste of the test item.
Other clinical signs noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the
conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted.
The statistically significantly lower body weight gain of females at 1000 mg/kg during the second week of the premating period was slight in nature and with continuation of treatment body weight gain was similar to controls for these females. As such, this change was considered to be of no toxicological significance.
Other variations in body weights and body weight gain among treated groups remained similar to control levels throughout treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters of selected rats were considered unaffected by treatment.
Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Females at 1000 mg/kg had a statistically significantly higher mean cholesterol level (approximately 42% higher than the control mean).
No further (statistically significant) changes in clinical biochemistry parameters occurred that were considered to be related to treatment. One control male had a very high alanine and aspartate aminotransferase activity (ALAT and ASAT). There were no morphological correlates that would support or explain this incidental finding.
Thyroid hormone analyses: Serum levels of T4 and TSH in F0 males were considered unaffected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Grip strength and motor activity showed no variations that were considered to be related to treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hypertrophy of follicular cells of the thyroid gland was recorded at a slightly increased severity in males and females at 1000 mg/kg.
Remaining histologic changes were considered to be incidental findings. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in one female at 100 mg/kg, i.e. the female for which mating was overlooked, and irregular cycles occurred in one female each at 300 and 1000 mg/kg which all had normal litters. The incidence of these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTION DATA
Reproductive parameters (mating and fertility indices, precoital time, and number of implantation sites) were not considered to have been affected by treatment.
All females showed evidence of mating. All females delivered offspring, except for two couples at 300 mg/kg: male no. 22/female no. 62 and male no.29/female no.69 (implantation sites only). No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. One of these females (no. 69) showed two early resorptions in the right uterus horn. Based on the fact that all couples at 1000 mg/kg had offspring, the occurrence of two infertile couples at 300 mg/kg was considered to be unrelated to treatment.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.
For control group female nos. 44 and 50, 100 mg/kg female nos. 58 and 60 and 1000 mg/kg female no. 71, the number of pups was slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed between Days 14-16 of lactation.

DEVELOPMENTAL DATA
Developmental parameters were considered unaffected by treatment. No changes in gestation index and duration, parturition, maternal care and early postnatal pup development (postnatal endpoints evaluated are given below) were observed that were considered to be related to treatment.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: Post-implantation survival index was considered unaffected by treatment.
- Early postnatal pup development: Number of dead and living pups at first litter check, postnatal loss, live birth, viability and lactation indices, anogenital distance, areola/nipple retention and sex ratio were unaffected by treatment, and clinical signs, body weight, external macroscopy and serum concentration of the thyroid hormone T4 (PND 4 and 13-15) did not reveal treatment-related findings.
The high postnatal loss and correspondingly low viability index for females at 1000 mg/kg was attributed to 7 missing pups of one litter (no. 71) on lactation Day 4. With exception for one missing pup for litter 76 on lactation Day 3, there was no further postnatal loss among these high dose females. As such, this incidence of postnatal loss was considered to be unrelated to treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of blue spots on the snout, abdomen or neck, scabs on the snout or head, black discolouration of the tail apex, missing tail apex, pallor, absence of milk in the stomach, alopecia on various body parts. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One pup each at 300 and 1000 mg/kg was found dead at first litter check. One control pup, 3 pups at 100 mg/kg and 8 pups at 1000 mg/kg were found dead or missing between lactation Days 0 and 4. Breeding loss (i.e. occurring between lactation Days 5 and 13) consisted of 1 control pup and 1 pup at 1000 mg/kg that were found missing on lactation Days 11 and 5, respectively. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered to have been unaffected by treatment.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in PND 4 pups and PND 13-15 pups were considered unaffected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Incidental macroscopic findings of pups that were found dead consisted of a wound on the left foreleg, and beginning or advanced autolysis. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio: Sex ratio was not affected by treatment.
Anogenital distance: Anogenital distance in male and female pups was not affected by treatment. The statistically significantly higher median anogenital distance of female pups at 100 mg/kg occurred without a dose related-trend and were therefore not considered to be related to treatment.
Areola/nipple retention: Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

other: Sex ratio; Anogenital distance; Areola/nipple retention

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-4370561-AAA (T002326) by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no adverse parental toxicity and no reproduction and developmental toxicity up to 1000 mg/kg.
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.
Therefore, the substance is not classified as a reproductive toxicitant according to the CLP Regulation.