4-[(6-chloro-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethylbenzonitrile

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-02 to 2017-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, OPPTS 870.3550- Reproduction/Developmental Toxicity Screening test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-({6-chloro-2-[(4-cyanophenyl)amino]pyrimidin-4-yl}oxy)-3,5-dimethylbenzonitrile

EC Number:

928-806-6

Cas Number:

1070377-34-2

Molecular formula:

C20H14ClN5O

IUPAC Name:

4-({6-chloro-2-[(4-cyanophenyl)amino]pyrimidin-4-yl}oxy)-3,5-dimethylbenzonitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): T002615
- Physical state: solid
- Appearance white solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16CC0982
- Expiration date of the lot/batch: 2019-03-23
- Purity test date: 2016-03-23

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature and 14 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL as part of the analytical method development and validation study (Test Facility Study No. 514641).

FORM AS APPLIED IN THE TEST (if different from that of starting mateiral): suspension

OTHER SPECIFICS: correction factor is 1

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has 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: Males approx. 10 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: Males: 267-301 g; Females: 201-249 g
- 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-one basis 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).
- 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: 2016-12-07 (start pretest); 2016-12-21 (start treatment); 2017-01-27/28/29/30/31 (delivery of litters)
To: 2017-01-19 (necropsy males); 2017-02-08/09/12/13/14 (necropsy pups); 2017-02-09/10/13/14/15 (necropsy females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400, specific gravity 1.125

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared weekly and were homogenized to a visually acceptable level. Formulations were filled out in daily portions and kept refrigerated protected from light until dosing. Adjustment was made for specific gravity of the vehicle. 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), 10 mg/mL (group 2), 40 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: 1:1 basis
- 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 Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
- 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 (04 January 2017, Day 15 of treatment) according to a validated method (Test Facility Study No. 514641). Duplicate samples were collected.
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 ±10.00% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%.

Duration of treatment / exposure:

29 days (males), 50-56 days (females that delivered), 41-43 days (nonpregnant females). Female nos. 45, 57 and 80 (Groups 1, 2 and 4, respectively) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation. Pups were not dosed directly but were potentially 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 group)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

200 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 selected based on a 28-day repeated dose toxicity study (data on file at Sponsor site), in which test item was administrated daily by oral gavage to Wistar rats at dose levels of 50, 200 and 1000 mg/kg. No test item-related clinical signs or differences in organ and body weights, food consumption, hematology and clinical biochemistry parameters, or macroscopic findings were noted by treatment up to 1000 mg/kg. Microscopic examination revealed minimal to slight signs of topical irritative effects in the stomach of animals dosed at 200 and 1000 mg/kg. These findings consisted of minimal to slight grades of hyperkeratosis, epithelial hyperplasia and basal cell hyperplasia of the limiting ridge. There was also a tendency to increased submucosal inflammatory infiltrates. These findings were considered to be non-adverse. Hence, the no observed effect level (NOEL) was established as being 50 mg/kg, and the no observed adverse effect level (NOAEL) as being 1000 mg/kg. Dose levels of 50, 200 and 1000 mg/kg 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) 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.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Limited to thyroid hormone analysis
- End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, non-pregnant females and all males after at least 4 weeks of treatment.
- Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of approximately 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retro-orbital sinus and collected into serum tubes.
- Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid stimulating hormone (TSH).
- Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).

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. For female no. 79 with no evidence of copulation (mating overlooked), vaginal lavage continued until conformation of pregnancy by palpation. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis: testis weight

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 post-natal day 1 (= day the litter was found completed)
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1:
- Mortality/viability: The numbers of live and dead pups were determined on PND1 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 were reported in the respective table of the study report.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND1 and 4.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND13, all males in each litter were examined for the number of areola/nipples.


GROSS EXAMINATION OF DEAD PUPS:
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals, on PND 14-16 (females which delivered), on days 25 or 26 (females which failed to deliver, with evidence of mating).

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 examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- For females which failed to deliver a complete nest, uterine contents (i.e. any fetuses, placenta and implantation sites) were fixed (if applicable), but was not examined histopathologically.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Cervix (F), Clitoral gland (F), Coagulation gland (M), (Cowper’s gland) (M), Epididymides (M), Mammary gland area (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Ovaries (F), Preputial gland (M), Pituitary gland (M/F), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (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.
- Reproductive organs were only examined by the pathologist for males that failed to sire and all females that failed to deliver healthy pups. Female mammary gland area was only examined by the pathologist for females with total litter loss.

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from all animals on the scheduled day of necropsy: Epididymides, Testes, Thyroid, Prostate gland, Seminal vesicles (including coagulation gland).

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 ovaries, testes, epididymides and thyroids of the animals of Groups 1 and 4; Additional slides of the testes of all males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- All abnormalities were described and included in the study 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
- On PND 4 (at culling), 2 pups per litter were killed by decapitation for blood collection.
- On PND 13-15, 2 pups per litter were anaesthetized using isoflurane for blood collection by aorta puncture followed by exsanguination.
- All remaining pups (PND 13-15) were sacrificed using Euthasol 20% by intraperitoneal injection.

GROSS NECROPSY
- All pups were sexed both externally and internally. 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, was preserved in 10% buffered formalin

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

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 mated) 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 implantation 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

Percentage of live males at first litter check (%)

Percentage of live females at first litter check (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation seen after dosing among animals of all groups, including the control group, during the dosing 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.

Incidental findings that were noted included swellings, scabs, alopecia and rales. As these findings were observed for single animals and 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, these were not considered to be signs of 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):

Body weights were slightly higher for females dosed at 1000 mg/kg during the post-coitum phase from Day 11 onwards. A slight increase in body weight was not considered to be adverse and as the changes were not statistically significant and remained within the range considered normal for rats of this age and strain, they were not considered to be toxicologically relevant. The statistically significantly lower body weight gain on Day 4 of lactation for females dosed at 200 mg/kg compared to controls, was not considered to be treatment related as this occurred in the absence of a dose-response relationship. One single male at 1000 mg/kg showed a remarkably lower body weight on Day 15 of the mating phase compared to Day 8 (relative difference of 7%). Terminal body weight recorded the next day after fasting overnight was increased by 8% compared to Day 15, which is in contrast with all other males of this study that lost weight during the fasting period prior to necropsy. In absence of any clinical signs, macroscopic findings or organ weight effects, the low body weight on Day 15 was considered to be due to a weighing error rather than body weight loss.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption before and after allowance for body weight was increased for females at 1000 mg/kg over Days 14-17 post-coitum. The differences from controls were 14% (absolute) and 12% (relative), reaching statistical significance for the absolute difference. This was related to the slightly increased body weights observed in this period. A slight increase in food consumption was not considered to be adverse and as it occurred on a single occasion, this was not considered to be toxicologically relevant.

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 levels of T4 in F0 males were not considered to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Recorded histologic changes were within the range of background pathology encountered in rats of this age and strain. 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:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment. All females of the treatment groups had regular cycles of 4 days. Irregular cycles occurred in three females of the control group. During the pretest phase, irregular cycles were noted for two females, followed by regular cycles during the premating phase. One female had two irregular cycles in the beginning of the premating phase followed by two regular cycles. Normal litters were observed for two females. As this was observed in the control group only, a relationship to test item can be excluded. One female was acyclic, as she showed three regular cycles of 4 days each followed by a period of 12 days without estrus. As this occurred in a single female in absence of an apparent correlation to pregnancy status (a normal litter was observed), this was not considered to be treatment related.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA
- Reproductive performance: There were 2/10 couples of the control group, 2/10 couples of the 50 mg/kg group, 3/10 couples of the 200 mg/kg group and 1/10 couple of the 1000 mg/kg group with no offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
- Mating index, Precoital time, Number of implantation sites and Fertility index were not affected by treatment. A total of eight females across the groups were not pregnant. Since these cases of non-pregnancy occurred in the control group as well, showed no dose-related incidence, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

DEVELOPMENTAL DATA
- Gestation index and duration: Gestation index and duration of gestation were not affected by treatment. All pregnant females delivered live offspring after 21 or 22 days of gestation.
- 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: The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The post-implantation survival indices across the groups ranged from 88 to 95%. For one female (Group 4), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 16 days of lactation. No toxicological relevance was attached to this finding in the current study.
- Litter size: Litter size was not considered affected by treatment. A statistically significantly higher mean number of living pups per litter was recorded at 1000 mg/kg (13.0 vs. 10.6 in the control group). As all values remained within the range considered normal for rats of this strain and age, this was not considered to be toxicologically relevant.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. There were no dead pups at first litter check noted in any of the groups.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not affected by treatment. One pup at 50 mg/kg and one pup at 1000 mg/kg were missing on PND 3 or 4, which were most likely cannibalised. No toxicological relevance was attributed to these 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.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Details on results (P0)

Parental results:
No parental toxicity was observed up to 1000 mg/kg. No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, T4 thyroid hormone analysis (males), macroscopic examination, organ weights and microscopic examination).

Reproductive results:
No reproduction toxicity was observed up to 1000 mg/kg. No treatment-related or toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Analysis of dose preparations:
- Accuracy of preparation: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). A small response at the retention time of the test item was observed in one of the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test item was detected.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes were noted in any of the parameters examined in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes were noted in any of the parameters examined in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not affected by treatment. One pup at 50 mg/kg and one pup at 1000 mg/kg were missing on PND 3 or 4, which were most likely cannibalised. No toxicological relevance was attributed to these 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 not considered to be affected 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 male and female PND 13-15 pups were not considered to be affected 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):

Macroscopic observations at necropsy did not reveal macroscopic findings in any of the pups.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Sex ratio: The statistically significant differences in sex ratio at 50 and 200 mg/kg compared to controls were considered to have arisen as a result of the atypical sex distribution in the control group (61% males over 39% females).
- Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
- Areola/nipple retention: Treatment up to 1000 mg/kg 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

Details on results (F1)

Developmental results
- Note: the evaluation of the developmental effects at 200 mg/kg was limited to seven pregnant females in this group, which is marginally lower than the aim for eight pregnant females per group. Sufficient pregnant females were available in the other groups, including the high dose group at 1000 mg/kg, for a proper toxicological evaluation of the developmental toxicity.
- No developmental toxicity was observed up to 1000 mg/kg.
- No treatment-related or toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone analysis (PND13-15) and macroscopy).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes were noted in any of the parameters examined in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-4098757-AAA (T002615) by oral gavage in male and female Wistar Han rats at dose levels of 50, 200 and 1000 mg/kg revealed no parental, reproduction or developmental toxicity up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg
Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.