Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-29 to 2016-11-16
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Dev elopmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA, OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA, OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-4754724-AAA (T002488)
- Physical state: solid
- Appearance: light brown powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15FC2164
- Expiration date of the lot/batch: 2017-06-23 (retest date)

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. 512673).

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

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 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. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 301-338 g (males) and 209-238 g (females)
- Fasting period before study: no
- Housing: Pretest: Females were housed in groups of a maximum 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 (MI II 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: To: 2016-07-29 To: 2016-11-16

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. A correction was made for the purity 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.
- Concentration in vehicle: 0 mg/mL (group 1); 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): I15FC2164
- 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, one female was cohabitated with one male of the same treatment group, avoiding sibling mating, 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 (27 September 2016, Day 7 of treatment) according to a validated method (Test Facility Study No. 512673). Duplicate samples were collected. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). 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 90.00-110.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Stability of formulations was determined as part of the analytical method development and validation study (Test Facility Study No. 512673).
Duration of treatment / exposure:
28 days (males); 50-56 days (females that delivered); 44 days (female with total litter loss); 41 or 42 (females which failed to deliver). 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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 514054) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, no clinical signs (indicative of toxicity) were observed. No mortality occurred, clinical appearance was considered normal, there were no effects on body weight and food consumption, no macroscopic abnormalities were noted, and kidney and liver weights were considered normal. Based on these results, dose levels of 100, 300 and 1000 mg/kg were selected for the main study. Therefore, clinical observations in the main study were conducted after dosing, and functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- 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, at least immediately after dosing. 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.

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 and calculated body weight gain were 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 on PND 1, 4, 7 and 13.
Both absolute food consumption and food consumption relative to body weight were reported.

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.

FUNCTIONAL OBSERVATIONS
- Time schedule: between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable)
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity

HAEMATOLOGY
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K3-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin, activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin
- parameters assessed: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- thyroid hormone analysis
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. 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)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND4; blood samples were collected from two of the surplus pups; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- 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 are presented in the respective table in 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 14.
- 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:
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 necropsied on the same day.

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), or on days post-coitum 25-27 (females which failed to deliver, with evidence of mating), or within 24 hours of litter loss (1 female with total litter loss), or in extremis (1 female was euthanized for humane reasons)

GROSS NECROPSY
- All animals surviving to the end of the observation period were deeply anaesthetized using i soflurane 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.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): 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 (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 (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 (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/ F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (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 on the scheduled day of necropsy: 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 of all males that failed to sire to examine staging of spermatogenesis; The preserved organs and tissues of one female at 1000 mg/kg (no. 72) that was euthanized in extremis; The mammary gland of one female at 100 mg/kg (no. 54) with total litter loss; All gross lesions of all animals (all dose groups); Thyroid gland of all selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in Group 4; 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
- Pups, younger than 7 days were killed by decapitation
- The F1 offspring were sacrificed at PND 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 (using Euthasol 20% by intraperitoneal injection).

GROSS NECROPSY
- All pups were sexed by both external and 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, 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 analyse 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 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs of toxicity were noted during the observation period.
- The only clinical sign noted was alopecia, observed for single females among the control and 100 mg/kg groups. As this finding 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, and absence of a dose-related incidence across the groups, this finding was not considered to be related to treatment.
- During the weekly arena observations, no additional treatment-related clinical signs were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- No mortality occurred during the study period that was considered to be related to treatment with the test item.
- One female at 1000 mg/kg was sacrificed in extremis on Day 9 of the premating period for humane reasons. Oesophageal perforation noted at necropsy indicated that this moribundity was due to a gavage accident and not related to treatment with the test item. Other related necropsy findings for this animal consisted of a gray-white discoloured and hard granulated oesophagus, hard, granulated gray-white content in the right axillary region and a gelatinous throat region. These findings correlated to microscopic findings, i.e. inflammation/hemorrhage/necrosis of the oesophagus and presence of amorphous material in the tissue surrounding the oesophagus and amorphous material in the subcutis of the axillary region.
- One female at 100 mg/kg was sacrificed on Day 4 of the lactation phase due to total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
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:
effects observed, treatment-related
Description (incidence and severity):
- At 300 mg/kg and 1000 mg/kg, a statistically significant higher mean activated partial thromboplastin time (APPT) was recorded in females. Means were approximately 26% and 30% higher than controls, respectively.
- All other haematological parameters of treated rats were not considered to be affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- At 1000 mg/kg, inorganic phosphate was statistically significantly higher in males. The mean was approximately 16% higher than the control mean. Any other statistically significant changes in clinical biochemistry parameters were not considered to be related to treatment as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.
- Thyroid hormone analyses Serum levels of total T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar 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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A slightly increased incidence and/or severity of thyroid follicular cell hypertrophy, up to a slight degree, was present in males treated at 1000 mg/kg and in females treated at 300 and 1000 mg/kg. The remainder of the recorded microscopic findings 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 considered to have been affected by treatment.
- All females surviving to planned necropsy had regular cycles. An irregular cycle was only noted for female no. 72 at 1000 mg/kg, which was sacrificed on Day 9 of the premating period. This female had 4 regular cycles followed by two cycles of two days each.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal (at least unilateral) for all males examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTION DATA
- There was one control couple, one couple at 100 mg/kg, one couple at 300 mg/kg and two couples at 1000 mg/kg without offspring. No abnormalities were seen in the reproductive organs of any of these animals which could account for their lack of offspring.
- One female at 100 mg/kg had a total litter loss. No abnormalities were seen which could explain the total litter loss. One female at 100 mg/kg/day had no offspring but had a mummified fetus in the left horn of the uterus. No abnormalities were seen in the reproductive organs of these couples.
- One female at 1000 mg/kg was sacrificed in extremis during the premating period. No abnormalities were seen in the reproductive organs of this female. As a result, reproduction and developmental data are available for only 9 females in the high dose group.
- Mating index: Mating index was not considered to be affected by treatment. All females showed evidence of mating.
- Fertility index: Fertility index was not considered to be affected by treatment. A total of five females (one control female, one female each at 100 and 300 mg/kg, and two females at 1000 mg/kg) were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.
- Number of implantation sites: Number of implantations were not considered to have been affected by treatment. At 1000 mg/kg, a statistically significantly lower number of implantation sites was recorded. However, it should be noted that due to the lower number of pregnant females at 1000 mg/kg, the lower number of implantation sites for one female had a more pronounced effect on the mean number of implantation sites at this dose. The number of implantation sites for the other females of this dose group was normal for rats of this age and strain, and within the range encountered for the control group. The number of implantations at 100 and 300 mg/kg was considered unaffected by treatment. The mean number of implantations at 100 mg/kg was even lower than recorded at 1000 mg/kg (but not statistically significantly different to the control group), which was due to two lower values for two females in the group. As this appeared not dose-related, this was considered unrelated to treatment.

DEVELOPMENTAL DATA
- Gestation index and duration: Gestation index and duration of gestation were not considered to be affected by treatment. One pregnant female at 100 mg/kg delivered no offspring, but had one mummified fetus in the uterus. This incidence showed no relationship to the dose, and was therefore
considered unrelated to treatment.
- Litter size: Litter size was not considered affected by treatment. A statistically significantly lower mean number of living pups was recorded at 1000 mg/kg. However, it should be noted that due to the lower number of pregnant females at 1000 mg/kg, the lower litter size of one female had a more pronounced effect on the mean litter size at this dose. Also, a dose-related response was absent, the mean remained within the range considered normal for rats of this age and strain and (with exception of female no. 78) within the range encountered for the control group.
- 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.
- 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. One pup of the control group and two pups at 300 mg/kg were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- 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 of the control group and one pup at 100 mg/kg were missing on Day 4. Pups missing 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 adverse effects on parental parameters were noted up to 1000 mg/kg. Microscopic examination of the thyroids revealed a slightly increased incidence and/or severity of follicular cell hypertrophy in males at 1000 mg/kg and in females at 300 and 1000 mg/kg. This was regarded to be an adaptive change and considered to be non-adverse at the incidences and severities recorded. A few changes in clinical pathology parameters were noted at 300 and 1000 mg/kg that were potentially related to treatment. These were not considered adverse in absence of any morphological correlates and since they essentially remained within the range considered normal for rats of this age and strain. These changes consisted of higher activated partial thromboplastin time in females at 300 and 1000 mg/kg and higher inorganic phosphate in males at 1000 mg/kg. No treatment-related effects on mortality, clinical appearance, functional observations, body weight, food consumption, organ weights and macroscopic appearance were observed.

Reproductive results:
No reproduction toxicity was observed up to 1000 mg/kg. No treatment-related 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 dose preparations: 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 90.00% and 110.00%). No test item was detected in the Group 1 formulation.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels (P0)

open allclose all
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.
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.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs occurred among pups that were considered to be related to treatment.
- For the pup of one female (100 mg/kg) who was missing on Day 4, absence of milk in the stomach and lean appearance were noted on Day 3. The nature and incidence of these and other clinical signs of pups 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:
mortality observed, non-treatment-related
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 of the control group and one pup at 100 mg/kg were missing on Day 4. Pups missing 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:
effects observed, non-treatment-related
Description (incidence and severity):
- Pup body weights were not considered to have been affected by treatment.
- At 1000 mg/kg, slightly higher mean pup body weights were recorded throughout lactation, achieving a level of statistical significance for female pups on Days 1 and 4 of lactation. This was attributed to a higher mean weight gain of the pups of one female which also had the lowest litter size of this group. Mean pup body weights of other litters of this group were within the range encountered for the control group. At 100 and 300 mg/kg, mean pup body weights remained similar to the control group.
- At 100 and 300 mg/kg, mean pup body weights remained similar to the control group.
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:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. Except for one control pup found dead at first litter check showing absence of milk in the stomach, no other macroscopic findings were noted.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment. At 100 mg/kg, anogenital distance normalized for body weight was statistically significantly higher than controls for male pups. Since this occurred without a dose related-trend, this variation was not considered to be related to 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 DATA
- Gestation index and duration: Gestation index and duration of gestation were not considered to be affected by treatment. One pregnant female at 100 mg/kg delivered no offspring, but had one mummified fetus in the uterus. This incidence showed no relationship to the dose, and was therefore
considered unrelated to treatment.
- Litter size: Litter size was not considered affected by treatment. A statistically significantly lower mean number of living pups was recorded at 1000 mg/kg. However, it should be noted that due to the lower number of pregnant females at 1000 mg/kg, the lower litter size of one female had a more pronounced effect on the mean litter size at this dose. Also, a dose-related response was absent, the mean remained within the range considered normal for rats of this age and strain and (with exception of female no. 78) within the range encountered for the control group.
- 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.
- 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. One pup of the control group and two pups at 300 mg/kg were found dead at first litter check. No toxicological relevance was attributed to these dead 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.


Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Developmental results:
- No developmental toxicity was observed up to 1000 mg/kg.
- No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, 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 levels (PND 13-15) 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.

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-4754724-AAA (T002488) by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no adverse parental, reproduction and 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.