Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-07 to 2016-03-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407
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: EPA OPPTS 870.3550
Deviations:
no
Qualifier:
equivalent or similar to
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. 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-1806792-AAA (T001325)
- Physical state: solid (powder)
- Appearance: brown powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15FB2768
- Expiration date of the lot/batch: 2017-07-01 (retest date)
- Purity test date: 2015-09-04

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. 509922).

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

OTHER SPECIFICS: correction factor: 1.00

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: females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment); males approx. 11 weeks (at start F0-treatment)
- Weight at study initiation: 313-351g (males) and 210-247g (females)
- 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).
- 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: 2016-01-06 (start pretest, females aged approx. 11 weeks); 2016-01-20 (start treatment, males aged approx. 11 weeks); 2016-02-26/27/28/29 and 2016-03-01/02/10/11 (delivery of litters)
To: 2016-02-18 (necropsy males); 2016-03-09/10/11/12/13/14/22/23 (necropsy pups); 2016-03-10/11/12/13/14/15/23/24 (necropsy females)

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 1.00 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): 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 Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating. Detection of mating was not confirmed for animal no. 71 which did deliver live offspring. The mating date of this animal 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 (2016-01-21; Day 2 of treatment), according to a validated method (Test Facility Study No. 509922). 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).
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 for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Once analytical results were approved by the Principal Scientist, the reserve samples were destroyed.
Duration of treatment / exposure:
29 days (males); 50-55, 63 or 64 days (females that delivered); 40-41 days (females that 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 / 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
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. 509919) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, treatment-related effects were confined to hunched posture and piloerection for all females at 1000 mg/kg between Days 3 and 10. No mortality occurred, there were no effects on body weight and food consumption, no macroscopic abnormalities were noted and kidney and liver weights were considered unaffected by treatment. Based on these results, dose levels of 100, 300 and 1000 mg/kg were selected for the main study 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 at 2 hours (±1 hour) 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 (prior to first 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.

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 hindlimb grip strength recorded as the 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 ditribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, 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. 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 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 PND 4; 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 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 14.
- 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 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 that delivered) or on post-coitum day 25 (females that failed to deliver).

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.
- Necropsy was conducted as soon as possibe after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected an d 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) an d 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 forma lin (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), Prepu tial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (F), 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, and females which failed 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 bu lbocavernosus 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), U terus (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, Pros tate, 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 bulbocavern osus 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 5 animals/ sex of Groups 1 and 4; The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Mesenteric lymph node of all selected 5 males and females of Groups 2 and 3, the kidneys of all selected 5 males of Groups 2 and 3, and the thyroid gland of the selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4; The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (two couples at 100 mg/kg (female no. 59 / male no. 19 and female no. 60 / male no. 20) and one couple at 300 mg/kg (female no. 65 / male no. 25).
- 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
- 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 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:
no effects observed
Description (incidence and severity):
No clinical signs or abnormalities during weekly arena observations were noted that were considered to be related to treatment.
No clinical signs were observed, except for one male at 300 mg/kg that showed a wound in the neck area. This 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, this was considered a sign 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:
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:
no effects observed
Description (incidence and severity):
Haematological parameters of treated rats were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, males had statistically significantly higher total bilirubin and bile acid levels (means were approximately 30 and 98% higher than control means, respectively).
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 dose-related trend.

Thyroid hormone analyses: Serum levels of T4 in F0 males were considered to have been unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Motor activity was considered unaffected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Grip strength was similar across the groups for both sexes.
The statistically significant higher grip strength of the hindlegs of males at 300 mg/kg occurred in the absence of a dose-related trend and was considered minor in nature. This variation was therefore considered to be unrelated to treatment.
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):
Males at 300 and 1000 mg/kg showed an increased incidence and severity of hyaline droplet accumulation in the kidneys (up to slight).
One 100 mg/kg female (no. 58) showed bilateral inflammatory cell infiltrate in the thyroid glands (slight or marked). This was regarded as an incidental finding and in absence of a dose-related incidence not considered to be related to treatment.
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.
Most females had regular cycles of 4 days. Extended di-estrus occurred in control female no. 43 with a regular cycle, and female no. 79 at 1000 mg/kg with an irregular cycle. An irregular cycle was also noted for female no. 76 at 1000 mg/kg (with normal litter). Given their incidental nature and absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted up to 1000 mg/kg.
- Mating index: Mating index was not considered affected by treatment. All females showed evidence of mating.
- Fertility index: Fertility index was not considered affected by treatment. A total of two females at 100 mg/kg and one female at 300 mg/kg were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment.
- Number of implantation sites: Number of implantation sites was not considered affected by treatment. For one female at 100 mg/kg (no. 58), the number of pups was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 14 of lactation. 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.

DEVELOPMENTAL DATA
No toxicologically relevant effects on developmental parameters were noted up to 1000 mg/kg. There were two couples (nos. 59/19 and 60/20) treated at 100 mg/kg and one couple (no. 65/25) at 300 mg/kg which had no offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. Based on this and the fact that all couples at 1000 mg/kg had offspring, these three infertile couples at 100 or 300 mg/kg were considered to be unrelated to the treatment.
- Gestation index and duration: Gestation index and duration of gestation were not considered to be affected by 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: The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The mean post-implantation survival index appeared slightly lower at 1000 mg/kg compared to controls. However, there were no treatment-related changes in either the number of offspring born or the number of implantation sites. Therefore, this apparent difference was not considered to be related to treatment.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. One pup at 100 mg/kg (litter no. 57), one pup at 300 mg/kg (litter no. 69) and one pup at 1000 mg/kg (litter no. 74) 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 considered affected by treatment. One pup of the control group (litter 43), one pup at 100 mg/kg (litter no. 55) and two pups at 300 mg/kg (litter nos. 61 and 69) were found missing between Days 1 and 4 of lactation. These pups 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 considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Details on results (P0)

Parental results:
A non-adverse increase in incidence and severity of hyaline droplet accumulation in the kidneys (up to slight) was recorded for males at 300 and 1000 mg/kg. This likely represents alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation. This finding was not accompanied by any test item-related degenerative change and was therefore considered to be a non-adverse finding. This male rat specific protein is not present in female rats nor in higher mammals, including man.
Higher total bilirubin and bile acid levels were recorded for males at 1000 mg/kg. In absence of any supportive morphological lesions, and given the minor magnitude of change of these parameters, these were not considered adverse in nature.

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (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 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% and 115%). No test item was detected in the group 1 formulations.
- homogeneity: the formulations of group 2 and group 4 were homogeneous (i.e. coefficient of variation <=10%).

Effect levels (P0)

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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:
other: No adverse effects were noted in any of the parameters examined in this study.
Remarks on result:
not determinable due to absence of adverse toxic effects
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:
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)

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.
Incidental clinical symptoms of pups consisted of pale, lean or cold appearance, a blue spot on the tail or snout, scabs or a wound on the snout, wound on the abdomen, discolouration of the abdomen, alopecia on the back, insufficient milk in the stomach and a missing tail apex. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Note: Only days on which clinical signs were present between first and last litter check are presented in the correspondng table of the study report.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
One pup of the control group (litter 43), one pup at 100 mg/kg (litter no. 55) and two pups at 300 mg/kg (litter nos. 61 and 69) were found missing between Days 1 and 4 of lactation. These pups 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):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Incidental macroscopic findings of pups that were found dead included autolysis and absence of milk in the stomach. Incidental macroscopic findings among surviving pups included alopecia on the back and a missing tail apex. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
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. The statistically significantly higher median anogenital distance of male and female pups at 100 and 300 mg/kg occurred without a dose related-trend. As such, these variations were 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

Details on results (F1)

Developmental results
- No developmental toxicity was observed up to the highest dose level tested (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 (PND1), areola/nipple retention (PND13 males), T4 thyroid hormone levels (PND13-15) and macroscopy.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
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)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with JNJ-1806792-AAA (T001325) 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.