Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-16 to 2017-01-12
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/Developmental Toxicity Screening Test)
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:
liquid
Details on test material:
- Name of test material (as cited in study reports): JNJ-118872-AAA (T000749)
- Physical state: liquid
- Appearance: Light yellow, light brown liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A16EB1463
- Expiration date of the lot/batch: 2017-09-12 (retest date)
- Purity test date: Certificate of Analysis was approved on 2017-06-29

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 for at least 3 hours at room temperature and 26 days in the refrigerator (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 512824).

FORM AS APPLIED IN THE TEST (if different from that of starting material): Light yellow solutions

OTHER SPECIFICS: correction factor is 1.04

Test animals

Species:
rat
Strain:
Wistar
Remarks:
rat
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: Crl:WI(Han) (outbred, SPF-Quality) from Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 10-12 weeks (at start F0-treatment); females approx. 10-12 weeks (at start pretest) and approx. 12-14 weeks (at start F0-treatment).
- Weight at study initiation: 293-296 g (males) and 220-225 g (females)
- Fasting period before study: The animals were deprived of food overnight (with a maximum of 24 hours, except for a number of non-selected males before blood sampling, but water was available.
- 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400, specific gravity 1.125
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 3 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test item. A correction was made for the purity/composition of the test item. A correction factor of 1.04 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:
From 2016-11-18 to 2016-11-23: 0 mg/mL (group 1), 11 mg/mL (group 2), 33 mg/mL (group 3), 100 mg/mL (group 4)
From 2016-11-24 to study end: 0 mg/mL (group 1), 2.2 mg/mL (group 2), 6.6 mg/mL (group 3)
- Amount of vehicle (if gavage): 1 mL/kg body weight (2016-11-18 to 2016-11-23); 5 mL/kg body weight (2016-11-24 to study end)
- 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.
- 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 (08 December 2016, Day 15 of treatment) according to the most recently approved validated method (Test Facility Study No. 512824, method approved on 01 December 2016). One set of duplicate samples were collected and analysed. 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%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%. Stability of formulations over 3 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 512824).
Duration of treatment / exposure:
6 days (Group 4); 31 days (Group 1-3 males); 52-55 days (Group 1-3 females that delivered); 41 days (female that 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 other: mg eq/kg/day
Remarks:
Group 1 (control group)
Dose / conc.:
11 other: mg eq/kg/day
Remarks:
Group 2
Dose / conc.:
33 other: mg eq/kg/day
Remarks:
Group 3
Dose / conc.:
100 other: mg eq/kg/day
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. 512824) in which animals were dosed for 10 days at 30, 100, 300 mg eq/kg/day. In summary, all animals in the 300 mg eq/kg group were sacrificed early on Day 6; no mortality was observed in the 100 mg eq/kg and 30 mg eq/kg groups. All animals in the 300 and 100 mg eq/kg groups displayed piloerection, hunched posture and salivation (mostly slight); lethargy was observed in all high dose animals and 1/3 mid-dose animals; ptosis was observed in all high dose animals. The only clinical sign in the 30 mg eq/kg group was slight salivation. All animals in the 300 mg eq/ kg group exhibited 5-8% loss of initial body weight, while all animals at 30 and 100 mg eq/kg gained less weight than expected. Food consumption was markedly reduced in the 300 mg eq/kg group, while it was normal at 30 and 100 mg eq/kg. Upon macroscopic examination, abnormalities in forestomach were observed in 1/3 animals with many reddish foci and irregular surface; no macroscopic abnormalities were observed at 30 and 100 mg eq/kg. Liver and kidney weights were normal for all animals in all dose groups. Based on the results of this range finding study, dose levels for the main study were selected to be 0, 11, 33 and 100 mg eq/kg. Clinical signs of toxicity at the highest dose level selected for the main study (100 mg eq/kg) generally started immediately after dosing and were still present about 3 hours after dosing (piloerection and hunched posture). Based on this finding, clinical observations (clinical signs and arena) and functional observation tests in the main study were started at least one hour (± 30 minutes) after dosing.

- 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. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

Note: Due to the early termination of Group 4 animals treated at 100 mg eq/kg, they were not subjected to any of the following investigations (except for body weight, post-mortem examination and limited estrous cycle evaluation).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals and were started at least at 1 hour (± 30 min) 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 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.


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.

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: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of 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 retroorbital sinus and collected into tubes with K3-EDTA for hematology parameters and with citrate for clotting tests.
- Parameters: 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 time, activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- 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 anesthesia 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 for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- Parameters: 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

FUNCTIONAL OBSERVATIONS:
- Time schedule: These tests were performed after observation for clinical signs (incl. arena observation, if applicable) 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.
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hindlimb grip strength recorded as the mean of three measurements per animal, locomotor activity
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 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 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 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.

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).
- Female animals: All surviving animals, on PND 14-16 (females that delivered) or on post-coitum day 26 (females that failed to deliver, with evidence of mating).
Group 4 animals - Study Day 7 - sacrificed for humane reasons.

GROSS PATHOLOGY: Yes
- 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 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 (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 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, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid (including parathyroid if detectable), Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Testes, Thyroid

HISTOPATHOLOGY: Yes
- 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 3 and all animals of Group 4.· Additional slides of the testes of the selected 5 males of Groups 1 and 3, all males of Group 4, and male no. 28 (Group 3) that failed to sire to examine staging of spermatogenesis. All gross lesions of all animals (all dose groups); Stomach and esophagus of all selected 5 animals of Group 2 (males and females), based on treatment-related changes in this organ in Groups 3 and 4. The reproductive organs of male no.28 that failed to sire and female no.68 that failed to deliver healthy pups: 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.

OTHER
Note: due to their early sacrifice animals treated at 100 mg eq/kg were not subjected to functional observations, haematology or clinical biochemistry examinations, nor was data collected for food consumption, organ weights or reproductive performance and developmental data.
Postmortem examinations (offspring):
SACRIFICE
- Pups, younger than 7 days were euthanized by decapitation.
- All remaining pups (PND 7-15), except those used for blood sampling on PND 13-15, were sacrificed using Euthasol by intraperitoneal (ip) injection.

GROSS NECROPSY
- All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, 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 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.
- 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 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
Percentage live males at First Litter Check (%) = (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%) = (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) 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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical signs of toxicity were noted at 100 mg eq/kg in both sexes. Findings in females at 100 mg eq/kg included hunched posture, piloerection and a lean appearance in up to all females, starting on Day 4, slight lethargy in half of the females at Day 6, and rales in a few females starting on Day 2. Males at 100 mg eq/kg were less severely affected. They showed hunched posture from Day 5 and one male had a lean appearance.

At 33 mg eq/kg, one female showed hunched posture and piloerection between Days 5-16, and diarrhoea on Days 8-10.

Salivation was noted after dosing at all dose levels with a dose-related trend in frequency and time of onset. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its severity and may be related to the irritancy of the test item.
No additional clinical signs of toxicity were noted during the weekly arena observations.

The only other findings were alopecia in two females (treated at 11 or 33 mg eq/kg) and single occasions of piloerection in a single female at 11 mg eq/kg and among a few females at 33 mg eq/kg. These findings were considered to be incidental and not related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female treated at 100 mg eq/kg was found dead on Day 7. Prior to death this animal showed clinical signs of toxicity including piloerection, hunched posture, lethargy, rales and a lean appearance. Similar signs of toxicity were noted in the surviving animals of this group, particularly in females. Moreover, these animals showed significant body weight loss (on average 10% or 15% of the initial weight for males and females, respectively). Therefore, all remaining animals treated at 100 mg eq/kg were sacrificed for humane reasons on the same day.

Erosion/ulceration in the esophagus and/or forestomach were considered to be the main contributors responsible for the moribund condition or death of these rats. Main test item related macroscopic findings were present in the esophagus in the form of thickened wall and grey white contents (microsco pic correlates erosion/ulceration squamous epithelium, hypertrophy squamous epithelium and/or lymphogranulocytic cell infiltrate), in the forestomach in the form of irregular surface (microscopic correlate hyperplasia squamous cell) and distended with gas and mucous/gelatinous/foamy contents (no microscopic correlate), and in various parts of the intestines in the form of distended with gas and gelatinous/foamy contents (no microscopic correlate). The macroscopic and microscopic changes in the esophagus and forestomach indicated irritancy of the test item.

In addition, one control male was sacrificed in extremis on Day 19 when it showed piloerection, a pale appearance, red discolouration of the penis, and a hardened abdomen with a nodule. The large malignant nephroblastoma in the right kidney was regarded as the main cause of moribundity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males and females treated at 100 mg eq/kg showed significant weight loss between Days 1-7 (on average 10 and 15%, respectively). Reduced mean body weight gain was observed in females at 33 mg eq/kg over the premating period, achieving levels of statistical significance on Day 8 and at start of the mating period when compared to controls. However, the reduction was mainly the results of marked body weight loss in one female . From start of mating onwards, body weight gain at 33 mg eq/kg was normal and body weights of these females were within the same range as controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption before or after allowance for body weight were noted for males and females treated at 11 and 33 mg eq/kg in comparison with controls.

No data for food consumption of Group 4 are available, because these animals were sacrificed within one week after start of treatment.
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):
Statistically significant differences in haematological parameters were limited to a lower percentage of neutrophils at 33 mg eq/kg in males. The mean value at 33 mg eq/kg was 35% lower than the concurrent control value and slightly below the historical control range. A concurrent increase in lymphocytes was observed in these male, not reaching a level of statistical significance but slightly above the historical control range.

Animals treated at 100 mg eq/kg were not subjected to haematology examinations due to their early sacrifice.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of rats treated up to 33 mg eq/kg. Isolated statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the lack of a dose-related response and/or slight magnitude of the difference from controls (values in treated rats remained within normal limits).

Thyroid hormone analyses: Serum levels of T4 in F0 males were not affected by treatment.

Animals treated at 100 mg eq/kg were not subjected to clinical biochemistry examinations due to their early sacrifice.
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 examined animals. Grip strength was not affected by treatment.
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.

Animals treated at 100 mg eq/kg were not subjected to functional observations due to their early sacrifice.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in organ weights up to 33 mg eq/kg. Organs of animals treated at 100 mg eq/kg were not weighed due to the early sacrifice of these animals. At all doses liver and kidney weights were considered to be normal.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals treated at 100 mg eq/kg (found dead or sacrificed moribund on Day 7): Main test item-related macroscopic findings were present in the esophagus (thickened wall and grey white contents), stomach (irregular surface of the forestomach and distended with gas and mucous/gelatinous/foamy contents) and various parts of the intestines (distended with gas and gelatinous/foamy contents).

Controls and animals treated at 11 or 33 mg eq/kg: There were no treatment-related gross observations up to 33 mg eq/kg. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment. In one control male that was sacrificed in extremis on day 19, an enlarged right kidney and an enlarged spleen were observed at necropsy. Histopathology revealed a large malignant nephroblastoma in the right kidney. This type of finding in a control animal occurred spontaneously and was considered non-study related, incidental finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals treated at 100 mg eq/kg (found dead or sacrificed moribund on Day 7): The test item-related findings in the stomach of males and females treated at 100 mg eq/kg consisted of lymphogranulocytic inflammation forestomach, hyperplasia squamous cell and ulcer forestomach, all up to moderate degree. The test item-related findings in the esophagus of males and females treated at 100 mg eq/kg consisted of erosion/ulceration squamous epithelium (up to massive degree), hypertrophy squamous epithelium (up to moderate degree) and lymphogranulocytic inflammatory cell infiltrate (up to marked degree).

There was a test item-related finding in the stomach of females treated at 33 mg eq/kg, consisting of hyperplasia squamous cell at minimal degree. There were no other test item-related histologic changes. 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 affected by treatment up to 33 mg eq/kg.
Most females had regular cycles of 4 days. One female at 33 mg eq/kg had an irregular cycle during the premating period, which coincided with the occurrence of significant weight loss and clinical signs of toxicity in this female. The irregular cycle might be the result of stress during this period. Since no effects on reproduction were observed in the female, it delivered a normal litter, the irregular cycle was considered not directly related to treatment.
For females treated at 100 mg eq/kg, the available estrous cycle data were insufficient for proper evaluation.
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
- Mating index: Mating index was not affected by treatment. All females treated up to 33 mg eq/kg showed evidence of mating.
- Precoital time: Precoital time was not affected by treatment up to 33 mg eq/kg.
- Number of implantation sites: Number of implantation sites was not affected by treatment up to 33 mg eq/kg.
- Fertility index: Fertility index was not affected by treatment up to 33 mg eq/kg. Except for one female at 33 mg eq/kg, all mated females were pregnant. This incidental non-pregnancy without related histopathology changes in reproductive organs was considered to be unrelated to treatment.

DEVELOPMENTAL DATA
- Gestation index and duration: Gestation index and duration of gestation were not affected by treatment up to 33 mg eq/kg.
- 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: A relatively low post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) of 82% was observed at 33 mg eq/kg, compared with 90% and 94% in the controls and 11 mg eq/kg groups, respectively. In the ten control group females, the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) varied between 0 and 3. In the 11 mg eq/kg treated group, there were nine females with 0-3 and one female with 4 unaccounted for sites. In the 33 mg eq/kg treated group, there were six females with 0-3 and two females with 4 and one female with 6 unaccounted for sites. These results indicated a trend of increased number of females with a higher number of unaccounted for sites, outside the range of that observed in controls, at higher doses. Although the mean number of implantation sites and living pups at 33 mg eq/kg were within normal limits, a relatively low post-implantation survival index of 82% was calculated for this group. For females no. 52 (11 mg eq/kg) and 69 (33 mg eq/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is occasionally observed and is caused by normal resorption of these areas during (the 14 days of) lactation. No toxicological relevance was attached to this finding in this study.
- Litter size: Litter size was not affected by treatment up to 33 mg eq/kg.
- Live birth index: Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment up to 33 mg eq/kg. The live birth indices across the groups ranged from 98 to 100%. One female at 11 mg eq/kg had two dead pups at first litter check. This incidental pup mortality was within normal limits and unrelated to treatment with the test item.
- Viability index: Viability index was not affected by treatment up to 33 mg eq/kg. The viability indices the across the groups ranged from 95 to 100%. Six pups at 11 mg eq/kg (of three litters) and one pup at 33 mg eq/kg went missing (presumably cannibalized) or were found dead between PND 2-4. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
- Lactation index: Lactation index was not affected by treatment up to 33 mg eq/kg. The lactation index was 99% in the control group (one pup was sacrificed for humane reasons on PND 11) and 100% in the other groups.

Details on results (P0)

Parental results:
Significant general toxicity was observed at 100 mg eq/kg in both sexes, most severely in females. Clinical signs of toxicity appeared after a few days of treatment and included hunched posture, piloerection, lean appearance, lethargy and rales. Additionally, salivation (up to moderate degree) occurred after dosing. One female was found dead in the morning of Day 7. The other animals showed significant weight loss (on average 10 and 15% of the initial weight in males and females, respectively). These findings led to the early sacrifice of the 100 mg eq/kg animals (on Day 7 prior to dosing) and adaptation of the dose volume (from 1 to 5 ml/kg bw) for the Group 1-3 animals from Day 7 onwards.

The 100 mg eq/kg animals had macroscopic and microscopic changes in the esophagus and stomach indicative of local toxicity resulting from the irritant properties of the test item. Main macroscopic findings in the esophagus included a thickened wall and grey white contents which correlated microscopically with erosion/ulceration of the squamous epithelium, squamous hyperplasia and/or lymphogranulocytic cell infiltrate. Gastric findings included an irregular surface of the forestomach at necropsy and histopathological changes consisting of lymphogranulocytic inflammation, squamous cell hyperplasia and/or ulceration in the forestomach. Additional macroscopic findings included distension of the stomach (with gas and mucous/gelatinous/ foamy contents) and various parts of the intestines (with gas and gelatinous/foamy contents).

The morbidity of the animals treated at 100 mg eq/kg was considered to have resulted from the local irritation in the esophagus and stomach.

Body weight loss, and related clinical signs, observed in an individual female rat at 33 mg eq/kg were likely to be secondary to local (concentration-dependent) irritant effects of the test item at the portal of entry. After reduction of the test item concentration in the dosing formulation (the dose volume was 5-fold increased from Day 7), the clinical signs disappeared, and body weight gain returned to normal.

At the microscopic level, local irritation by the test item was present in the form of minimal squamous cell hyperplasia in the forestomach of females treated at 33 mg eq/kg. Based on its minimal severity this finding was considered to be non-adverse.

Haematological investigation showed a decrease (by 35%) in the percentage of neutrophils in males treated at 33 mg eq/kg. Values at 33 mg eq/kg were slightly below the normal range. In the absence of accompanying changes in total white blood cells or other types of white blood cells, the decrease in neutrophils was regarded as non-adverse.


Reproductive and developmental results:
No reproduction toxicity was observed at 11 and 33 mg eq/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).
Developmental toxicity: higher prenatal mortality and concurrently reduced post-implantation survival index were observed at 33 mg eq/kg.

Analysis of dose preparations:
- Accuracy of preparation: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were considered to be 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
Effect level:
33 other: mg eq/kg/day in a dose volume of 5 ml/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Based on local adverse effects in the esophagus and stomach, resulting in moribundity or death, at 100 mg eq/kg in a dose volume of 1 ml/kg.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 33 other: mg eq/kg/day in a dose volume of 5 ml/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other:
Remarks:
No reproduction toxicity was observed at 11 and 33 mg eq/kg. At these dose levels 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). Histopathological examination of reproductive organs of the animals treated at 100 mg eq/kg for 6 days showed no test item-related changes. Their reproductive performance was not examined due to the early sacrifice.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 other: mg eq/kg/day
System:
gastrointestinal tract
Organ:
oesophagus
stomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

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.

The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

The pup of the control group which was sacrificed for humane reasons (pup no. 2 of litter no.50) had an unusual malformation (scoliosis) which hampered its normal gait.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Viability index was not affected by treatment up to 33 mg eq/kg. The viability indices the across the groups ranged from 95 to 100%. Six pups at 11 mg eq/kg (of three litters) and one pup at 33 mg eq/kg went missing (presumably cannibalized) or were found dead between PND 2-4. This post-natal loss was considered to be unrelated to treatment as the incidence showed no 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 affected by treatment up to 33 mg eq/kg.
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 affected by treatment up to 33 mg eq/kg.
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):
The nature and incidence of the macroscopic findings noted remained within the range considered normal for pups of this age, and were therefore considered incidental and not related to treatment.

The pup of the control group which was sacrificed for humane reasons had an unusual malformation (scoliosis)
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment up to 33 mg eq/kg.

- Areola/nipple retention: Treatment up to 33 mg eq/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

- Post-implantation survival index: A relatively low post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) of 82% was observed at 33 mg eq/kg, compared with 90% and 94% in the controls and 11 mg eq/kg groups, respectively. In the ten control group females, the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) varied between 0 and 3. In the 11 mg eq/kg treated group, there were nine females with 0-3 and one female with 4 unaccounted for sites. In the 33 mg eq/kg treated group, there were six females with 0-3 and two females with 4 and one female with 6 unaccounted for sites. These results indicated a trend of increased number of females with a higher number of unaccounted for sites, outside the range of that observed in controls, at higher doses. Although the mean number of implantation sites and living pups at 33 mg eq/kg were within normal limits, a relatively low post-implantation survival index of 82% was calculated for this group. For females no. 52 (11 mg eq/kg) and 69 (33 mg eq/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is occasionally observed and is caused by normal resorption of these areas during (the 14 days of) lactation. No toxicological relevance was attached to this finding in this study.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The prenatal mortality, expressed as the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for sites) showed a trend towards increase with dose level. One (out of ten) 11 mg eq/kg treated females showed 4 unaccounted for sites and three (out of nine) 33 mg eq/kg treated females showed 4 or 6 unaccounted for sites, whereas the unaccounted for sites in all ten control females varied between 0 and 3. Although the mean number of implantation sites and living pups at 33 mg eq/kg were within normal limits, a relatively low post-implantation survival index (the total number of offspring born as percentage of the total number of uterine implantation sites per group) of 82% was calculated from these results at 33 mg eq/kg. The higher number of females with an increased prenatal mortality and concurrent relatively low post implantation survival index at 33 mg eq/kg might indicate a relationship to treatment. No treatment-related changes were noted in any of the other 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), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
11 other: mg eq/kg/day in a dose volume of 5 ml/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on higher prenatal mortality and concurrently reduced post-implantation survival index at 33 mg eq/kg/day.
Remarks on result:
other:
Remarks:
No data were available for developmental toxicity assessment at 100 mg eq/kg.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
33 other: mg eq/kg/day
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with JNJ-118872-AAA (T000749) by oral gavage in male and female Wistar Han rats at dose levels of 11, 33 and 100 mg eq/kg resulted in macroscopic and microscopic changes in the oesophagus and stomach in the 100 mg eq/kg animals indicative of local toxicity resulting from the irritant properties of the test item. No reproduction toxicity was observed at 11 and 33 mg eq/kg. Higher prenatal mortality and concurrently reduced post-implantation survival index were observed at 33 mg eq/kg.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 33 mg eq/kg in a dose volume of 5 ml/kg
Reproduction NOAEL: at least 33 mg eq/kg (no data were available for reproductive performance assessment at 100 mg eq/kg).
Developmental NOAEL: at least 11 mg eq/kg (no data were available for developmental toxicity assessment at 100 mg eq/kg).

Therefore, the substance is classified as a reproductive toxicant category 2 according to the CLP Regulation.