Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-29 to 2016-11-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA, OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Deviations:
no
Principles of method if other than guideline:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Test Facility Study No. 512673).

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

OTHER SPECIFICS: Correction factor was 1

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 301-338 g (males) and 209-238 g (females)
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of a maximum of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one basis in Macrolon plastic cages (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-hours light/12-hours dark cycle.

IN-LIFE DATES: From: 2016-07-29 To: 2016-11-16

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Method: Oral gavage, using a plastic feeding tube.
Frequency: 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
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity of the test item. A correction factor of 1 was used. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 20 mg/mL (group 2); 60 mg/mL (group 3); 200 mg/mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according to the latest body weight)
- Lot/batch no. (if required): I15FC2164
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (27 September 2016, Day 7 of treatment) according to a validated method (Test Facility Study No. 512673). Duplicate samples were collected. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90.00-110.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Stability of formulations was determined as part of the analytical method development and validation study (Test Facility Study No. 512673).
Duration of treatment / exposure:
28 days (males); 50-56 days (females that delivered); 44 days (female with total litter loss); 41 or 42 (females which failed to deliver). Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 514054) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, no clinical signs (indicative of toxicity) were observed. No mortality occurred, clinical appearance was considered normal, there were no effects on body weight and food consumption, no macroscopic abnormalities were noted, and kidney and liver weights were considered normal. Based on these results, dose levels of 100, 300 and 1000 mg/kg were selected for the main study. Therefore, clinical observations in the main study were conducted after dosing, and functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- Rationale for animal assignment (if not random): randomized
Positive control:
no

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on PND 1, 4, 7 and 13.
Both absolute food consumption and food consumption relative to body weight were reported.

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

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

CLINICAL BIOCHEMISTRY: 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 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 for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- parameters checked: 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: between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable)
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: All surviving animals, on PND 14-16 (females which delivered), or on days post-coitum 25-27 (females which failed to deliver, with evidence of mating), or within 24 hours of litter loss (1 female with total litter loss), or in extremis (1 female was euthanized for humane reasons)

GROSS 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 possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes- mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glanspenis (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)

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 4; Additional slides of the testes of the selected 5 males of Groups 1 and 4 and of all males that failed to sire to examine staging of spermatogenesis; The preserved organs and tissues of one female at 1000 mg/kg (no. 72) that was euthanized in extremis; The mammary gland of one female at 100 mg/kg (no. 54) with total litter loss; All gross lesions of all animals (all dose groups); Thyroid gland of all selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in Group 4; The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- All abnormalities were described and included in the study report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.
Other examinations:
ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus, bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavern osus muscle complex (LABC), Testes, Thyroid
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs of toxicity were noted during the observation period.
- The only clinical sign noted was alopecia, observed for single females among the control and 100 mg/kg groups. As this finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study, and absence of a dose-related incidence across the groups, this finding was not considered to be related to treatment.
During the weekly arena observations, no additional treatment-related clinical signs were noted.
Mortality:
no mortality observed
Description (incidence):
- No mortality occurred during the study period that was considered to be related to treatment with the test item.
- One female at 1000 mg/kg was sacrificed in extremis on Day 9 of the premating period for humane reasons. Oesophageal perforation noted at necropsy indicated that this moribundity was due to a gavage accident and not related to treatment with the test item.
Other related necropsy findings for this animal consisted of a gray-white discoloured and hard granulated oesophagus, hard, granulated gray-white content in the right axillary region and a gelatinous throat region. These findings correlated to microscopic findings, i.e. inflammation/hemorrhage/necrosis of the oesophagus and presence of amorphous material in the tissue surrounding the oesophagus and amorphous material in the subcutis of the axillary region.
- One female at 100 mg/kg was sacrificed on Day 4 of the lactation phase due to total litter loss.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- At 300 mg/kg and 1000 mg/kg, a statistically significant higher mean activated partial thromboplastin time (APPT) was recorded in females. Means were approximately 26% and 30% higher than controls, respectively.
- All other haematological parameters of treated rats were not considered to be affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- At 1000 mg/kg, inorganic phosphate was statistically significantly higher in males. The mean was approximately 16% higher than the control mean.
- Any other statistically significant changes in clinical biochemistry parameters were not considered to be related to treatment as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.
- Thyroid hormone analyses Serum levels of total T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
- The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no test item-related gross observations.
- All recorded macroscopic findings noted among surviving animals were within the range of background gross observations encountered in rats of this age and strain. One female at 1000 mg/kg was sacrificed in extremis on Day 9 of the premating period for humane reasons. Oesophageal perforation noted at necropsy indicated that this moribundity was due to a gavage accident and not related to treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- A slightly increased incidence and/or severity of thyroid follicular cell hypertrophy, up to a slight degree, was present in males treated at 1000 mg/kg and in females treated at 300 and 1000 mg/kg.
- The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
- No adverse effects on parental parameters were noted up to 1000 mg/kg.
- Microscopic examination of the thyroids revealed a slightly increased incidence and/or severity of follicular cell hypertrophy in males at 1000 mg/kg and in females at 300 and 1000 mg/kg. This was regarded to be an adaptive change and considered to be non-adverse at the incidences and severities recorded.
- A few changes in clinical pathology parameters were noted at 300 and 1000 mg/kg that were potentially related to treatment. These were not considered adverse in absence of any morphological correlates and since they essentially remained within the range considered normal for rats of this age and strain. These changes consisted of higher activated partial thromboplastin time in females at 300 and 1000 mg/kg and higher inorganic phosphate in males at 1000 mg/kg.
- No treatment-related effects on mortality, clinical appearance, functional observations, body weight, food consumption, organ weights and macroscopic appearance were observed.
- The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e., mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation <= 10.00%).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with JNJ-4754724-AAA (T002488) by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no adverse toxicity up to 1000 mg/kg.
Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be 1000 mg/kg. Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.