Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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/Deve lopmental 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
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 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: 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: 313-351 g (males) and 210-247 g (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).
- 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); 2016-03-10/11/2/13/14/15/23/24 (necropsy females); 2016-03-09/10/11/12/13/14/22/23 (necropsy pups)

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 1.00 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
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

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 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.

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 anesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived from 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, differnetial leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated 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 aminotrasnferase, aspartate aminotrasnferase, 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 hindlimb grip strength recorded as the 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 that delivered) or on post-coitum day 25 (females that failed to deliver, with evidence of mating).

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 exa mination, 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) 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), 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 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 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.
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 bulbocavernosus muscle complex (LABC), Testes, Thyroid
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 co ntrol 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 in tergroup 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:
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.
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). 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.
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
Description (incidence and severity):
There were no test item-related alterations in organ weights.
The statistically significant higher liver weight of females at 100 mg/kg occurred in the absence of a dose-related trend. As such, this variation was not considered to be related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
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). 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.
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
Details on results:
- analysis of dose preparations: the concentrations analysed in the formulations of group 2, group 3 and group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the group 1 formulations. The formulations of group 2 and group 4 were homogeneous (i.e. coefficient of variation <=10%).

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.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical 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 toxicity up to 1000 mg/kg.
Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be at least 1000 mg/kg.
Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.