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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-27 to 2017-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
other: OECD Guideline 421 (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:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
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 Guideline 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: crystalline
Details on test material:
- Name of test material (as cited in study reports): JNJ-42808389-AAA (T003421)
- Physical state: crystalline powder
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16FB2273
- Expiration date of the lot/batch: 2017-06-14 (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 determined as part of the analytical method development and validation study (Test Facility Study No. 513680).

FORM AS APPLIED IN THE TEST
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. No correction was made for the purity/composition of the test item.

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. 10 weeks (at start F0-treatment); females approx. 10 weeks (at start pretest) and approx. 12 weeks (at start F0-treatment).
- Weight at study initiation: 289-293 g (males) and 224-230 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: 2016-12-27 To: 2017-04-25

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube
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. No correction was made for the purity/composition of the test item

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), 80 mg/mL (group 2), 250 mg/mL (group 3), 750 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 (22 February 2017, Day 1 of treatment) according to a validated method (Test Facility Study No. 513680). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed. 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 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Stability of formulations over 6 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. 513680).
Duration of treatment / exposure:
29 days (males); 49-62 days (females that delivered); 42 days (non preagnant females). Female nos. 45, 48 (Group 1), nos. 56, 59 (Group 2), nos. 67, 70 (Group 3) and nos. 77 and 80 (Group 4) were left out from treatment for one day as they were littering at the moment of dosing. 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
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
750 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. 513676) in which animals were dosed for 10 days at 500 and 1000 mg/kg/day
- 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: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals after treatment. 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.
Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
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: Yes
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 25 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3- EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). The remnant of the Li-heparin sample was stored at ≤-75°C for possible future clinical biochemical analysis.
An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids, and a further blood sample (0.5 mL) was collected into a serum tube for possible future clinical biochemical analysis. After clotting and centrifugation, serum samples were stored at ≤-75°C; these samples were discarded prior to report finalization.
- Parameters checked : 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 anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 25 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:
T4: F0-males: after at least 28 days of treatment

FUNCTIONAL OBSERVATIONS
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, 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), on days 25-27 (females which failed to deliver, with evidence of mating) or euthanized in extremis (when pain, distress or discomfort was considered not transient in nature or was likely to become more severe).

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 with bone marrow including joint (M/F), 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), Epididymides (M), Mammary gland area, inguinal region with skin (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
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 all males that failed to sire.
• The preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanized in extremis.
• All gross lesions of all animals (all dose groups).
• The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups, i.e. non-pregnant couples 51/11 (Group 2), 65/25 and 69/29 (Group 3) and 79/39 (Group 4).
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, 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, Prostate, Semincal vesicles, including coagulation glands, Testes, Thyroid including parathyroid if detectable
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were noted among surviving animals during the observation period that were considered to be related to treatment.
Observed clinical signs among surviving animals 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, these were not considered to be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment.
One female at 250 mg/kg (no. 61) was sacrificed in extremis on PND 1 (i.e. after 40 days of treatment) due to a severe vaginal prolapse. At necropsy, this animal also showed pale discolouration of the whole body, and reddish discolouration of the thymus. Secondary to vaginal prolapse, distension of both uterine horns and serosal inflammation in the uterus were observed histopathologically.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated animals were not considered affected by treatment.Incidental occurrences of statistically significantly lower mean body weights or body weight gain of females at 80 and 250 mg/kg during the treatment period occurred in the absence of a dose-related trend, and as such were not considered to be related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was not considered affected by treatment. The lower mean food consumption of females at 80 mg/kg during lactation (statistically significant on Days 1-4 and 7-13) was ascribed to a low food intake of two females (nos. 53 and 54), one of which only delivered a single pup. This variation in food intake as well as other statistically significantly lower mean absolute and/or relative food consumption values on several occasions during post-coitum at 250 mg/kg occurred in the absence of a dose-related trend. Therefore, these variations were not considered to be related to treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 750 mg/kg, activated partial thromboplastin time (APTT) was elongated for females (approximately 34% increase compared to the control mean); the mean remained within the normal range for rats of this age and strain.
Other statistically significant changes in haematological parameters were not considered related to treatment in the absence of a dose-related trend, and/or concurrent changes in other red blood cell parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were not considered to be affected by treatment.
Thyroid hormone analyses:
Serum levels of T4 in F0 males were not considered to be affected by treatment. The statistically significantly lower T4 values at 80 and 750 mg/kg occurred in the absence of a dose-related trend and means remained within the normal range for rats of this age and strain. As such, these variations in T4 were not considered to be related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were not considered to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.
Fore- and hindlimb grip strength was not considered affected by treatment. The statistically significantly lower forelimb grip strength of females at 80 mg/kg occurred in the absence of a dose-related trend. The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with high activity in the first interval 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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Any differences, including those that reached statistical significance were considered not to be test item-related due to the lack of dose-related pattern.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations.
One male rat at 750 mg/kg (no. 34) showed moderate chronic progressive nephropathy. This is an unusual finding in rats of this age, but based on the presence in only one animal and the absence of test item-related findings in the kidneys of the other animals, the finding was considered to be incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Analysis of Dose Preparations:
Accuracy of preparation:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%).
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%).

Parental results:
No adverse parental effects were observed up to the highest dose level tested (750 mg/kg).
An elongated activated partial thromboplastin time was recorded for females at 750 mg/kg. However, the mean remained within the normal range for rats of this age and strain, and there were no concurrent changes in other (clotting) parameters in this study. Therefore, this change was not considered to be adverse.
No treatment-related changes were noted in any of the other parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical biochemistry investigations, macroscopic examination, organ weights, and microscopic examination).
In contrast to the range finding study (Test Facility Study no. 513676) where intermittently occurring clinical signs were observed at 500 and 1000 mg/kg including hunched posture, uncoordinated movements and/or piloerection, no such clinical signs were recorded for animals in the main study at 750 mg/kg. This may be related to the time point on which these signs were observed in the main study (i.e. shortly after dosing). Although no clear peak effect of occurrence of clinical signs were noted in the range finding study for the 500 and 1000 mg/kg dose levels combined, clinical signs recorded at 500 mg/kg were noted at 1 hour post-dose.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
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-42808389-AAA (T003421) by oral gavage in male and female Wistar rats at dose levels of 80, 250 or 750 mg/kg/day revealed no parental toxicity up to 750 mg/kg. Based on these results a No Observed Adverse Effect Levels (NOAEL) of 750 mg/kg was derived.