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Administrative data

Description of key information

Repeated dose toxicity - oral: In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test performed according to the OECD Guideline 422 ted dose and US EPA OPPTS 870.3650, no adverse parental effects were observed up to the highest dose level tested (750 mg/kg) (van Otterdijk, 2018). The following parental NOAEL was derived 750 mg/kg bw/day.


In a 90-day repeated dose toxicity study performed according to OECD 408 guidelines, no test item-related effects were observed up to the highest tested dose level (1000 mg/kg bw/day). The NOAEL was considered to be at least 1000 mg/kg/day (Lourens, 2022). 


Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.


Repeated dose toxicity - dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-05-19 to 2021-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: M21AB0732
- Chemical Name: 2,3,4,6-TETRAKIS-O-(2,2-DIMETHYLPROPANOYL)-ALPHA-D-GLUCOPYRANOSYL BROMIDE
- Molecular formula: C26H43BrO10
- Molecular weight: 579.53
- Physical appearance: white crystals
- Expiration date of the lot/batch: 2022-01-29 (retest date)
- Purity: 100.0 % (w/w)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with argon desiccated
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: stable formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) in propylene glycol.

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

OTHER SPECIFICS
- Other relevant information: pH 8-8.3 at concentration of 0.02 mg/L
- Correction factor: 1.00
- Vapour pressure: 3.8 x 10E-7 Pa at 20°C; 1.1 x 10E-6 Pa at 25 °C
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 163 g to 209 g (males), 123 g to 159 g (females)
- Fasting period before study: no
- Housing: Polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS -J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate case (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. Cages are arranged on the racks according to a Latin-square model.
Animals will be socially housed for psychological/environmental enrichment and may be provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): Ad libitum, pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals will not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water; freely available to each animal via water bottles
- Acclimation period: 15 days before the commencement of dosing.

DETAILS OF FOOD AND WATER QUALITY:
Food: Pellets (alternate diet may be provided on individual animal basis). Results of analysis for nutritional components and environmental contaminants. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Water: Periodic analysis of the water is performed, and results of these analyses show that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (actual: 21°C)
- Humidity (%):40 to 70% (actual: 44 to 77%)
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-05-21 To: 2021-09-02
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 for a minimum of 13 weeks.
Justification of route: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
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. No correction was made for the purity/composition of the test item (correction factor is 1.00). A correction factor of 1.036 was used to correct for the specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the test facility.
- Concentration in vehicle: 0 mg/mL (group 1), 22 mg/mL (group 2), 66 mg/mL (group 3), 200 mg/mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg
- 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:
- Sample analysis: Dose formulation samples will be collected for analysis. Additional samples may be collected.
- week 1, 6 and 12
- concentration (middle) all groups, 2x approx 500 mg;
- homogeneity (top, middle, bottom) groups 2 and 4, 2x approx 500 mg;
- sampling from dosing container
All samples are transferred (at room temperature) to the analytical laboratory at the test facility for same day analysis.
- Analytical method: analyses are performed by using a validated analytical procedure
- Storage conditions of formulation samples: room temperature
- Acceptance Criteria:
- for concentration: mean sample concentration results within or equal to ± 15 % of theoretical concentration.
- For homogeneity: relative standard deviation (RSD) of concentrations of
< or =10% for each group.
- In addition to the criteria mentioned in the validated analytical method, each calibration curve were 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. In the event that the criteria were not met, results were discussed with the technician and new analysis were performed.
Duration of treatment / exposure:
a minimum of 13 weeks
Frequency of treatment:
7 Days a week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
110 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
330 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 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Justification of routes:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
- Justification of dose levels: The dose levels were selected based on results of a 10-day repeated dose toxicity study (500 and 1000 mg/kg/day) and a 28-day OECD422 study (80, 250 and 750 mg/kg/day) with oral exposure of JNJ-42808389-AAA (T003421) in rats, Test Facility Study No. 513675, and in an attempt to produce graded responses to the test item. In the 10-day study, clinical signs included hunched posture on Day 3 for animals dosed at 500 mg/kg/day and on Day 1 for animals dosed at 1000 mg/kg/day, and uncoordinated movements on Days 1 and 7-10 in animals dosed at 500 and 1000 mg/kg/day. Females dosed at 1000 mg/kg/day were also observed with piloerection on Days 4-5 and one female was observed with labored respiration rate on Days 3 and/or 4. No effect on body weight or food consumption was noted, and no macroscopic findings or effects on organ weights were noted. In the 28-day OECD 422 study, no clinical signs were noted. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
Positive control:
no
Observations and examinations performed and frequency:
MORTALITY:
- Time schedule: at least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily
- all animals are observed within their cage unless necessary for identification or confirmation of possible findings

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1at 0 to 1 hours postdose
- all animals are observed within their cage unless necessary for identification or confirmation of possible findings. For observations that cannot be attributed to an individual animals due to social housing (eg watery feces), the observation will be recorded to each animal in the socialized group

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.
- all animals
- Animals are removed from the cage.

ARENA OBSERVATIONS: Yes
-Time schedule: Once before the first administration of the test item and weekly during the Treatment Period.
- all animals are observed for clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs are recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study.
In order to monitor the health status, animals may have be weighed more often.
- all animals
- study animals surviving until scheduled euthanasia have a terminal body weight recorded (fasted weight) on the day of necropsy

FOOD CONSUMPTION : Yes
- Time schedule: Weekly; from at least Day 1 and throughout the study.
- all animals
- quantitatively measured per cage

WATER CONSUMPTION: No
- Time schedule for examinations: regular basis throughout the study
- all animals
- Water consumption was monitored by visual inspection of the water bottles. If inter-group differences are noted, consumption may be assessed by weight

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
Pretreatment Period - All animals once (including spare animals)
Dosing Period - All Group 1 and 4 animals during Week 13. If treatment-related findings are noted, the other animals were also examined.
- Procedure: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%)

FUNCTIONAL OBSERVATION: Yes
-Time schedule: Once during the Dosing Period. The first 5 animals per sex per group during Week 12-13, performed after clinical observations (including arena observation, if applicable).
- Procedure: The following tests were performed:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength were recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy, sampled between 7.00 and 10.30 from the retro-orbital sinus.
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes, overnight
- How many animals: All animals
- Parameters checked: White Blood Cell (WBC), neutrophils (absolute), lymphocytes (absolute), monocytes (absolute), eosinophils (absolute), basophils (absolute), Large Unstained cells (LUC) (absolute), Red Blood Cell (RBC), reticulocytes (absolute), Red Blood Cell Distribution Width (RDWG), hemoglobin, hematocrit, Mean corpuscular volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelets.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy, sampled between 7.00 and 10.30 from the retro-orbital sinus.
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked in table: Alanine aminotransferase (ALT), Triglycerides, Aspartate aminotransferase (AST), HDL and LDL Cholesterol, Alkaline Phosphatase (ALP), Sodium, Total protein, Potassium, Albumin, Chloride, Total Bilirubin, Calcium, Urea, Inorganic Phosphate (Inorg. Phos), Creatinine, Triiodothyronine (T3), Glucose, Thyroxine (T4), Cholesterol, Thyroid-Stimulating Hormone (TSH).
- Procedure: After receipt of the serum for T3, T4 and TSH analysis, it was divided in two aliquots. One aliquot was used for measurement of thyroid hormones TSH using the IMMULITE® 1000 analyser. The aliquot for TSH were stored in an ultra-low freezer set to maintain -80°C until analysis. Any remaining sample after TSH analysis was discarded. The other aliquot was used for measurement of T3 and T4 using LC-MS. The aliquot for T3 and T4 was collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and stored in a freezer set to maintain -20°C until analysis. Measurement of T3 and T4 was performed according to the bioanalytical method validated in Test Facility Study No. 20213516. Any samples remaining after the LC-MS analysis was returned to storage for the retention period.

COAGULATION: Yes
- Time of blood sample collection: on the day of necropsy, sampled between 7.00 and 10.30 from the retro-orbital sinus.
- Animals fasted: Yes, overnight
- How many animals: All animals
- Parameters checked in table: Prothrombin time (PT), Activated partial thromboplastin time (APTT)

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

ESTROUS STAGE DETERMINATION: Yes
- Time schedule: End of Treatment - on the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all female animals. This was done for all females except for females that had to be euthanized in extremis or died spontaneously.
- Procedure: Estrous stage was evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.
Sacrifice and pathology:
SACRIFICE:
Animals were anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

GROSS PATHOLOGY: Yes
Complete post mortem examinations were performed on all animals which included an evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
At the time of necropsy, the following tissues and organs were collected and placed in 10% neutral-buffered formalin fixative or initially in Modified Davidson’s solution: artery, aorta, body cavity, nasal, Bone marrow, sternum, bone femur, bone sternum, brain, epididymis, esophagus, eye, gland adrenal, gland clitoral, gland harderian, gland lacrimal, gland mammary, gland parathyroid, gland pituitary, gland preputial, gland prostate, gland salivary submandibular, gland salivary sublingual, gland salivary parotid, gland seminal vesicle including coagulation gland and fluid, gland thyroid, gut-associated lymphoid tissue, heart, kidney, large intestine cecum, large intestine colon, large intestine rectum, larynx, liver, lungs, lymph node mandibular, lymph node mesenteric, muscle, skeletal, never optic, nerve sciatic, never tibial, ovary, pancreas, skin, small intestine duodenum, small intestine ileum, small intestine jejunum, spinal cord, spleen, stomach, testis, thymus, tongue, trachea, urinary bladder, uterus, cervix, vagina.

ORGAN WEIGHT:
Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair may be taken and entered as a tissue comment. Organ weight as a percent of body weight (using the terminal body weight) and organ weight as a percent of brain weight were calculated.

HISTOPATHOLOGY: Yes
Microscopic examination of routinely prepared hematoxylin-eosin stained paraffin sections was performed on all tissues collected at necropsy from all control group and 1000 mg/kg/day treated animals. Gross lesions were examined from all animals and correlated to microscopic findings if possible. Tissues that were supposed to be microscopically evaluated per protocol but were not available on the slide were therefore not evaluated. These missing tissues did not affect the outcome or interpretation of the pathology portion of the study because sufficient numbers from each group were available for evaluation. The animal data and macroscopic findings were electronically recorded in the Provantis® computer system. Stained histologic sections were examined by light microscopy in the period 21 October-1 November 2021 and the microscopic findings were recorded. Severity grades were assigned to non-neoplastic histopathologic diagnoses. Severity grades were assigned based on the severity of alterations in the examined histologic sections and may not reflect the overall severity of the pathologic process in the entire tissue, organ, or animal. Histopathological changes were described according to distribution, severity (minimal, mild, moderate, marked, severe) and morphological character. The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions (INHAND) was used as guidance for the description of histopathological changes.

Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions where analyzed according to sex and occasion.
Descriptive statistics number, mean and standard deviation were reported whenever possible. Values may also be expressed as a percentage of pretreatment or control values when deemed appropriate. Inferential statistics were performed according to the matrix below when possible, but were exclude semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Incidence
An overall Fisher’s exact test were used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant. Additional methods of statistical analysis may have been used at the discretion of the Study Director.


Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical observations were noted during daily detailed clinical observations or weekly arena observations.
Incidental findings noted during the Dosing Period included decreased respiratory rate and abnormal breathing sounds in one male at 1000 mg/kg/day on Days 3-5. Furthermore, abnormal breathing sounds and/or sneezing were noted in one female at 330 mg/kg/day and two females at 1000 mg/kg/day on Days 3-4 or on Days 61-63. No toxicological relevance was attached to these findings as they were only seen in individual animals and occurred in absence of a relation to duration of treatment. These findings are likely to be related to the dosing procedure.
Other findings (e.g. fur loss, scabs, lesions and soft feces) noted during the Dosing Period 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 as in this study, occurred in control animals only, and/or did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment with the test item up to 1000 mg/kg/day.
Body weight was minimally lower in males at 110, 330 and 1000 mg/kg/day, reaching statistical significance only for males at 110 mg/kg/day on Day 71. Incidental statistically significant differences on body weight gain were noted between Days 22 and 92 (males at 110 mg/kg/day) and between Days 57 and 64 for all dose levels, compared to control animals.
Overall mean body weight gain was slightly lower for males dosed with the test item compared to control animals but did not reach statistical significance. No toxicological relevance was attached to this finding, as the difference in body weights and body weight gain occurred in absence of a clear dose response and body weight and body weight gain were within the range of normal values for rats of this age and strain. The apparent lower body weights observed in females at 1000 mg/kg/day during the Dosing Period, was due to the fact that these high dose females already had a lower body weight on Day 1 when compared to the control group. Overall, mean body weight gains were in the same range as for the concurrent control group and therefore no relevance was attached to this finding.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was considered to have been unaffected by treatment with the test item up to 1000 mg/kg/day.
A general trend (not statistically significant) towards slightly lower mean food consumption was observed in males at 110 mg/kg/day and females at 330 mg/kg/day from start of dosing onwards when compared to controls. This finding was considered to be unrelated to treatment with the test item since no relationship with dose could be established.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted during the Pre-treatment Period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings in Week 13 were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of males and females were considered unaffected by treatment up to 1000 mg/kg/day.
Platelets clumps were seen in one control female (No. 48), two males at 110 mg/kg/day (Nos. 12 and 18) and one male and two females at 330 mg/kg/day (Nos. 26, 67 and 69). As this finding lacked a dose-related response and was observed at a low incidence, this was considered to be not test item-related.
Coagulation parameters of treated males and females were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry parameters of males and females were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day. At an individual level, Female No. 75 (1000 mg/kg/day) was noted with high alanine aminotransferase (ALT, 131 U/L) and aspartate aminotransferase (AST, 136 U/L) activities. In absence of any corroborative findings, and considering the single incidence, this finding was considered unrelated to treatment with the test item. Remaining differences in clinical chemistry parameters between treated groups and the concurrent control group, regardless of achieving statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid stimulating hormone (TSH) concentration was lower in males at 110, 330 and 1000 mg/kg/day (0.78, 0.55 and 0.70x, respectively; not statistically significant), but occurred without a clear dose response. TSH concentration in females was decreased at 110 mg/kg/day (0.66x; not statistically significant) and increased at 330 and 1000 mg/kg/day (1.95 and 2.02, respectively; not statistically significant). This high TSH value was caused by one animal of each group (Nos. 66 and 73). Without these animals, the group mean would be 0.0615mU/L (1.09x of control) and 0.0751mU/L (1.33x of control), respectively. These differences in TSH concentration in males and females were considered not test item-related based on the absence of any statistical significance, absence of a dose response, the general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment with the test item up to 1000 mg/kg/day in week 12.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The apparent statistically significant decrease in grip strength of the hind leg in males at 330 and 1000 mg/kg/day, can be attributed to the relatively high value observed in the control animals. Mean grip strength in males at 330 and 1000 mg/kg/day was within the range of historical control values. Therefore, this is considered to be not test item-related.
The statistically significant increased mean number of ambulations in males at 1000 mg/kg/day can be attributed to the relatively low mean number of ambulations observed in the control animals, and the high number of ambulations observed in a single male at 1000 mg/kg/day (No. 35). Also, the control animals showed an abnormal motor activity habituation profile, with a low activity at the first 5-minute interval. Mean number of ambulations of males dosed at 110, 330 and 1000 mg/kg/day were all in the same range and within the range of historical control values.
Therefore, this is considered to be not test item-related.
All groups dosed with the test item showed a similar motor activity 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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related organ weight differences.
There were individual organ weight values that were different from their respective controls.
There were, however, no patterns or correlating data to suggest these values were test item related, and they were therefore considered incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross findings.
Gross findings observed were of the nature commonly observed in this strain and age of rat and/or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
Microscopic findings observed were of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Analytical verification


Stability analysis: Stability analyses performed previously in conjunction with the method development and validation demonstrated that the test item formulation at low target level (1 mg/ml) were unstable when stored in the refrigerator for at least 15 days, and formulations at high target level (200 mg/ml) were stable when in the refrigerator for at least 15 days. Formulations at low and high target level were stable when stored at room temperature under normal laboratory light conditions.


Concentration: the concentrations analyzed in the formulations of Groups 2-4 prepared for use in Weeks 1, 6 and 12 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration).


Homogeneity: The formulations of Group 2 and Group 4 prepared for use in Weeks 1, 6 and 12 were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Body weight (g) and body weight gain (g)





















































































































































Sex Male



Day(s) Relative to start date



1



8



15



22



29



36



43



50



57



64



71



78



85



91



0 mg/kg/day


10 animals



Mean



190.3



231.7



270.9



303.3



329.4



351.0



367.1



383.3



396.4



413.2



426.4



430.0



435.7



437.0



110 mg/kg/day


10 animals



Mean



184.1



221.6



258.5



286.5



305.3



326.9



339.3



355.7



367.1



380.0



387.3*



394.3



399.0



400.7



% Diff



-3.3



-4.4



-4.6



-5.5



-7.3



-6.9



-7.6



-7.2



-7.4



-8.0



-9.2



-8.3



-8.4



-8.3



330 mg/kg/day


10 animals



Mean



189.9



230.5



268.0



296.6



318.3



335.6



353.4



368.2



381.9



394.3



403.6



412.8



417.7



419.5



%Diff



-0.2



-0.5



-1.1



-2.2



-3.4



-4.4



-3.7



-3.9



-3.7



-4.6



-5.3



-4.0



-4.1



-4.0



1000 mg/kg/day


10 animals



Mean



189.5



228.5



268.7



300.3



322.0



340.2



354.1



369.6



380.8



393.4



402.5



408.8



417.5



417.5



%Diff



-0.4



-1.4



-0.8



-1.0



-2.2



-3.1



-3.5



-3.6



-3.9



-4.8



-5.6



-4.9



-4.2



-4.5



Anova & Dunnett: * = p ≤ 0.05


Food consumption - Daily food cons per animal (g)





















































































































































Sex Male



Day(s) Relative to animal start date



1-8



8-15



15-22



22-29



29-36



36-43



43-50



50-57



57-64



64-71



71-78



78-85



85-91



1-91



0 mg/kg/day


2 animals



Mean



21.3



22.0



23.2



23.2



22.9



22.4



22.5



21.8



21.5



21.1



21.2



21.6



21.8



22.0



110 mg/kg/day


2 animals



Mean



20.3



20.9



21.4



21.5



21.4



20.8



21.0



19.9



19.9



19.6



19.4



19.5



19.3



20.4



%Diff



-4.8



-5.2



-7.8



-7.5



-6.4



-7.1



-6.6



-8.6



-7.5



-7.0



-8.2



-10.0



-11.5



-7.5



330 mg/kg/day


2 animals



Mean



21.3



21.8



22.5



22.9



23.0



22.1



21.9



20.9



20.6



20.4



20.8



20.9



20.4



21.5



%Diff



-0.2



-1.0



-3.0



-1.2



0.2



-1.5



-2.7



-4.3



-3.9



-3.5



-1.6



-3.4



-6.3



-2.4



1000 mg/kg/day


2 animals



Mean



21.6



22.7



23.3



23.8



23.6



23.2



22.4



21.0



21.0



21.0



20.8



21.5



20.8



22.1



%Diff



1.5



3.0



0.5



2.4



2.9



3.5



-0.5



-3.5



-2.0



-0.6



-1.8



-0.8



-4.4



0.1



 





















































































































































Sex Female



Day(s) Relative to animal start date



1-8



8-15



15-22



22-29



29-36



36-43



43-50



50-57



57-64



64-71



71-78



78-85



85-91



1-91



0 mg/kg/day


2 animals



Mean



14.6



15.0



16.0



17.4



15.7



15.5



16.1



15.9



15.4



15.0



15.5



15.4



15.0



15.6



110 mg/kg/day


2 animals



Mean



14.7



15.1



15.7



16.6



15.6



16.1



16.3



15.3



15.1



15.0



15.4



15.2



15.0



15.4



%Diff



0.9



0.6



-1.7



-7.8



-0.5



4.1



1.1



-3.7



-2.0



-0.2



-0.6



-1.4



-0.4



-1.0



330 mg/kg/day


2 animals



Mean



14.0



14.1



14.8



15.0



14.8



15.1



15.2



14.9



14.2



14.0



14.3



14.4



14.0



14.5



%Diff



-3.8



-6.0



-7.5



-13.5



6.0



-2.8



-5.8



-6.5



-7.8



-6.9



-7.6



-6.1



-7.0



-6.8



1000 mg/kg/day


2 animals



Mean



14.0



14.7



15.5



16.0



15.8



15.5



15.7



15.3



15.0



14.5



14.8



14.6



14.6



15.1



%Diff



-4.0



-1.8



-2.9



-7.8



0.2



0.1



-2.9



-3.9



-2.4



-3.5



-4.3



4.8



-3.0



-3.2



Functional Test Males



















































 



 



Group 1



Group 2



Group 3



Group 4



 



0 MG/KG/DAY



110 MG/KG/DAY



330 MG/KG/DAY



1000 MG/KG/DAY



Grip fore gram


5 animals



Mean



1321



1332



1379



1064



SD



208



92



239



159



Grip hind gram


5 animals



Mean



944



813



692**



678**



SD



107



142



70



78



+/++ Steel-test significant at 5% (+) or 1% (++) level


*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level




































 



 



Group 1



Group 2



Group 3



Group 4



 



0 MG/KG/DAY



110 MG/KG/DAY



330 MG/KG/DAY



1000 MG/KG/DAY



Ambulation


5 animals



Mean



322



589



592



666*



SD



106



311



35



365



MEAN and STDEV values are calculated per group, from each animal's total Ambulations over all intervals


* indicates a p-value <0.05, ** indicates a p-value <0.01


 


Thyroid-stimulating hormones (TSH) - Day: 92 and 93 Relative to Start Date for males and females respectively






































































 



Males


(mU/L)


G1



Females


(mU/L)


G1



0 mg/kg/day


10 animals



Mean



0.1162



0.0562



SD



0.0899



0.0635



110 mg/kg/day


10 animals



Mean



0.0907



0.0371



SD



0.0814



0.0273



tCtrl



0.78



0.66



330 mg/kg/day


10 animals



Mean



0.0636



0.1098



SD



0.0371



0.1587



tCtrl



0.55



1.95



1000 mg/kg/day


10 animals



Mean



0.0815



0.1137



SD



0.0781



0.1361



tCtrl



0.70



2.02



[G1] - Kruskal-Wallis & Dunn


 


 


 


 


 


 


 


 


 

Conclusions:
In conclusion, administration of JNJ-42808389-AAA (T003421) by once daily oral gavage was well tolerated in Wistar Han rats at dose levels up to 1000 mg/kg/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was considered to be a least 1000 mg/kg/day.
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
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
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
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. QA statement)
Limit test:
no
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.

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 or test system 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
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.
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
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.
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.
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
Key result
Critical effects observed:
no
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP-compliant study, performed according to OECD guideline

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated toxicity: oral


A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 80, 250, 750 mg/kg bw/ day via gavage (OECD 422, Van Otterdijk, 2018). The vehicle used was propylene glycol and the test solutions were prepared daily and administered within 6 hours after preparation.


No adverse parental effects were observed up to the highest dose level tested (750 mg/kg).


There were no test item-related premature decedents in the study. However, one female (no. 61 dosed at 250 mg/kg/day) was sacrificed after 40 days of treatment. The animal had a vaginal prolapse with subsequently dilation of the uterus horns and serosal inflammation in the uterus at microscopy. 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.


Based on the abovementioned considerations, the NOAEL was considered to be 750 mg/kg bw/day (nominal dose received).


 


In a key, subchronic repeated dose oral toxicity study (OECD 408; Lourens, 2022), the test item was administered via gavage to male and female Wistar Han rats for at least 90 days (13 weeks) at dose levels of 0, 110, 330 and 1000 mg/kg/day (10 rats/sex/group). The test item was formulated in propylene glycol. Chemical analyses of formulations were conducted in Weeks 1, 6 and 12 to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously. There were no premature decedents in this study. No test item-related or toxicologically relevant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, functional observations, ophthalmoscopy, clinical laboratory investigations (i.e., hematology, coagulation and clinical chemistry parameters), and pathological examinations (i.e., organ weight changes, necropsy or microscopic findings)).


In conclusion, administration of the test item by once-daily oral gavage for at least 90 days was well tolerated in Wistar Han rats at dose levels up to 1000 mg/kg/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was considered to be a least 1000 mg/kg/day.


 


Repeated toxicity: inhalation/dermal


A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation or dermal route of exposure.

Justification for classification or non-classification

As no adverse effects are observed in the oral OECD 422 study (Van Otterdijk, 2018) up to 750 mg/kg/d, or in the oral subchronic repeated dose toxicity study (OECD408; Lourens, 2022) up to a 1000 mg/kg/day, the substance doesn't meet the criteria for classification as STOT RE according to EU CLP.