D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetrakis(2,2-dimethylpropanoate), (1S)-

Endpoint summary

Description of key information

Repeated dose toxicity - oral:

In a combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the test substance was administered daily to rats up to a dose level of 1000 mg/kg body weight/day (OECD 422; Peter, 2017). The NOAEL is established to be at least 1000 mg/kg.

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 bw/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

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-12 to 2022-03-23

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:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I20IB2949
- Chemical name: (1S)-1,5-ANHYDRO-2,3,4,6-TETRAKIS-O-(2,2-DIMETHYLPROPANOYL)-1-(3-([5-(4-FLUOROPHENYL)THIOPHEN-2-YL]METHYL)-4-METHYLPHENYL)-D-GLUCITOL
- CAS Number: 1283129-18-9
- Physical appearance: white powder
- Expiration date of the lot/batch: 2022-09-19 (retest date)
- Purity, including information on contaminants, isomers, etc.: 98.9 %
- Molecular weight: 781.0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: stability for at least 6 hours at room temperature under normal laboratory light conditions, and for 26 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL.

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

OTHER SPECIFICS
- pH 7.7 at concentration of <0.01 g/L
- correction factor: 1.00

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

Justification of test system: At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 140 - 219 grams (males), 127 - 166 grams (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 to 5 animals of the same sex and same dosing group together.
During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
Animals were 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, (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.
It is considered that there are no known contaminants that would interfere with the objectives of the study.
- Water (e.g. ad libitum): municipal tap water, freely available to each animal via water bottles
- Acclimation period: 15 days

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

IN-LIFE DATES: From:2021-05-18 to 2021-08-27

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.

Vehicle:

propylene glycol

Remarks:

Specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared weekly (storage condition 4 °C) and were homogenized to a visually acceptable level. The dose formulations were be stirred continuously during dosing. 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 (study Nos 512709 and 512704).
- Concentration in vehicle: 0 mg/mL (group 1- control), 22 mg/mL (group 2), 66 mg/mL (group 3) and 200 mg/mL (group4).
- 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:

Dose formulation samples are collected for analysis as follows:
- week 1, 6 and 12
- concentration (middle): all groups, 6x approx 500 mg
- homogeneity (top, middle, bottom): groups 2 and 4, 6x 500 mg
All samples to be analysed will be transferred (at room temperate) to the analytical laboratory for same day analysis, where possible or stored for analysis within known formulation stability period.
Additional samples for homogeneity analysis are collected in week 3 (top, middle, bottom) for groups 2 and 4 (6x approx 500 mg), samples from bulk container (weekly volume)
The analyses are performed by using a validated analytical procedure.
- storage conditions: room temperature set to maintain 21 °C
- 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.

Stability analysis: performed previously in conjunction with the method development and validation demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability for at least 6 hours at room temperature under normal laboratory light conditions, and for 26 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL.

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 - control

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 number of animals: the total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
- 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.
- Justification of dose level selection: The dose levels were selected based on information provided by the Sponsor and results of a OECD 422 study with oral exposure of JNJ-42808415-AAA (T003422) in rats (Test Facility Study No. 512704) and in an attempt to produce graded responses to the test item. In this OECD 422 study only mild symptoms were noted only on Day 1 (hunched posture in animals at 500 mg/kg and uncoordinated movements in animals at 1000 mg/kg). Slight, temporal, body weight loss was observed in some animals at 500 and 1000 mg/kg, but all animals recovered on the days thereafter. Food consumption was normal, no macroscopic findings were noted and no effects on liver and kidney weight was observed.
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: Yes
- Time scheduled: 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
- animals will be 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 1 at 0 to 1 hours postdose.
- all animals
- animals will be 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 socialised 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
- animals will be observed for clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs will be 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 be weighed more often.
- all animals
- Animals surviving until scheduled euthanasia have a terminal body weight recorded

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 will be 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: 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 will be 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 at end of treatment, sampled between 7.00 and 10.30 from the retro-orbital sinus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours
- How many animals: All animals
- samples: 0.5 mL with anticoagulant (K3)EDTA (not processed)
- Parameters evaluated: 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.
- A blood smear was prepared from each hematology sample. Blood smears are labeled, stained and stored. These smears were not examined but could be evaluated when required to confirm analyzer results.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy at end of treatment, sampled between 7.00 and 10.30 from the retro-orbital sinus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours
- How many animals: all animals
- Samples: 0.5 mL (plasma) and 1.0 mL (serum, thyroid hormone measurement) with anticoagulant Li-heparin (not for serum) , processed to plasma or serum
- Parameters evaluated: 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 schedule for collection of blood: on the day of necropsy at end of treatment, sampled between 7.00 and 10.30 from the retro-orbital sinus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours
- How many animals: all animals
- Samples: 0.9 mL with anticoagulant Citrate , processed to plasma
- Parameters evaluated: 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:
Unscheduled euthanasia: Animals were anesthetized using isoflurane and subsequently exsanguinated. If necessary, animals were refrigerated to minimize autolysis.
Scheduled euthanasia: Animals were deeply 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.

ORGAN WEIGHT
- organs are weighed at necropsy
- 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 will be calculated
- list of organs weighed: brain, epididymis (2), adrenal gland (2), pituitary gland, prostate gland, seminal vesicle gland including coagulation gland and fluid (2), thyroid gland (2), heart, kidney (2), liver, ovary (2), spleen, testis (2), thymus, uterus/cervix
(2) = both sides

TISSUE COLLECTION AND EVALUATION:
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, nasal body cavity, bone marrow of sternum, femur bone, sternum bone, brain, epididymis (2), esophagus, eye (2), adrenal gland (2), clitoral gland (2), harderian gland (2), lacrimal gland (2) (extra-orbital), mammary gland, parathyroid gland (2), pituitary gland, preputial gland (2), prostate gland, salivary submandibular gland (2), salivary sublingual gland (2), salivary parotid gland (2), seminal vesicle gland (2) including coagulation gland and fluid, thyroid gland (2), gut-associated lymphoid tissue, heart, kidney (2), large intestine cecum, large intestine colon, large intestine rectum, larynx, liver, lung, mandibular lymph node (2), mesenteric lymph node, skeletal muscle (2), optic nerve (2), sciatic nerve (2), tibial nerve (2), ovary (2), pancreas, skin, small intestine duodenum, small intestine ileum, small intestine jejunum, spinal cord, spleen, stomach, testis (2), thymus, tongue, trachea, urinary bladder, uterus/cervix, vagina.
(2) = both sides

HISTOPATHOLOGY: Yes
- histology: tissues will be embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin
- the following tissues were processed: artery, aorta, bone marrow of sternum, sternum bone, brain, epididymis (2), esophagus, eye (2), adrenal gland (2), mammary gland, parathyroid gland (2), pituitary gland, prostate gland, salivary submandibular gland (1), salivary sublingual gland (1), seminal vesicle gland (2) including coagulation gland and fluid, thyroid gland (2), gut-associated lymphoid tissue, heart, kidney (2), large intestine cecum, large intestine colon, large intestine rectum, liver, lung, mandibular lymph node (1), mesenteric lymph node, skeletal muscle (1), optic nerve (1), sciatic nerve (1), ovary (2), pancreas, skin, small intestine duodenum, small intestine ileum, small intestine jejunum, spinal cord, spleen, stomach, testis (2), thymus, trachea, urinary bladder, uterus/cervix, vagina

- 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.
- the following tissues were examined microscopically: artery, aorta, bone marrow of sternum, sternum bone, brain, epididymis (2), esophagus, eye (2), adrenal gland (2), mammary gland, parathyroid gland (2), pituitary gland, prostate gland, salivary submandibular gland (1), salivary sublingual gland (1), seminal vesicle gland (2) including coagulation gland and fluid, thyroid gland (2), gut-associated lymphoid tissue, heart, kidney (2), large intestine cecum, large intestine colon, large intestine rectum, liver, lung, mandibular lymph node (1), mesenteric lymph node, skeletal muscle (1), optic nerve (1), sciatic nerve (1), ovary (2), pancreas, skin, small intestine duodenum, small intestine ileum, small intestine jejunum, spinal cord, spleen, stomach, testis (2), thymus, trachea, urinary bladder, uterus/cervix, vagina

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 labored breathing (one female at 110 mg/kg/day), abnormal breathing sounds (one male at 110, 330 and 1000 mg/kg/day each and one female at 330 mg/kg/day), erected fur (one male at 1000 mg/kg/day), and slight to moderate salivation (two males at 330 mg/kg/day and two females at 110 mg/kg/day). 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 and/or dose.
Other findings (e.g. fur loss, skin lesions and scabs) 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 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 treatment with 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 the test item up to 1000 mg/kg/day.
A slightly lower mean body weight gain was noted in males and females at 1000 mg/kg/day when compared to controls from Days 43 to 50 (not statistically significant in females). No toxicological relevance was attached to this finding, as it was a temporally change followed by completely normal values of body weight and body weight gain in the subsequent weeks.
The apparent (not statistically significant) lower mean body weights observed in females at 1000 mg/kg/day during the complete 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 the test item up to
1000 mg/kg/day.
A general trend (not statistically significant) towards slightly lower mean food consumption was observed in females at 100 and 1000 mg/kg/day from start of dosing onwards, when compared to controls. This finding was considered to be unrelated to 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 specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to the test item.
The nature and incidence of ophthalmology findings noted during the Pretreatment 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 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.
The slightly lower mean corpuscular volume (MCV) in females at 1000 mg/kg/day was considered unrelated to treatment with the test item as the difference was minimal only (0.97x of control) and occurred in absence of changes in correlating parameters.
In females at 110 mg/kg/day, lower mean counts of lymphocytes (LYMPH), monocytes (MONO), basophils (BASO; not statistically significant) and large unstained lymphocytes (LUC) were noted when compared to concurrent control, resulting in a lower mean total white blood cell (WBC) count white blood cell count. This finding was considered unrelated to administration of the test item due to absence of a dose response.
Platelets clumps were seen in one control male (No.5), and in one male at 110, 330 and 1000 mg/kg/day (No. 11, 21 and 32, respectively). As this finding lacked a dose-related response and occurred without any other corroborating findings, this was considered to be not test item -related.

Coagulation 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 the individual level, two females (Nos. 72 and 76) at 1000 mg/kg/day were noted with slightly longer prothrombin times (PT: 1.16 and 1.18x of the group mean, respectively). In addition, activated partial thromboplastin time (APTT) was slightly longer in these females (1.20 and 1.14x of the group mean, respectively), but remained within the concurrent control range or was only marginally higher. Considering the low incidence, relatively small magnitude of change and in absence of any indications for internal bleedings, no toxicological relevance was attached to this observation.

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.
No toxicological relevance was attached to the slightly higher mean urea concentration (UREA; 1.21x of control) observed in males at 1000 mg/kg/day, since individual values in these high dose males had great overlap with those of low dose males.
Remaining differences in clinical chemistry parameters, including TSH, T3 and T4 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:

no effects observed

Description (incidence and severity):

No test item-related effects were observed on TSH, T3 and T4 parameters, nor in thyroid gland organ weight determination.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment with the test item up to 1000 mg/kg/day. Hearing ability, pupillary reflex and static righting reflex and grip strength were normal in all examined animals.The variation in motor activity did not indicate a relation with treatment with the test item. All groups 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:

no effects observed

Description (incidence and severity):

There were no test item-related organ weight changes following the oral (gavage) administration of test substance for at least 90 days up to 1000 mg/kg/day to Wistar Han rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related organ necropsy following the oral (gavage) administration of the test substance for at least 90 days up to 1000 mg/kg/day to Wistar Han rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related organ microscopic findings following the oral (gavage) administration of the test substance for at least 90 days up to 1000 mg/kg/day to Wistar Han rats.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

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

Dose formulation analyses

Accuracy: the concentrations analyzed in the formulations of Groups 2-4 prepared for use in weeks 1, 3, 6 and 12 were in agreement with target concentrations (ie mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulations.

Homogeneity: The formulations of Groups 2 and 4 prepared for use in weeks 1, 3, 6 and 12 were homogeneous (ie coefficient of variation <=10.00%)

 

Summary of Body Weight Gains (g)

Sex Male		Day(s) Relative to animal start date
		1-8 [G]	8-15 [G1]	15-22 [G]	22-29 [G]	29-36 [G]	36-43 [G]	43-50 [G]	50-57 [G]	57-64 [G]	64-71 [G]	71-78 [G]	78-86 [G]	86-91 [G]	1-91 [G1]
0 mg/kg/day 10 animals	Mean	43.0	38.0	29.2	23.0	22.0	15.0	13.4	13.3	12.8	11.6	9.4	7.4	3.6	241.7
	SD	4.2	3.6	5.9	4.3	3.4	3.8	4.2	4.0	4.6	4.4	2.5	5.5	4.0	19.9
110 mg/kg/day 10 animals	Mean	44.0	38.4	31.8	24.8	19.3	15.1	15.5	16.2	11.9	13.3	10.5	6.8	3.9	251.5
	SD	10.9	7.3	6.8	6.4	5.4	7.0	3.1	5.5	4.5	4.1	5.3	5.4	3.0	48.6
330 mg/kg/day 10 animals	Mean	44.0	37.9	27.9	21.7	19.2	14.0	15.2	12.0	13.4	10.6	12.3	6.8	2.6	237.6
	SD	3.1	2.6	3.0	2.9	3.9	4.4	2.5	3.2	3.4	3.2	3.9	3.7	2.5	11.8
1000 mg/kg/day 10 animals	Mean	43.6	40.5	27.3	25.3	19.7	13.1	8.9*	13.6	11.2	13.8	12.0	7.1	2.4	238.5
	SD	4.9	3.9	4.2	6.1	4.2	3.8	4.5	2.5	4.8	4.7	3.5	3.7	2.9	26.8

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

Sex Female		Day(s) Relative to animal start date
		1-8 [G]	8-15 [G1]	15-22 [G]	22-29 [G]	29-36 [G]	36-43 [G]	43-50 [G1]	50-57 [G]	57-64 [G]	64-71 [G]	71-78 [G]	78-86 [G]	86-91 [G]	1-91 [G1]
0 mg/kg/day 10 animals	Mean	20.5	15.6	7.5	14.3	8.1	2.6	2.1	8.3	5.4	5.7	-0.5	2.0	5.9	97.5
	SD	3.6	5.6	7.8	6.3	4.7	3.7	5.4	4.4	5.2	4.5	5.4	2.8	6.5	9.4
110 mg/kg/day 10 animals	Mean	21.1	17.1	11.2	9.9	10.5	0.0	6.4	4.7	4.6	3.2	4.2	2.3	1.0	98.1
	SD	4.8	7.2	6.2	6.7	4.4	3.0	3.6	2.7	4.0	5.1	3.6	3.7	4.1	9.8
330 mg/kg/day 10 animals	Mean	20.4	15.4	10.4	11.0	10.8	2.7	2.0	7.2	6.1	4.5	2.2	2.4	2.5	97.6
	SD	2.8	4.9	4.7	5.2	4.5	4.8	4.2	4.3	5.4	3.7	4.5	2.4	4.6	8.7
1000 mg/kg/day 10 animals	Mean	20.4	16.1	8.5	13.6	9.4	1.3	0.1	8.9	4.7	5.2	1.8	0.9	3.0	93.9
	SD	2.5	2.4	3.8	4.8	2.9	3.1	3.0	7.1	4.1	1.9	5.8	2.3	5.0	7.9

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

Summary of Body Weights (g)

Sex Female		Day(s) Relative to animal start date
		1	8	15	22	29	36	43	50	57	64	71	78	86	91
0 mg/kg/day 10 animals	Mean	143.1	163.6	179.2	186.7	201.0	209.1	211.7	213.8	222.1	227.5	233.2	232.7	234.7	240.6
	SD	9.2	8.1	11.2	12.2	11.3	11.7	10.8	11.4	11.3	12.5	13.0	14.6	15.2	14.0
110 mg/kg/day 10 animals	Mean	140.4	161.5	178.6	189.8	199.7	210.2	210.2	216.6	221.3	225.9	229.1	233.3	235.6	239.4
	SD	9.6	12.3	12.3	13.5	13.1	15.5	15.5	16.8	16.1	16.6	17.8	15.9	17.0	15.1
	%diff	-1.9	-1.3	-0.3	1.7	-0.6	0.5	-0.7	1.3	-0.4	-0.7	-1.8	0.3	0.4	-0.5
330 mg/kg/day 10 animals	Mean	146.9	167.3	182.7	193.1	204.1	214.9	217.6	219.6	226.8	232.9	237.4	239.6	242.0	244.5
	SD	9.6	10.7	10.5	10.5	11.3	10.8	10.9	10.3	11.3	11.8	13.6	13.3	13.1	14.6
	%diff	2.7	2.3	2.0	3.4	1.5	2.8	2.8	2.7	2.1	2.4	1.8	3.0	3.1	1.6
1000 mg/kg/day 10 animals	Mean	135.3	155.7	171.8	180.3	193.9	203.3	204.6	204.7	213.6	218.3	223.5	225.3	226.2	229.2
	SD	6.8	7.4	8.2	8.0	7.6	8.4	8.0	7.7	9.2	8.1	8.1	9.4	10.3	11.4
	%diff	-5.5	-4.8	-4.1	-3.4	-3.5	-2.8	-3.4	-4.3	-3.8	-4.0	-4.2	-3.2	-3.6	-4.7

Anova & Dunnett

 

Summary of Food Consumption - Daily Food Cons Per Animal (g)

 

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	78-86	86-91	1-91
0 mg/kg/day 2 animals	Mean	14.6	15.1	15.2	16.4	14.8	15.6	15.1	15.7	15.1	15.5	14.6	15.7	14.4	15.2
	SD	0.0	0.1	0.1	0.2	0.1	0.1	0.1	0.2	0.2	0.5	0.8	0.7	0.7	0.3
110 mg/kg/day 2 animals	Mean	13.9	14.7	15.3	16.1	14.8	15.1	14.6	14.8	14.5	14.7	13.6	14.5	13.8	14.7
	SD	0.5	0.9	0.4	1.3	1.1	1.1	1.6	0.7	1.1	1.4	1.2	1.5	1.4	1.1
	%diff	-5.1	-2.7	0.8	-1.6	0.1	-3.4	-3.5	-6.0	-4.2	-5.1	-6.6	-7.7	-4.6	-3.8
330 mg/kg/day 2 animals	Mean	14.6	15.1	15.8	16.9	15.3	15.6	14.6	15.3	15.4	15.0	14.2	15.3	14.4	15.2
	SD	0.8	0.7	1.4	1.1	0.9	0.4	1.3	1.6	1.2	0.8	0.9	0.9	1.3	1.0
	%diff	0.1	-0.4	3.5	2.9	3.4	0.0	-3.5	-2.8	1.8	-3.1	-3.0	-2.5	0.0	-0.3
1000 mg/kg/day 2 animals	Mean	13.7	14.3	14.6	16.1	14.2	14.7	13.5	14.9	14.1	14.4	13.8	14.4	13.9	14.4
	SD	0.2	0.1	0.2	0.3	0.0	0.0	0.5	1.1	0.4	0.4	0.9	0.4	0.2	0.2
	%diff	-6.2	-5.5	-4.0	-1.9	-3.6	-6.1	-10.3	-5.4	-6.8	-6.8	-5.2	-8.5	-3.7	-5.8

 

Summary of Hematology Values - Day: 92 Relative to Start Date

Sex Female		Reporting Hematology
		WBC 10^9/L [G]	LYMPH 10^9/L [G]	MONO 10^9/L [G]	BASO 10^9/L [G]	LUC 10^9/L [G]	MCV (fL) [G]
0 mg/kg/day 10 animals	Mean	4.301	3.649	0.094	0.004	0.031	54.41
	SD	0.908	0.855	0.037	0.005	0.010	0.85
110 mg/kg/day  10 animals	Mean	3.123*	2.484*	0.054**	0.002	0.014**	53.48
	SD	0.773	0.624	0.023	0.004	0.007	1.31
	tCtrl	0.73	0.68	0.57	0.50	0.45	0.98
330 mg/kg/day  10 animals	Mean	3.542	2.910	0.076	0.003	0.026	53.40
	SD	0.877	0.833	0.028	0.005	0.016	0.50
	tCtrl	0.82	0.80	0.81	0.75	0.84	0.98
1000 mg/kg/day 10 animals	Mean	4.276	3.550	0.090	0.006	0.028	52.86**
	SD	1.249	1.096	0.023	0.007	0.012	1.35
	tCtrl	0.99	0.97	0.96	1.50	0.90	0.97

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Summary of Clinical Chemistry Values - Day: 92 Relative to Start Date

Sex male		Reporting biochemistry
		UREA (mmol/L) [G]	T3 (ng/mL) [G1]	T4 (ng/mL) [G]	TSH (mU/mL) [G]
0 mg/kg/day 10 animals	Mean	6.61	0.317	39.99	0.1375
	SD	0.91	0.062	3.85	0.0520
110 mg/kg/day  10 animals	Mean	7.47	0.401*	38.42	0.1074
	SD	1.17	0.052	8.13	0.0639
	tCtrl	1.13	1.26	0.96	0.78
330 mg/kg/day  10 animals	Mean	6.94	0.388	44.45	0.0772
	SD	0.86	0.133	10.02	0.0458
	tCtrl	1.05	1.22	1.12	0.56
1000 mg/kg/day 10 animals	Mean	8.02**	0.309	39.75	0.1577
	SD	0.75	0.077	5.42	0.0827
	tCtrl	1.21	0.97	0.99	1.15

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

Conclusions:

In conclusion, administration of the test substance 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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-29 to 2016-12-20

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: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developm ental Toxicity Screening Test

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 No.of test material: I15AB0305
- Expiration date of the lot/batch: 2017-01-23


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature


OTHER SPECIFICS: Yes, correction factor is 1

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. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 311-315 grams
- 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): 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube

Vehicle:

propylene glycol

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 adjustment was made for specific gravity/density of the test item. Adjustment was made for specific gravity of the vehicle (1.036). A correction
was made for the purity/composition of the test item. A correction factor of 1 was used.


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: 5 mL/kg body weight
- Amount of vehicle (if gavage): 0, 110, 330, 1000 mg/kg

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 (26 October 2016, Day 2 of treatment) according to a validated method (Test Facility Study No. 512709).
Sextuplicate samples (i.e. 3 sets of duplicate samples) were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% 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%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.

Duration of treatment / exposure:

Males-29 days ; 50-56 days (females that delivered); 39 days (females with total litter loss). 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:

Control

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/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of the acute oral
toxicity study (LD50>2000 mg/kg; data on file at Sponsor site)
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Once prior to start of treatment and at weekly intervals during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour ( ± 15 minutes) after treatment (on the peak period of anticipated effects after treatment).

BODY WEIGHT: Yes
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. 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.


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


HAEMATOLOGY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K2-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential white blood 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. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, thyroid hormone analysis

FUNCTIONAL OBSERVATIONS:
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability, pupillary reflex, and static righting reflex, fore- and hind-limb grip strength, locomotor activity. Total movements and ambulations are reported.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes
- mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M),
Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve ( M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)


HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- Additional slides of the testes of the selected 5 males of Groups 1 and 4
- The mammary gland area of female no. 59 with total litter loss
- All gross lesions of all animals (all dose groups)
- The reproductive organs of the male that failed to sire and the female that failed to deliver healthy pups ( cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina)

Other examinations:

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus, bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavern osus muscle complex (LABC), Testes, Thyroid

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test 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.
In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
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 of toxicity were noted during the observation period.
Incidental findings that were noted included rales, scabs, alopecia, salivation and enophthalmos. As these 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, were seen in control animals only or did not show any apparent dose-related trend, they were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 110 mg/kg (no. 59) was sacrificed on Day 2 of the lactation phase due to total litter loss.
All remaining animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were not considered to be affected by treatment.
Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

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.
The statistically significantly lower creatinine level in males at 110 mg/kg was not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
One female at 110 mg/kg (no. 55) showed a notably high bile acid level of 384 umol/L. All other parameters for liver function and liver morphology were normal in this female. This together with the fact that other individual bile acid values of this group remained in the range considered normal for rats of this age and strain and as no dose-response relationship was observed, it was 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, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. At 330 mg/kg, statistically significantly increased grip strength of the foreleg for males (1543 vs. 1316 gram for controls) and total ambulations for females (1077 vs. 611 counts for controls) were noted. These changes were not considered to be toxicologically relevant as they occurred in absence of a dose-related trend.

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 statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

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. The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

There was 1/10 couples (male no. 19 and female no. 59) of the 110 mg/kg group with total litter loss. At histopathology inflammation of the uterine endometrium was noted. This finding was considered to be related to the early post-natal phase at the time of total litter loss and not test item-related. All other tissues (including mammary gland) were normal.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Parental results: No parental toxicity was observed up to 1000 mg/kg. No treatment-related effects were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes were noted in any of the parameters examined in this study.

Key result

Critical effects observed:

no

Conclusions:

In conclusion, treatment with JNJ-42808415-AAA (T003422) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no adverse toxicity up to 1000 mg/kg.
Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be of at least 1000 mg/kg. Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.

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 guidelines

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 0 (vehicle), 110, 330, and 1000 mg/kg bw/day via gavage (OECD 422; Peter, 2017). The vehicle used was propylene glycol and the test solutions were prepared daily within 6 hours prior to dosing.

No parental toxicity was observed up to 1000 mg/kg bw/d. No treatment-related effects were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Based on these results, the NOAEL was concluded to be at least 1000 mg/kg bw/d.

 

In a key, subchronic repeated dose oral toxicity study (OECD 408; Lourens, 2022) the test item was administrated via gavage to male and female Wistar rats for at least 90 days (13 weeks) at the dose levels of 0, 110, 330 and 1000 mg/kg/day/day (10 rats/sex/group). The test item was formulated in propylene glycol. Chemical analyses of formulations were conducted in Weeks 1, 3, 6 and 12 to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously. Test item was not detected in the Group 1 control samples.

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. mortality, clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, 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 substance 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 NOAEL was considered to be a least 1000 mg/kg/day.

 

Repeated 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 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.

 

 

Justification for classification or non-classification

 

Based on the available data and according to the criteria of the CLP Regulation, T003422 should not be classified as STOT RE via the oral route.