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Administrative data

Description of key information

In a combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed according to OECD 422 and GLP principles, the parental NOAEL was set at 100 mg/kg bw/day based on the decreased body weigh (gain) in females at 300 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 November 2019 - 18 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
• EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability at higher temperatures: Yes, maximum temperature: 220°C
- Test item handling: No specific handling conditions required

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Administered as received
Species:
rat
Strain:
other: 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 toxicity testing by regulatory agencies. 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
- Age at study initiation: 10-11 weeks (males); 13-14 weeks (females)
- Weight at study initiation: 299 - 335 g (males); 188 - 248 g (females)
- Fasting period before study: no
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (pretest (females only) and pre-mating period); males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (mating phase); males were housed in their home cage with a maximum of 5 males/cage, females were individually housed in Macrolon plastic cages (post-mating phase); females were housed in Macrolon plastic cages (lactation phase). Pups were housed with the dam, except during locomotor activity monitoring of the dams.; The cages contained appropriate bedding and were equipped with water bottles. Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: ad libitum (except during designated procedures); Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum; Municipal tap water. Periodic analysis of the water was performed and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 23°C (actual range)
- Humidity: 36 to 51% (actual range, The value that was outside the targeted range (40-70%) occurred for 1 day with a minimum of 36% and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study.)
- Air changes: Ten or greater air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 13 December 2019 To: 13 February 2020
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was administered as received. An adequate amount of the test item was dispensed into daily aliquots, which were stored the same as for the bulk test item until use. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.
Duration of treatment / exposure:
Administered for 7 days a week for a minimum of 28 days. Males were treated for 32 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 52-62 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41-42 days.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day Dose Range Finder with oral administration of SHR 1396 in rats (Test Facility Reference No. 20197619), and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:once daily

BODY WEIGHT: Yes
- Time schedule for examinations: first day of treatment (prior to 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.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption: quantitatively measured 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.

WATER CONSUMPTION: Yes
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters: White blood cells (WBC), Reticulocytes (absolute), Neutrophils (absolute), Red Blood Cell Distribution Width (RDW), Lymphocytes (absolute), Haemoglobin, Monocytes (absolute), Haematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular haemoglobin (MCH), Large unstained cells (LUC) (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos).

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. On PND 4 at culling, blood was collected from two surplus pups per litter. On PND 14-16, separate blood samples were collected from two pups per litter.
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
- Parameters: Measurement of total T4 was conducted for F0-males and PND 14-16 pups.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during Week 4 of treatment and the females during the last week of lactation (i.e. PND 6-13)
- How many animals: 5 animals/sex/group
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

ESTROUS CYCLE: Yes
- Time schedule: daily, for all females beginning 14 days prior to treatment and the first 14 days of treatment and during mating until evidence of copulation was observed
- Examination: examining the vaginal cytology of samples obtained by vaginal lavage

COHABITATION/MATING PROCEDURE
- Cohabitation: 1:1 basis within the same treatment group
- Detection of mating: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: Yes
- How many animals: Selected animals (5/sex/group) and non-selected (remaining) animals
- Organs weighed for all selected animals: Brain, Cervix, Epididymis, Adrenal gland, Coagulation gland, Parathyroid gland, Prostate gland, Seminal vesicle gland, Thyroid gland, Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Uterus
- Organs weighed for all remaining animals: Epididymis, Coagulation gland, Parathyroid gland, Prostate gland, Seminal vesicle gland, Thyroid gland, Testes

HISTOPATHOLOGY: Yes
- Selected animals: Nasopharynx body cavity, Bone marrow, Femur bone, Sternum bone, Brain (eight levels), Cervix, Epididymides, Eye, Adrenal gland, Coagulation gland, Mammary gland, Parathyroid gland, Pituitary gland, Prostate gland, Seminal vesicle gland, Thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Cecum, Colon, Rectum, Liver, Lung, Lymph node (mandibular and mesenteric site), Skeletal muscle, Sciatic nerve, Ovaries, Duodenum, Ileum, Jejunum, Spinal cord, Spleen, Stomach, Testes, Thymus, Trachea, Urinary bladder, Uterus, Vagina
- Males that failed to sire (except for males which were selected) and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Remaining animals: Gross lesions/masses.
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. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

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.
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related salivation was observed during daily detailed clinical observations at 100, 300 and 1000 mg/kg bw/day.
Any other clinical signs noted during the treatment 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. No findings were noted during the weekly arena observations in this study.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain were considered affected by treatment with the test item in females at 300 and 1000 mg/kg bw/day.
At 1000 mg/kg bw/day in females, mean body weights were decreased to 0.88x of control on post-coitum Day 20 and to 0.92x of control on lactation Day 1. In addition, mean body weight gain was decreased to 0.72x of control at 1000 mg/kg bw/day in females at post-coitum Day 20.
At 300 mg/kg bw/day in females, mean body weights and body weight gain were decreased to 0.85x and 0.78x of control, respectively on post-coitum Day 20. Mean body weights were also statistically significant decreased to 0.87-0.91x of control from post-coitum Day 0-17 and lactation Day 1-4. However, since mean body weight gain was decreased at post-coitum Day 20 only, the statistical significant changes in mean body weight that were recorded before post-coitum Day 20 were attributed to the lower mean body weight at 300 mg/kg/day compared to controls at pre-mating Day 1 and were considered unrelated to treatment with the test item.
At 100 mg/kg/day in females, mean body weight was decreased to 0.92x of control at postcoitum Day 20. This change was attributed to the lower mean body weight at 100 mg/kg bw/day compared to control at pre-mating Day 1 and was therefore considered to be unrelated to treatment with the test item.
In males, mean body weight gain was slightly decreased at 1000 mg/kg/day on pre-mating Day 8. This relative change was attributed to the higher mean male body weight at 1000 mg/kg bw/day compared to controls at pre-mating Day 1 and was therefore considered to be unrelated to treatment with the test item.
Any other statistically significant changes in body weights and body weight gain were considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and duration of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption after correction for body weight was considered affected by treatment with the test item at 1000 mg/kg bw/day.
Absolute food consumption was unaffected by treatment with the test item up to 1000 mg/kg/day.
At 1000 mg/kg bw/day, mean relative food consumption was considered lower compared with controls from pre-mating Days 1-8 for males and females (0.86x and 0.78x of control, respectively) and recovered to normal levels in the following days. In addition, mean relative food consumption was lower in females between lactation Days 7-13 (0.81x of control) at 1000 mg/kg bw/day.
The decrease in mean food consumption observed for females during post-coitum Days 0-7 at 300 mg/kg bw/day was considered unrelated to treatment with the test item in absence of a doserelated response.
The increase in mean relative food consumption for females during post-coitum Days 17-20 at 300 mg/kg bw/day was considered to be unrelated to treatment with the test item since no trend was apparent regarding dose and duration of treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, mean percentage red blood cell distribution width (RDW) was higher (1.14x) in females compared to controls. As the mean value remained within the historical control range, the mean value of the concurrent control was relatively low (based on the historical control range) and no other concurrent findings were observed, this change was considered not treatment related.
The observed decreases in mean reticulocyte concentration in males at 100, 300 and 1000 mg/kg bw/day were considered unrelated to treatment with the test item in absence of a dose related response and as all values remained within the historical control range.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered affected by treatment with the test item at 300 and 1000 mg/kg bw/day.
In males, mean bile acid concentrations were decreased at 100, 300 and 1000 mg/kg bw/day (0.41, 0.39 and 0.46x of control, respectively) and in females, mean cholesterol concentration was decreased (0.67x of control) at 1000 mg/kg bw/day. As all mean values remained within the historical control range, these changes were considered not toxicologically relevant.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
A treatment-related decrease was observed in mean serum T4 concentrations at 300 and 1000 mg/kg bw/day in F0-males (0.74 and 0.78x of control, respectively).
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 were normal in all examined animals up to 1000 mg/kg/day. Grip strength and motor activity was similar between control and high dose animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and/or relative to body weights) were noted in the 100, 300 and 1000 mg/kg bw/day group (Mean percent weight differences: 16%, 15%, 48% (absolute) and 17%, 26%, 53% (relative) in males and -8%, 9%, 19% (absolute) and 3%, 16%, 22% (relative) in females, respectively) and test item-related higher kidney weights (11% (absolute) and 16% (relative) in males) or adrenal gland weights (17% (absolute) and 22% (relative) in females) were noted in the 1000 mg/kg bw/day group.
Any other differences, including those that reached statistical significance (absolute brain, and heart weight of 100 mg/kg bw/day females; absolute heart weight of 300 mg/kg bw/day males and absolute spleen and uterus weight of 300 mg/kg bw/day females; absolute prostate gland weight of 1000 mg/kg bw/day males and relative spleen weight of 1000 mg/kg bw/day females) were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related enlargement of the liver was observed in 3/10 males treated at 1000 mg/kg bw/day.
Findings of note were present in the liver consisting of an accentuated pattern. This finding was present in 1/10 females of the control group, in 1/10 males at 100 mg/kg bw/day, 1/10 males and 1/10 females at 300 mg/kg bw/day and in 4/10 males at 1000 mg/kg bw/day. The somewhat higher incidence of this finding in 1000 mg/kg bw/day males was without microscopic correlate and since this finding can be found in control rat as well, regarded as unrelated to treatment with the test item.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test item were noted in the liver, stomach and kidneys of the 300 and/or 1000 mg/kg bw/day group males and/or females.
Liver: Hepatocellular hypertrophy was observed in 300 mg/kg bw/day males at minimal degree (3/10 animals) and in 1000 mg/kg bw/day rats of both sexes up to slight degree (7/7 males slight degree; 1/5 females minimal and 4/5 females slight degree). The distribution pattern in males was centrilobular and in females diffuse.
Liver: An increased incidence and severity of single cell necrosis (up to slight) was observed in 1000 mg/kg bw/day males (4/7 males minimal and 2/7 males slight degree). The minimal degrees of single cell necrosis noted in a few rats of the control (2/5 males), 100 (1/5 males) or 300 mg/kg bw/day group (2/5 males) were regarded to be within background range and unrelated to treatment with the test item.
Stomach: A single male and a single female of the 1000 mg/kg/day group showed test item related findings in the forestomach (i.e. non-glandular stomach) in the form of squamous cell hyperplasia with hyperkeratosis and subepidermal lymphogranulocytic infiltrate.
Kidneys: Increased incidences and severity of hyaline droplet accumulation (up to moderate) were noted in 300 and 1000 mg/kg bw/day males (1/5 minimal and 4/5 slight at 300 mg/kg bw/day and 1/5 minimal, 1/5 slight and 3/5 moderate at 1000 mg/kg bw/day, respectively. Controls: 3/5 males minimal degree).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycle: Length and regularity of the estrous cycle were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no

Historical control data for Wistar Han rats; F0-animals (period 2017-2019):

Red blood cell distribution width (%) – females: mean = 12.6; P5 – P95 = 10.60 – 14.70 (n=308).

Reticulocytes (109/L) – males: mean = 220.7; P5 – P95 = 163.70-289.20 (n=331)

Bile acids (μmol/L) – males: mean = 26.20; P5 – P95 = 9.70 – 54.00 (n=348).

Cholesterol (mmol/L) – females: mean = 2.05; P5 – P95 = 1.43 – 2.81 (n=325).

Summary of the Dose Range Finder (DRF)

A dose range finder (Test Facility Reference No. 20197619) was conducted to select dose levels for the main study (Test Facility Study No. 20197624), and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only.

If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study.

Dose Formulations

The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.

Test System

Female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. Females were 11-12 weeks old and weighed between 204 and 220 g at initiation of dosing.

On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages. At study assignment, each animal was identified using earmark and tailmark.

The actual daily mean temperature during the study period was 20 to 21°C with an actual daily mean relative humidity of 49 to 54%.

Experimental Design

Group No.

Dose Level

(mg/kg/day/day)a

Dose Volume

(mL/kg)

Number of

Females

Animal

Numbers

 1

 500

 0.583

 3

 1-3

 2

 1000

 1.167

 3

 4-6

a Dose volume was calculated as dose level (g/kg) / specific gravity (factor: 0.857)

The test item was administered to the appropriate animals by once daily oral gavage for 14 consecutive days.

The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg/day, Test Facility Study No. 20197470).

In-life procedures

Mortality: Twice daily throughout the study.

Clinical Observations: At least daily from Days 1-14, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.

Body Weights: On Day 1 prior to dosing and on Days 5, 10, 12 and 14.

Food Consumption Over Days 1-5, 5-10, 11-12 and 12-14.

Terminal Procedures

All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy, for deviations, see Appendix 7). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed.

Results

 Parameter  500 mg/kg bw/day  1000 mg/kg bw/day
 Mortality  No mortality No mortality 
 Clinical appearance

Hunched posture was recorded from

Day 2 of treatment onwards for all

animals. Piloerection was recorded

between Days 1-13 for all animals.

Uncoordinated movements, quick

breathing and salivation were

incidentally recorded for all animals.

Hunched posture and piloerection were

recorded from Day 2 onwards for all

animals. Quick breathing was recorded

between Days 3-13 for all animals.

Uncoordinated movements and

salivation were incidentally recorded for

some or all animals throughout the

study.

 

 Body weight

In 1/3 animals, a lower body weight

(0.86x) was recorded at Day 5 which

recovered to 0.98x at Day 14

compared to Day 1 of treatment.

In 1/3 animals, body weight did not

change between Days 1-12 and a slight

body weight loss (0.99x) was recorded

at Day 14 of treatment.

Body weight loss (0.97x) was recorded

in 2/3 females on Day 5 of treatment,

followed by recovery of body weight

gain (1.05-1.07x) on Day 14 of

treatment.

 

 Food consumption

Absolute and relative food

consumption was increased over Days

1-5 of treatment and recovered to

normal levels over Days 5-14.

Absolute and relative food consumption

was increased over Days 1-5 of

treatment and recovered to normal levels

over Days 5-14.

 Macroscopic

examination

No abnormalities noted.

No abnormalities noted. 

 Organ weights

Increased absolute and relative liver

and kidney weights.

Increased absolute and relative liver

weights. Kidney weights considered to

be normal.

Conclusion

Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg/day.

Since no clear peak effect of occurrence of clinical signs was observed, clinical observations were conducted and functional observations were started in the main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

Conclusions:
Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed according to OECD 422 and in accordance with GLP principles, the NOAEL of SHR 1396 is considered to be 100 mg/kg bw/day based on the decreased body weigh (gain) in females at 300 mg/kg bw/day.
Executive summary:

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD 422 and in accordance with GLP principles. Male and female rats (10/dose) were exposed via gavage to 0 (control), 100, 300 and 1000 mg/kg bw/day.

There were no unscheduled deaths. Parental toxicity was observed at 300 and 1000 mg/kg bw/day. In females, body weight was decreased at 300 mg/kg/day at post-coitum Day 20 and lactation Days 1-4 and at 1000 mg/kg bw/day at post-coitum Day 20 and lactation Day 1. In addition, body weight gain was decreased at 300 and 1000 mg/kg bw/day at post-coitum Day 20. At the magnitude of the effect, these changes were considered adverse. At 1000 mg/kg bw/day in males, an increase in liver weight, enlargement of liver and centrilobular hepatocellular hypertrophy up to slight degree, which was often accompanied by single cell necrosis in the same area, were observed and the combination of these findings was regarded to be adverse.

In this study, a decrease of total T4 was observed at 300 and 1000 mg/kg/day in parental males which was considered to be test item-related. However, under the conditions of this screening study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental NOAEL.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for SHR 1396 were established: Parental NOAEL 100 mg/kg bw/day (based on the decreased body weight (gain) in females at 300 mg/kg bw/day), Reproduction NOAEL 100 mg/kg bw/day (based on the lower number of implantation sites at 300 and 1000 mg/kg bw/day), Developmental NOAEL 100 mg/kg bw/day (based on the lower litter size, which was linked to the lower number of implantation sites, at 300 and 1000 mg/kg bw/day).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD 422 and in accordance with GLP principles. Male and female rats (10/dose) were exposed via gavage to 0 (control), 100, 300 and 1000 mg/kg bw/day. There were no unscheduled deaths. Parental toxicity was observed at 300 and 1000 mg/kg bw/day. In females, body weight was decreased at 300 mg/kg/day at post-coitum Day 20 and lactation Days 1-4 and at 1000 mg/kg bw/day at post-coitum Day 20 and lactation Day 1. In addition, body weight gain was decreased at 300 and 1000 mg/kg bw/day at post-coitum Day 20. At the magnitude of the effect, these changes were considered adverse. At 1000 mg/kg bw/day in males, an increase in liver weight, enlargement of liver and centrilobular hepatocellular hypertrophy up to slight degree, which was often accompanied by single cell necrosis in the same area, were observed and the combination of these findings was regarded to be adverse.

Reproduction toxicity was observed at 300 and 1000 mg/kg bw/day, and consisted of a decreased number of implantation sites. Litter size was decreased at 300 and 1000 mg/kg/day, which was considered the result of the lower number of implantation sites and was considered an adverse finding. No treatment-related or toxicologically relevant changes were noted in any of the remaining reproductive parameter investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental toxicity was observed at 1000 mg/kg bw/day and consisted of lower pup body weights. No treatment-related or toxicologically relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for SHR 1396 were established: Parental NOAEL 100 mg/kg bw/day (based on the decreased body weight (gain) in females at 300 mg/kg bw/day), Reproduction NOAEL 100 mg/kg bw/day (based on the lower number of implantation sites at 300 and 1000 mg/kg bw/day), Developmental NOAEL 300 mg/kg bw/day (based on the lower pup body weights at 1000 mg/kg bw/day).

Justification for classification or non-classification

Based on the available study results, SHR 1396 does not have to be classified and has no obligatory labelling requirement according to Regulation (EC) No 1272/2008 and its amendments.