Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Deviations:

no

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

Deviations:

no

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

Vehicle:

unchanged (no vehicle)

Details on 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.

Details on mating procedure:

- M/F ratio per cage:1:1 basis within the same treatment group
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged: individually

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 prod uce 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.

Parental animals: Observations and examinations:

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.

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
- How many animals: 5 animals/sex/group
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limbgrip strength, Locomotor activity.

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Parameters examined in F0 male parental generation (control and high dose): testes weight, epididymis weight, spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter 4/sex/litter (as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: postnatal mortality, clinical signs, body weights (PND 1, 4, 7 and 13), sex (PND 1 and 4), Anogenital distance (AGD), presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS:
- The pup that died before scheduled termination was examined externally and sexed (both externally and internally).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after a minimum of 28 days of administration
- Maternal animals: All surviving animals, PND 14-16 or after failure to deliver

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS
- 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
- 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.

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.
- All remaining pups were euthanized on PND 14-16 by an intraperitoneal injection of sodium pentobarbital. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

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).

Reproductive indices:

Mating index (%): (Number of females mated/Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100
Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100

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 postcoitum 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 attri
buted 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 tobe 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

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 300 mg/kg bw/day (with normal litter) during the premating period. Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Precoital time was considered not to be affected by treatment with the test item up to 1000 mg/kg bw/day. All females showed evidence of mating within 4 or 13 days.
Number of implantation sites was considered affected by treatment with the test item from 300 mg/kg bw/day. The number of implantation sites were 13.4, 13.4, 9.8 and 9.9 for the control, 100, 300 and 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 1000 mg/kg bw/day.
For one pup (100 mg/kg bw/day) who was missing on Day 2, absence of milk in the stomach and a pale appearance was noted on Day 1. For one litter (300 mg/kg bw/day), 8 out of 9 pups were dehydrated at first litter check.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item up to 1000 mg/kg bw/day.
Viability indices were 99, 99, 100 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One pup of the control group, one pup at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found missing on PND 2 or 3. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead or missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered affected by treatment with the test item at 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, mean body weights of male and female pups were statistically significantly lower from PND 1 until 13 and were decreased to 0.77-0.81x of control in males, 0.76-0.80x in females and to 0.77-0.82x for males and females combined.
At 100 mg/kg/day, mean body weights of pups were statistically significantly decreased to 0.88x of control in males, females, and males and females combined at PND 13. These changes were considered to be unrelated to treatment with the test item as they occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum T4 concentrations in male PND 14-16 pups was increased by treatment with the test item (1.20x of control) at 1000 mg/kg bw/day.
Serum T4 levels in female PND 14-16 pups were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 1000 mg/kg bw/day.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item up to 1000 mg/kg bw/day.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Litter size was considered affected by treatment with the test item at 300 and 1000 mg/kg bw/day.
Live litter sizes were 12.5, 10.4, 8.8 and 9.1 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The relatively small mean litter size at 100 mg/kg/day could be attributed to the small litter size of one female (i.e. 2 pups). Exclusion of this female results in a mean litter size of 11.6 at 100 mg/kg bw/day. Therefore, the low mean litter size at 100 mg/kg/day was considered not related to treatment with the test item. The lower litter size at 300 and 1000 mg/kg bw/day was considered the result of the lower number of implantation sites and was considered an adverse finding.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

not specified

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 parental 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. The reproduction NOAEL was determined to be 100 mg/kg bw/day based on based on the lower number of implantation sites at 300 and 1000 mg/kg bw/day and the developmental NOAEL was 300 mg/kg bw/day based on lower pup body weights at 1000 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.

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 bw/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).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

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:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

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

Deviations:

no

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 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

Vehicle:

unchanged (no vehicle)

Details on 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.

Details on mating procedure:

- M/F ratio per cage:1:1 basis within the same treatment group
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged: individually

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.

Maternal examinations:

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.

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-females were not fasted overnight.
- How many animals: 5/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-females were not fasted overnight.
- How many animals: 5/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).

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during Week 4 of treatment and the females during the last week of lactation
- How many animals: 5 females/group
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hindlimbgrip strength, Locomotor activity

OESTROUS CYCLE
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

SACRIFICE
- Maternal animals: All surviving animals, PND 14-16 or after failure to deliver

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

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

HISTOPATHOLOGY
- Selected animals: Nasopharynx body cavity, Bone marrow, Femur bone, Sternum bone, Brain (eight levels), Cervix, Eye, Adrenal gland, Mammary gland, Parathyroid gland, Pituitary 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, Thymus, Trachea, Urinary bladder, Uterus, Vagina
- Females that failed to deliver pups: Cervix, ovaries, uterus and vagina.
- Remaining animals: Gross lesions/masses

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter 4/sex/litter (as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: postnatal mortality, clinical signs, body weights (PND 1, 4, 7 and 13), sex (PND 1 and 4), Anogenital distance (AGD), presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS:
- The pup that died before scheduled termination was examined externally and sexed (both externally and internally).

SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.
- All remaining pups were euthanized on PND 14-16 by an intraperitoneal injection of sodium pentobarbital. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

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).

Indices:

Mating index (%): (Number of females mated/Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100
Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100

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 postcoitum 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 bw/day compared to controls at pre-mating Day 1 and were considered unrelated to treatment with the test item. At 100 mg/kg bw/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. 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 bw/day.
At 1000 mg/kg bw/day, mean relative food consumption was considered lower compared with controls from pre-mating Days 1-8 for 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.

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 1000 mg/kg bw/day.
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.

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: -8%, 9%, 19% (absolute) and 3%, 16%, 22% (relative) in females, respectively) and test item-related higher 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 spleen and uterus weight of 300 mg/kg bw/day females; 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 1/10 females of the control group and 1/10 females of the 300 mg/kg bw/day group.

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.
Liver: Hepatocellular hypertrophy was observed in 1000 mg/kg bw/day females up to slight degree (1/5 females minimal and 4/5 females slight degree). The distribution pattern in was females diffuse.
Stomach: 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.
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):

Length and regularity of the estrous cycle were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 300 mg/kg bw/day (with normal litter) during the premating period. Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.Precoital time was considered not to be affected by treatment with the test item up to 1000 mg/kg bw/day. All females showed evidence of mating within 4 or 13 days.
Number of implantation sites was considered affected by treatment with the test item from 300 mg/kg bw/day. The number of implantation sites were 13.4, 13.4, 9.8 and 9.9 for the control, 100, 300 and 1000 mg/kg bw/day.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Number of implantation sites was considered affected by treatment with the test item from 300 mg/kg/day.
The number of implantation sites were 13.4, 13.4, 9.8 and 9.9 for the control, 100, 300 and 1000 mg/kg/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

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered affected by treatment with the test item at 1000 mg/kg bw/day. At 1000 mg/kg bw/day, mean body weights of male and female pups were statistically significantly lower from PND 1 until 13 and were decreased to 0.77-0.81x of control in males, 0.76-0.80x in females and to 0.77-0.82x for males and females combined. At 100 mg/kg bw/day, mean body weights of pups were statistically significantly decreased to 0.88x of control in males, females, and males and females combined at PND 13. These changes were considered to be unrelated to treatment with the test item as they occurred in the absence of a dose related trend.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item up to 1000 mg/kg bw/day.
A slightly skewed sex ratio (41% males / 59% females) was recorded at 300 mg/kg bw/day, however, this was considered to be unrelated to treatment as it occurred in the absence of a dose-related trend.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size was considered affected by treatment with the test item at 300 and 1000 mg/kg/day.
Live litter sizes were 12.5, 10.4, 8.8 and 9.1 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively. The relatively small mean litter size at 100 mg/kg bw/day could be attributed to the small litter size of one female (i.e. 2 pups). Exclusion of this female results in a mean litter size of 11.6 at 100 mg/kg bw/day. Therefore, the low mean litter size at 100 mg/kg bw/day was considered not related to treatment with the test item.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Viability indices were 99, 99, 100 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Serum T4 concentrations in male PND 14-16 pups was increased by treatment with the test item (1.20x of control) at 1000 mg/kg bw/day.
Serum T4 levels in female PND 14-16 pups were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 1000 mg/kg bw/day.
Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

not specified

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).

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 parental 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. The reproduction NOAEL was determined to be 100 mg/kg bw/day based on based on the lower number of implantation sites at 300 and 1000 mg/kg bw/day and the developmental NOAEL was 300 mg/kg bw/day based on lower pup body weights at 1000 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.

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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

(octan-2-yl)benzene

EC Number:

950-576-0

Cas Number:

31047-57-1

Molecular formula:

C14H22

IUPAC Name:

(octan-2-yl)benzene

Test material form:

liquid

Details on test material:

Identification: SHR 1396
Physical Description: Clear colorless liquid
Storage Conditions: At room temperature

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

group 2

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

group 3

Doses / concentrations 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.

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).