tert-dodecanethiol

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and developmental immunotoxicity (Cohorts 1A, 1B with extension, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 June 2019 to 1 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Remarks:

Study performed in accordance with ECHA Decision No. TPE-D-2114394439-33-01/F. The decision to include an F2 generation was based on observations in the study (see section "Justification for study design").

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks before mating: In ECHAs decision on a testing proposal (TPE-D-2114394439-33-01/F), ECHA noted that ten week predating exposure duration is required because there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7a. Ten weeks exposure duration is supported also by the lipophilicity of the substance (log Kow of 7.43) to ensure that the steady state in parental animals has been reached before mating.
- Basis for dose level selection: Dose levels were based on the results of previous toxicology studies performed in the same species by gavage. Based on these data, the selected dose levels were: 25, 75 and 200 mg/kg bw/day.
- Inclusion of extension of Cohort 1B: In ECHAs decision on a testing proposal (TPE-D-2114394439-33-01/F), ECHA concludes that Cohort 1B must not be extended to include mating of the animals and production of the F2 generation. However, in the study report it is justified that "Given the effects noted on the body weight evolution in pups at birth and throughout the lactation period, the decision was taken to include an F2 generation in the study"

- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: Because the criteria as described in column 2 of Section 8.7.3 of Annex X and detailed in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a, Section R.7.6 (version 6.0, July 2017) were not met (TPE-D-2114394439-33-01/F). As such, there was no particular concern on (developmental) neurotoxicity.
- Inclusion of developmental immunotoxicity Cohort 3: In ECHAs decision on a testing proposal (TPE-D-2114394439-33-01/F), ECHA concluded that the developmental immunotoxicity Cohort 3 needs to be conducted because there is a particular concern on (developmental) immunotoxicity based on the results from a sub-chronic toxicity (90-day) inhalation study (2017) with the registered substance. This study showed evidence of toxicity in thymus and spleen. Specifically, a significant decrease in the thymus weight (-35%) in females, was reported for the high-dose group (100 ppm). ECHA noted that in the females of the high-dose group there was no exposure-related body weight changes. Hence, the 35% reduction in the thymus weight indicates concern for developmental immunotoxicity.
- Route of administration: The oral route was selected as it is the appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 6.0, July 2017) Chapter R.7a. Since the substance to be tested is a liquid, ECHA concludes that testing should be performed by the oral route.
- Other considerations: The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected as background data from previous studies are available at Charles River Laboratories Evreux.

Test material

Test material information

Constituent 1

Reference substance name:

tert-dodecanethiol

EC Number:

246-619-1

EC Name:

tert-dodecanethiol

Cas Number:

25103-58-6

Molecular formula:

C11H24S to C13H28S

IUPAC Name:

2-methylundecane-2-thiol

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males and females approximately 6 and 5 weeks old, respectively
- Weight at study initiation: Males: 220 to 262 g; Females: 153 to 190 g
- Fasting period before study: no
- Housing: The P and Cohort 1B animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (Le Comptoir des Sciures, Meyzieu, France).
Cohort 1A and 3 animals were group-housed in 2000P polycarbonate cages (2035 cm2) with stainless steel lids (4/sex per cage for cohort 1A and 5/sex per cage for cohort 3) containing autoclaved sawdust (Le Comptoir des Sciures, Meyzieu, France).
Individual housing was chosen as it is preferable for pregnant, littering and lactating females in order not to jeopardize the gestation, littering and lactation phases, and to avoid aggressive behavior around mating in males.
Toward the end of gestation and during lactation, autoclaved wood shavings (Le Comptoir des Sciures, Meyzieu, France) were provided as nesting material for the females and their litters.
Each cage contained a rat hut and a cocoon (P generation) or a rat hut and a nylabone (Cohorts 1A, 1B and 3) for environmental enrichment. The cages were placed in numerical order on the racks.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly, ad libitum. No contaminants were present in the diet at levels could have been expected to interfere with or prejudice the outcome of the study.
- Water (e.g. ad libitum): Tap water (filtered with a 0.22 µm filter), ad libitum. No contaminants were present in the drinking water at levels could have been expected to interfere with or prejudice the outcome of the study.
- Acclimation period: 8 days before treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (target)
- Humidity (%): 50 ± 20% (target)
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h
IN-LIFE DATES: From: 20 June 2019 To: 21 Febuari 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) Carboxymethylcellulose (400-800 cps) / 0.5% (v/v) Tween 80 in drinking water treated by reverse osmosis

Details on exposure:

FORMULATION PROCEDURE
Type of test item formulation (visual observation): Emulsion in the vehicle (0, 5, 15 and 40 mg/mL)
Preparation procedure: According to Charles River Laboratories Evreux/Study No. 46439 AHS (homogeneity testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.
Frequency of preparation: Test item dose formulations: on the days of treatment; Control dose formulation: according to the vehicle expiry date
Delivery conditions (control and test item dose formulations): At room temperature and protected from light. Test item dose formulations were used within 4 hours after the end of their preparation

ADMINISTRATION
The dose formulations were administered by gavage using a plastic syringe fitted with a plastic gavage tube (juvenile gavage tubes from Days 22 to 33 p.p. and adult gavage tubes from Day 34 p.p. onwards), once a day, at approximately the same time of day.
The quantity of dose formulations administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage volume of 5 mL/kg/day was used. Control animals (group 1) received the vehicle only.
The dose formulations were maintained under delivery conditions (at room temperature, protected from light) throughout the administration procedure. The control and test item dose formulations were stirred for at least 15 minutes before administration and throughout the administration procedure. The test item dose formulations were administered within 4 hours after the end of their preparation.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until mating, with a maximum of 14 consecutive days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD0)
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: -

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical technique: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Determination of test item concentrations in dose formulations:
Nine analyses: two during the P-premating period and one during each of the following periods: P mating, P gestation, P-lactation, F1-postweaning, F1 premating, F1-gestation and F1-lactation. A sample was taken from control and test item dose formulations and analyzed using the validated method.
Acceptance criteria: Measured concentration = nominal concentration ± 15%

Duration of treatment / exposure:

P generation and F1 lactating offspring:
Daily for 10 weeks prior to pairing, during pairing (males and females), and through gestation and lactation until weaning of the F1 pups [Day 21 post partum (p.p.)]. A control group of 24 males and 24 females received the vehicle only, under the same experimental conditions. (Females with no evidence of mating or no delivery were euthanized 24-26 days after the last day of the mating period).
F1 generations:
• Cohort 1A: daily from weaning (Day 22 p.p.) until euthanasia (on Days 91 to 100 p.p.),
• Cohort 1B: daily from weaning (Day 22 p.p.) until euthanasia. Males were treated from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks) and until after euthanasia of F2 pups (on Day 4 p.p.). Females were treated from weaning (Day 22 p.p.) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation and during lactation (until Day 4 p.p. inclusive) or until euthanasia for females with no evidence of mating or no delivery (25 days after the last day of the mating period),
• Cohort 3: daily from weaning (Day 22 p.p.) until 5 days after Keyhole Limpet Hemocyanin (KLH) injection (between Day 53 and Day 57 p.p.), when these animals were euthanized (between Days 58 and 62 p.p.).

Frequency of treatment:

Daily

Details on study schedule:

in the P males (at least 18 weeks of treatment):
- 10 weeks before mating,
- during the mating period (up to 2 weeks),
- until euthanasia (after euthanasia of the P females),

in the P females (at least 16 to 18 weeks of treatment):
- 10 weeks before mating,
- during the mating period (up to 2 weeks),
- during gestation,
- during lactation until Day 21 p.p. inclusive (or Day 22 p.p., after hematology, coagulation,
blood chemistry and urinalysis on Day 23 p.c. for the selected females) or until
euthanasia,
- until euthanasia for females with no evidence of mating or no delivery (24-26 days after
the last day of the mating period).

Day 1 corresponds to the first day of the treatment period.
Day 0 p.c. corresponds to the day of confirmed mating.
Day 0 p.p. corresponds to the day on which parturition occurs.
Day 1 p.p. corresponds to the day on which parturition is completed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

P: 24/sex/dose
F1: 20/sex/dose (Cohort1A and 1B); 10/sex/dose (Cohort 3)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a two-week study, the test item was administered daily by gavage to five males and five females at dose levels of 100, 300 or 1000 mg/kg bw/day. The test item induced ptyalism at 300 and 1000 mg/kg bw/day, hard/swollen abdomen at 1000 mg/kg bw/day (last day of treatment only), lower body weight gain (-50% vs. controls) and lower food consumption ( 35% vs. controls) mainly at 1000 mg/kg bw/day between Days 1 and 4. At = 100 mg/kg bw/day, squamous cell hyperplasia and hyperkeratosis in the forestomach and centrilobular hepatocellular hypertrophy in the liver were observed in both sexes, and tubular hyaline droplets accumulation and degeneration/regeneration were observed in the kidneys in males only. Findings in the forestomach and kidneys were considered to be adverse at = 300 mg/kg bw/day. At 1000 mg/kg bw/day, hepatocellular hypertrophy associated with subcapsular hepatocellular necrosis was considered to be adverse, and adverse ulceration was present in the stomach. The No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg bw/day.

In the OECD 422 study, Tert-dodecanethiol was administered by gavage to SD rats at 0, 50, 100 or 200 mg/kg bw/day for 8 to 10 (females) or 10 (males) weeks (before mating, during mating, gestation and lactation), and the following observations were made:
F0 animals:
No test item-related deaths were reported. Ptyalism was observed at 100 and 200 mg/kg bw/day throughout the study. No effects were noted in the functional observation battery/motor activity, body weight, food consumption, estrus cycle, mating, fertility, delivery data or laboratory investigations. Increased liver weights were noted in males and/or females at = 50 mg/kg bw/day, and increased kidney weights were noted in males at = 100 mg/kg bw/day. No test item-related macroscopic post-mortem changes were noted. Test item-related microscopic observations included adverse renal changes in males at = 50 mg/kg bw/day (mainly degeneration/necrosis, accumulation of hyaline droplets, tubular vacuolation), non-adverse hepatocellular hypertrophy in the liver, follicular hypertrophy in the thyroid glands, hyperkeratosis and squamous cell hyperplasia in the forestomach at = 50 mg/kg bw/day in males and at = 100 mg/kg bw/day in females, atrophy of stomach glands in males at 20 mg/kg bw/day and increased vaginal mucification at = 100 mg/kg bw/day in females.
Pups:
No effects on mortality, viability, clinical signs, sex ratio or anogenital distance were observed from birth until Day 21 p.p. in the pups. The only observation consisted of a lower body weight at birth in male and female pups at 100 and 200 mg/kg bw/day (-11 to -7%), followed by lower body weight gain throughout the lactation period, resulting in a lower body weight on Day 21 p.p. (around -10%). Thyroid hormone analysis did not reveal any disturbances on Day 21 p.p. in pups.
- Fasting period before blood sampling for clinical biochemistry: Prior to blood sampling and urine collection at termination, the first 10 surviving males and lactating females per P generation group and the first 10 surviving Cohort 1A animals per sex per group were deprived of food for an overnight period of at least 14 hours.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals once a week until the end of the study
- Observations: included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male was weighed once during the acclimation period, on the first day of treatment, then at least once a week until euthanasia
Each female was weighed as follows once during the acclimation period, on the first day of treatment, once a week until mated (or until euthanasia for females with no evidence of mating), and then on Days 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period and then after the mating period until euthanasia.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14 21 p.p.
During the mating period, food consumption was not measured for males or females

WATER CONSUMPTION: No

HEMATOLOGY: Yes
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Red blood cell count, Mean cell volume, Packed cell volume (hematocrit), Hemoglobin, Mean cell haemoglobin concentration, Mean cell haemoglobin, Trombocyte (platelet) count, Total leucocyte count, Reticulocytes and Differential white cell count with cell morphology (Neutrophils, Eosinophils, Basophils, Lymphocytes, Large unstained cells, Monocytes).

COAGULATION: Yes
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Prothrombin time (blood clotting time), Fibrinogen, and Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total cholesterol, Triglycerides, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Total proteins, Albumin, Albumin/globulin ratio, and Bile acids.

URINALYSIS: Yes
For urine collection, the animals were individually placed in metabolism cages an overnight period of at least 14 hours. The urine was collected onto thymol crystals.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Appearance, Color, Volume, pH, Specific gravity, Proteins, Glucose, Ketones, Bilirubin, Nitrites, Blood (hemoglobin), Urobilinogen (non-GLP), and Cytology of sediment (Leucocytes, Erythrocytes, Cylinders, Magnesium ammonium phosphate crystals, Calcium phosphate crystals, Calcium oxalate crystals, Epithelial cells).

THYROID HORMONE ANALYSIS: Yes
Blood samples were taken from the orbital sinus of 10 male and 10 lactating female animals (between 07:30 and 10:00 a.m.) under isoflurane anesthesia into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: No, except for animals also sampled for hematology, blood biochemistry and urinalysis.
Parameters analysed: thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning as follows:
• during the last 2 weeks of the premating period,
• during the mating period, until the females were mated or the mating period had ended.

Sperm parameters (parental animals):

Parameters examined in P and Cohort 1A male parental generations from the control and high-dose groups (groups 1 and 4): testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, and sperm morphology.
Sperm motility and morphology investigations of both the testis and epididymis were also performed on all surviving P and Cohort 1A males from the low- and intermediate-dose groups (groups 2 and 3).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To obtain as nearly as possible 5 males and 5 females per litter; partial adjustment (for example 6 males and 4 females) was permitted; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
detailed clinical observations at least once daily, including gross external examination (e.g. external visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture; presence of umbilical cord, lack of milk in the stomach, presence of dried secretion), qualitative assessment of body temperature, activity and reaction to handling, body with on 1, 4, 7, 14, 21 p.p., anogenital distance (AGD, on 1 p.p.), and number of nipples and areolae in male pups on 12 p.p.

GROSS EXAMINATION OF DEAD PUPS:
Yes, included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes
On Days 50-57 p.p., a subcutaneous injection of Keyhole Limpet Hemocyanin (KLH) was performed and specific IgM anti-KLH antibodies were measured using specific ELISA methods to evaluate the primary antibody response five days after immunization (Cohort 3).

F1 GENERATION FROM WEANING ONWARD: Cohorts 1A, 1B (20 animals/sex/dose), Cohort 3 (10 animals/sex/dose). Day 22 p.p. was designated Day 1 of the F1 generation.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity at least twice a day during the treatment period, including weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals once a week until the end of the study
- Observations: included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also evaluated.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male (Cohorts 1A, 1B and 3) was weighed on the first day of treatment, then at least once a week until euthanasia
Each female was weighed on the first day of treatment, once a week (Cohorts 1A, 1B and 3) until mated (Cohort 1B) or until euthanasia for female(s) with no evidence of mating, and then on Days 0, 4, 7, 10, 14, 17 and 20 p.c. and on Days 1 and 4 p.p. (Cohort 1B).

FOOD CONSUMPTION: Yes
The quantity of food consumed by each male was measured once a week from the first day of treatment (Cohorts 1A, 1B and 3). For cohorts 1A and 3, food consumption was measured until euthanasia. For cohort 1B, food consumption was measured until the start of the mating period and after the mating period until euthanasia.
The quantity of food consumed by each female was measured once a week from the first day of treatment (Cohorts 1A, 1B and 3). For cohorts 1A and 3, food consumption was measured until euthanasia. For Cohort 1B, food consumption was measured until the start of the mating period, and then during gestation for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the interval: Days 1-4 p.p.
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION: No

SEXUAL DEVELOPMENT: Yes
All males were observed each day from Day 35 p.p. (i.e. Day 14 of the F1 generation) until cleavage of the balanopreputial groove (preputial separation) was observed or until scheduled euthanasia if no cleavage was observed (cohorts 1A and 3) or until mating (Cohort 1B).
All females were observed each day from Day 25 p.p. (i.e. Day 4 of the F1 generation) until vaginal opening was observed or until scheduled euthanasia if no opening was observed (cohorts 1A and 3) or until mating (Cohort 1B).

HEMATOLOGY: Yes (Cohort 1A)
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Red blood cell count, Mean cell volume, Packed cell volume (hematocrit), Hemoglobin, Mean cell haemoglobin concentration, Mean cell haemoglobin, Trombocyte (platelet) count, Total leucocyte count, Reticulocytes and Differential white cell count with cell morphology (Neutrophils, Eosinophils, Basophils, Lymphocytes, Large unstained cells, Monocytes).

COAGULATION: Yes (Cohort 1A)
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Prothrombin time (blood clotting time), Fibrinogen, and Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes (Cohort 1A)
Blood samples were taken from the orbital sinus of 10 male and 10 female animals (under light isoflurane anesthesia) into tubes containing the appropriate anticoagulant.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total cholesterol, Triglycerides, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Total proteins, Albumin, Albumin/globulin ratio, and Bile acids.

URINALYSIS: Yes (Cohort 1A)
For urine collection, the animals were individually placed in metabolism cages an overnight period of at least 14 hours. The urine was collected onto thymol crystals.
Time point: Day of necropsy
Fasting: yes, overnight period of at least 14 hours
Parameters analysed: Appearance, Color, Volume, pH, Specific gravity, Proteins, Glucose, Ketones, Bilirubin, Nitrites, Blood (hemoglobin), Urobilinogen (non-GLP), and Cytology of sediment (Leucocytes, Erythrocytes, Cylinders, Magnesium ammonium phosphate crystals, Calcium phosphate crystals, Calcium oxalate crystals, Epithelial cells).

THYROID HORMONE ANALYSIS: Yes (F1 Culled Pups, Cohort 1A Animals and F2 Pups)
Blood samples were collected between 07:30 and 10:00 a.m.
Fasting: No, except for animals also sampled for hematology, blood biochemistry and urinalysis.
From F1 culled pups and from euthanized F2 pups (as much blood as possible was collected by decapitation under isoflurane anesthesia and then pooled per litter) for measurement of thyroid hormone (T4) levels.
Time point: day 4 p.p.
Parameters analysed: thyroid hormone (T4)
From F1 pups not selected for cohorts samples were collected from the vena cava immediately after euthanasia and then pooled per litter
Time point: day 22 p.p.
Parameters analysed: thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels.

Postmortem examinations (parental animals):

SACRIFICE
Prematurely euthanized males and females were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
On completion of the treatment period, all surviving P generation animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
- Male animals: All surviving animals after weaning of the F1 progeny.
- Female animals: All surviving animals which delivered on Day 23 p.p.; Animals that dit not deliver on days 24-26 p.c. or 25 days after the end of the mating period if no evidence of mating; Animals with total litter loss as appropriate.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all P animals.
- Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of implantation sites were recorded for females euthanized on Day 23 p.p.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissue collection and preservation according to guideline.

Postmortem examinations (offspring):

SACRIFICE
Prematurely euthanized males and females were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
On completion of the treatment period, all surviving F1 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
• F1 males-Cohort 1A: on Days 91-99 p.p.,
• F1 males-Cohort 1B: after euthanasia of the F2 progeny,
• F1 females-Cohort 1A: on Days 91-100 p.p.,
• F1 females-Cohort 1B: on Day 4 p.p.,
• F1 animals-Cohort 3: 5 days after KLH injection and after blood sampling (i.e. between Day 58 and Day 62 p.p.),
• any Cohort 1B females which did not deliver: on Day 26 p.c. (after body weight recording to check for a possible unnoticed delivery),
• Cohort 1B females with no evidence of mating: 25 days after the end of the mating period,
• any Cohort 1B females with total litter loss: as appropriate.
F2 pups were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
• any pups whose dam died: as soon as possible,
• pups: on Day 4 p.p.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all F1 animals (including F1 pups culled on Day 4 p.p. and non-selected F1 pups on Day 22 p.p.), all F2 pups, and any prematurely euthanized or found dead pups.
- Gross necropsy consisted of examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of implantation sites were recorded for females euthanized on Day 23 p.p. (P generation) or on Day 4 p.p. (Cohort 1B).

HISTOPATHOLOGY / ORGAN WEIGTHS
Tissue collection and preservation according to guideline.

Statistics:

Body Weight, Food Consumption and Reproductive data were compared by one-way analysis of variance and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
Statistical analysis of organ weight data (level of significance of 0.05 or 0.01) was performed according to a decision logic as indicated in sections 6.16.2 in the study report.
Statistical analysis of Splenic Lymphocyte Immunophenotyping was performed according to a decision logic as indicated in sections 6.16.3 in the study report.
Statistical analysis according to a decision logic as indicated in sections 6.16.4 in the study report was used for: 6.16.4. Ano-genital Distance, Number of Nipples and Areolae, Time of Preputial Separation/Vaginal Opening, Time to First Estrous after Vaginal Opening/Patency, Seminology, Hematology, Coagulation, Blood Biochemistry, Urinalysis, Thyroid Hormones, Anti KLH IgM, Primary Follicles and Post-Implantation Loss.

Reproductive indices:

The following parameters were calculated:
post-implantation loss:
(Number of implantation sites - Number of live pups / Number of implantation sites) x 100
mating index:
(Number of mated animals / Number of paired animals) x 100
fertility index:
(Number of pregnant female partners / Number of mated pairs) x 100
gestation index:
(Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

live birth index:
(Number of live pups on Day 1 p.p. / Number of delivered pups) x 100
viability index on Day 4 p.p.:
(Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups) x 100
lactation index:
(Number of surviving pups on Day 21 p.p. / Number of surviving pups on Day 4 p.p. (after culling)) x 100
AGD/cube root of body weight ratio (calculated with Excel):
AGD / ³vBody weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was noted after treatment in all groups (including controls) with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect. On rare occasions, ptyalism was also noted before treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

None of the deaths were attributed to the test item treatment because reproductive issues were observed in control and treated females or because no evident cause of death was identified.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant effects were noted on mean body weight or mean body weight change at any dose level during the treatment period in males or before the mating period in females.
The few statistically significant differences between controls and test item-treated animals were not attributed to the test item treatment as the differences were occasional/of low magnitude and/or not dose-related, and they did not impact the terminal body weight.
Also no relevant effects were noted on mean body weight or mean body weight change at any dose level during the pregnancy period nor during the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No relevant effects were noted on mean food consumption at any dose level during the treatment period in males and before the mating period in females.
The few statistically significant differences between controls and test item-treated animals were not attributed to the test item treatment as the differences were occasional/of low magnitude (males at 75 or 200 mg/kg bw/day) and/or not dose-related (females at 75 mg/kg bw/day).
Also, no effects were noted on mean food consumption at any dose level during the pregnancy period nor during the lactation period.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, when compared to controls, mean red blood cell parameters (including erythrocyte count, hemoglobin concentration and packed cell volume) were lower in females (-13, -10% and -12%, respectively). While a relationship to the test item treatment could not be excluded, these differences were of low magnitude and observed in only one sex, and were therefore considered to be non-adverse.
The other statistically significant differences from controls were considered to be incidental as they were isolated (no changes in the other white blood cell parameters) and of low magnitude with mean values included in the Historical Control Data (neutrophil count) or, poorly dose-related and because of the direction of the change (APTT).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, when compared to controls, mean glucose and triglyceride levels were lower in males (same tendency at 75 mg/kg bw/day for the glucose level). While a relationship to the test item treatment could not be excluded, these differences were within/close to our HCD and observed in only one sex, and were therefore considered to be non-adverse.
Changes observed in biomarkers of liver function (including total bilirubin and bile acid levels and alkaline phosphatase, aspartate amino transferase and alanine amino transferase activities) were considered to be incidental because of the direction and low magnitude of these changes.
The other statistically significant differences from controls (i.e. sodium, chloride, urea, total protein, albumin, albumin to globulin ratio) were not attributed to the test item treatment as they were slight/isolated and/or not dose-related and/or remained within the range of the historical control data.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONES
A summary of the thyroid hormone analysis results in P generation animals, P generation Females and Non-Selected F1 Offspring is presented in Table 1, 2 and 3, respectively, in section "Any other information on results incl. tables".

At 200 mg/kg bw/day, when compared to controls, the mean T4 level was significantly lower in P generation males (-22%). This change was attributed to the test item treatment and correlated with the thyroid follicular cell hypertrophy observed at microscopy. Higher mean TSH levels were noted in males at 75 or 200 mg/kg bw/day. As the dose relationship was not well defined, an effect of test item treatment remained unlikely. No other relevant differences from controls were noted in males (Table 1).
The thyroid hormones were considered to be unaffected by the test item treatment in P generation females. The statistically significant, higher mean TSH level in females at 75 mg/kg bw/day was considered to be incidental as this difference was not dose related and did not correlate with any alteration in thyroid follicles (Table 2).
No effects were noted on T4 or TSH levels in non-selected F1 pups at any dose level (Table 3).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects were noted on the quantitative or qualitative parameters at any dose level.
The higher mean volume recorded in females at 75 mg/kg bw/day was considered as incidental as this finding was isolated, not dose-related and included in the Historical Control Data range.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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 changes were noted in the kidneys (and ureters), liver, thyroid glands, forestomach and adrenal glands.

Kidneys and ureters
Hematoxylin-eosin-stained slides
There was a spectrum of dose-related degenerative and regenerative renal lesions in males treated at = 25 mg/kg bw/day and, at a lower extent in females. These lesions included:
• minimal to slight degeneration/necrosis of tubular cells at = 75 mg/kg bw/day in males,
• minimal to marked granular casts located in the outer medulla/inner cortex at = 75 mg/kg bw/day in males,
• increased severity and incidence of tubular basophilia, graded up to marked and consistent with regeneration following cellular alterations at = 25 mg/kg bw/day in males,
• minima to moderate tubular dilatation at = 25 mg/kg bw/day in males and females,
• minimal to marked hyaline droplet accumulation in the cortical tubules at = 25 mg/kg bw/day in males,
• increased severity and incidence of pelvic dilatation, at = 25 mg/kg bw/day in males only, graded up to marked, and probably secondary to renal dysfunction,
• minimal tubular vacuolation with an isolated occurrence of 3/24 males and 4/22 females treated at 200 mg/kg bw/day and in 1/24 females treated at 25 mg/kg bw/day.

Alpha-2 microglobulin-stained slides
When compared to controls, there were increased severity of granular staining of alpha-2 microglobulinein the tubules of kidneys from males treated at 200 mg/kg bw/day, associated with presence of staining in dilated tubules (i.e. those containing granular casts) and in the medulla (cytoplasm of collecting ducts and/or granular material in lumen). At a lower extent, the severity of staining was lower in males treated at 200 mg/kg bw/day than controls in the large aggregates. Altogether, these stainings suggested that there was an increase in the amount of
alpha-2uglobulin deposit in the kidneys of males treated at 200 mg/kg bw/day.
The ureters from 2 males treated at 200 mg/kg bw/day were dilated. This correlated with the macroscopic findings and was considered to be secondary to the renal/pelvic dysfunction.

Liver
Dose-related minimal to slight hepatocellular hypertrophy was noted in males and females treated at = 25 mg/kg bw/day.

Thyroid glands
Dose-related minimal to slight follicular cell hypertrophy was noted in males at = 75 mg/kg bw/day and in females treated at 200 mg/kg bw/day.

Forestomach
Dose-related minimal to moderate squamous cell hyperplasia, hyperkeratosis and edema were noted in males and females treated at = 25 mg/kg bw/day. This was associated focally with hemorrhage and/or mixed cell infiltrates in occasional high-dose animals. It is noteworthy that no ulceration/degeneration was associated with these changes.

Adrenal glands
There were increased incidence and severity of cortical hypertrophy in the adrenal glands from females treated at 200 mg/kg bw/day, together with slight degeneration/necrosis in one female.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included a malignant fibrous histiocytoma in one male treated at 75 mg/kg bw/day that was considered to be part of the spontaneous background.
There were test item-related changes neither in the male or female reproductive organs.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.

The main microscopic lesions (incidences and severity) observed in the P generation are presented in Table 8 of the section "Any other information on results incl. tables".

Enumeration of corpora lutea in HE-stained slides
At enumeration of corpora lutea in the ovary of high-dose group and control group, there were decreased numbers of corpora lutea in females treated at 200 mg/kg bw/day when compared to controls, with a mean number of corpora lutea of 7.41 (± 4.23) and 11.26 (± 4.87) per animal respectively (-35%; p<0.01).

Enumeration of the number of primordial follicles on PCNA-stained slides
There were no differences between the control and the high-dose groups, with a mean number of 6.31 (± 4.45) and 7.68 (± 5.30) primordial follicles per animal on PCNA-stained slides respectively. However, this difference did not reach statistical significance and thus was considered to be unrelated to the test item administration.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The histopathological findings were of non-neoplastic nature.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were noted on the estrous cycle parameters at any dose level.
The lower mean percentage of females with all stages at 200 mg/kg bw/day (83.3 vs. 95.8 in controls) was due to the contribution of four females in which proestrus or metestrus was not detected over the 15-day examination period (these animals mated within 5 days and 2/4 were pregnant). As the incidence of affected females remained low and the difference was not statistically significant, a relationship to treatment with the test item was considered to be unlikely.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test item-related effects were observed on sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate) in P generation males at 200 mg/kg bw/day.
The lower mean number of testicular sperm heads and lower daily sperm production rate in males given 200 mg/kg bw/day when compared to controls, were not attributed to the test item treatment as the differences were low and not statistically significant.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

MATING AND FERTILITY DATA:
No effects were noted on mating (including the mean number of days taken to mate) or fertility at any dose level.
The fertility indexes were lower compared to Historical Control Data resulting in a number of pregnant females less than 20 pregnant females per group, except for females treated at 25 mg/kg bw/day. However, the number of pregnant females and pups delivered was similar especially in controls and females treated at 75 or 200 mg/kg bw/day and the number of pups was sufficient for the constitution of the cohorts. The number of pregnant females less than 20 females per group was therefore considered to have no consequences on the interpretation of the data.

There were also no test item-related effects were observed on the gestation index, duration of gestation, number of implantation sites, percentage of post-implantation loss, live birth index or pup sex ratio at any dose level.
The lower mean live birth index (not statistically significant) recorded in the 200 mg/kg bw/day group (88.8 vs. 93.2 % in controls) was considered to be incidental and not attributed to the test item treatment as the difference was mainly due to the contribution of one female for which 13 pups were found dead on Day 1 p.p. (mean value of 94.2% excluding this female).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/ developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Absence of adverse effects on estrous cycle, mating, fertility, duration of gestation, number of implantation sites or live birth index

3

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Severe kidney toxicity was recorded in the kidneys at 75 and 200 mg/kg bw/day in males, associated with the accumulation of hyaline droplets. This accumulation is a toxic effect specific of male rat with no relevance for human risk assessment.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A, Cohort 1B, Cohort 3 - Post-Weaning
Ptyalism was noted after treatment in all groups (including controls) (Cohort 1A and 1B), and after treatment from 75 mg/kg bw/day (Cohort 3) with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect. On rare occasions, ptyalism was also noted before treatment.
The other findings, i.e. thin appearance, piloerection, hypoactivity, loud/abdominal breathing, chromodacryorrhea, chromorhynorrhea, half-closed eyes, alopecia/thinning of hair, scabs/wounds, shortened tail, abnormal growth of teeth and tail bent, were considered to be unrelated to the test item treatment as they were present with a similar incidence in controls and/or were reported sporadically in only very few animals and/or are commonly observed in untreated laboratory rats.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort 1A - Post-Weaning:
No test item-related deaths occurred in Cohort 1A males or females.
One male given 25 mg/kg bw/day died after episodes of convulsions on Day 50 (after observation to check for preputial separation). Scabs on the neck were recorded from Day 37, corresponding to the microscopic finding of a slight ulcer with crusts. The cause of death was not determined, but as this death occurred at the lowest dose level, and no unscheduled deaths were recorded in the other groups, a relationship to the test item treatment was excluded.

Cohort 1B - Post-Weaning:
None of the deaths were attributed to the test item treatment.
One male given 75 mg/kg bw/day was prematurely euthanized for humane reasons on Day 38. Signs of poor clinical condition (hunched posture, thin appearance, dyspnea, abdominal breathing and piloerection) were observed from Days 37/38, associated with body weight loss (-3%, when compared to its last body weight) and reduced food consumption (19 g/day on Days 29-36 vs. 31 g/day in controls). The cause of morbidity was not determined at microscopic examination.
One female (control group) given 0 mg/kg bw/day was prematurely euthanized for human reasons on Day 25 p.c. Signs of poor clinical condition (piloerection, hunched posture, pallor of extremities, red discolored urogenital region and cold to the touch) were noted from Days 24/25 p.c., together with difficulties to deliver. Eleven live pups and one dead and one cannibalized pup were found in the bedding while at hysterectomy, one dead fetus and eight placentas were found in the horns. The cause of death/morbidity was attributed to severe tubular degeneration/necrosis in the kidneys and moderate degeneration/necrosis in the adrenal glands, as well as the dead fetus in the uterus.
One female given 25 mg/kg bw/day was prematurely euthanized due to the death of its litter on Day 1 p.p. In addition, signs of poor clinical condition (piloerection and generalized pallor) were observed on the day of death. The cause of death was reproduction problems associated with focal necrosis in the uterus along with thrombus in an adjacent vessel.

Cohort 3 - Post-Weaning:
No unscheduled deaths occurred in Cohort 3 animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean body weight was slightly lower in males and females on Day 1 (-5 and -7%, respectively). This was considered to have resulted from the lower mean body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 until sacrifice due to mean body weight gains similar or higher to those of controls, reaching statistical significance on Days 15-22 and 29-36 (+5% for the whole period), indicating the reversibility of this finding. While in males, the mean body weight was continuously lower between Days 1 and 71 (-4% for the whole period) due to mean body weight gains generally lower than those of controls. These effects on body weight and body weight change in males were attributed to the test item treatment,but considered as non-adverse given their low magnitude.
At 25 or 75 mg/kg bw/day, no test item-related effects were noted on mean body weight or mean body weight change in males or females.

Cohort 1B - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean body weight was minimally lower in males and females on Day 1 (-6 and-3%, respectively). This was considered to have resulted from the lower mean body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 until sacrifice due to mean boy weight gains generally similar to those of controls or higher (+4% for the whole period), reaching statistical significance on
Days 22-29 and 57-64 (+4% for the whole period), indicating the reversibility of this finding. The statistically significant, lower mean body weight gain recorded on Days 71-78 was considered to be unrelated to the test item treatment as this difference was of low magnitude and isolated.
In males, the mean body weight was continuously lower between Days 1 and 113, but particularly between Days 8 and 29 (-18 to -7%, vs. controls), mainly due to statistically significant, lower mean body weight gain on Days 1-8 (-34%), while therea fter, the difference from controls was minimal (-2% in terms of body weight on Day 113 and body weight gain on Days 1-113). These effects on body weight and body weight change in males were attributed to the test item treatment, but considered as non-adverse given their low magnitude.
At 25 or 75 mg/kg bw/day, no test item-related effects were noted on mean body weight or mean body weight change in males or females. The few statistically significant differences observed in mean body weight gain were not attributed to the test item treatment as they were occasional and/or not dose-related.
No effects were noted on mean body weight or mean body weight change at any dose level during the pregnancy period.
No effects were noted on mean body weight or mean body weight change between Days 1 and 4 p.p. of the lactation period.

Cohort 3- Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean body weight was slightly lower in males and females on Day 1 (-6 and -9%, respectively). This was considered to have resulted from the lower mean body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 until sacrifice due to mean body weight gains similar or higher to those of controls, reaching statistical significance on Days 22-29 (+7% for the whole period), indicating the reversibility of this finding.
In males, the mean body weight was continuously lower between Days 1 and 36, but the difference from controls was minimal from Day 29 due to higher mean body weight gain from Day 22 (-1% for the whole period). These effects on body weight and body weight change in males were attributed to the test item treatment, but considered as non-adverse given their low magnitude
At 75 mg/kg bw/day, when compared to controls, mean body weight was slightly lower in males and females from Day 1 (-8 and-6%, respectively, not statistically significant) until Day 8 in females (-6%; statistically significant) or Day 15 in males (-5%, not statistically significant). Thereafter, mean body weights were similar to those of controls, due to similar or higher mean body weight gains. A relationship to the test item treatment was considered to be unlikely as these effects on body weight were of low magnitude, not dose-related (males only), and were not reported in Cohort 1A or 1B.
At 25 mg/kg bw/day, no effects were noted on mean body weight or mean body weight change in males or females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
Food consumption was unaffected by the test item treatment in males or females at all dose levels.
Some differences were reported in test item-treated males when compared to controls (not statistically significant), but as they were occasional and/or not dose-related and/or noted with an opposite trend during the course of the treatment period, they were considered to be unrelated to the test item treatment.

Cohort 1B - Post-Weaning:
At 200 m/kg/day, when compared to controls, mean food consumption was significant lower in males on Days 1-8 (-15%) and 8-15 (-17%). This effect was attributed to the test item treatment and correlated with the lower body weight gain recorded during this period (see above). The higher mean food consumption recorded in females on days 22-29 was not attributed to the test item treatment as this difference was isolated and of low magnitude.
At 75 mg/kg bw/day, mean food consumption was significantly lower in males on Days 8-15. Although a relationship to the test item treatment could not be excluded, this finding was isolated and noted in only one sex, and was therefore considered to be non-adverse.
The higher mean food consumption recorded in males during the postmating period was not attributed to the test item treatment as this difference was of low magnitude and not dose-related.
At 25 mg/kg bw/day, no relevant effects were noted on food consumption.
No test item-related effects were noted on mean food consumption at any dose level during the pregnancy period.
The statistically significant higher mean food consumption recorded at 75 or 200 mg/kg bw/day on Days 14-17 p.c. was not attributed to the test item treatment as these differences were isolated and/or not dose-related and rather considered to be the result of individual episodes of spillage.
No effects were noted on mean food consumption at any dose level during the lactation period.

Cohort 3 - Post-Weaning:
Food consumption was unaffected by the test item treatment in males or females at all dose levels.
Some differences were reported in test item-treated males and females when compared to controls, but as they were occasional and/or not dose-related and/or noted with an opposite trend during the course of the treatment period, they were considered to be unrelated to the test item treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean red blood cell parameters (including hemoglobin concentration, mean cell hemoglobin concentration and reticulocyte count) were minimally to slightly lower in males (-6, -5% and -15%, respectively). While a relationship to the test item treatment could not be excluded, these differences were of low magnitude and observed in only one sex, and were therefore considered to be non-adverse.
The other statistically significant differences from controls (i.e. higher thrombocyte count and fibrinogen level in females given 200 mg/kg bw/day) were considered to be incidental as they were of low magnitude and/or remained within the range of historical control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean glucose and creatinine levels were lower in females and males, respectively, and mean triglyceride level was higher in females. While a relationship to the test item treatment could not be excluded, these differences were within/close to our HCD and observed in only one sex, and were therefore considered to be non-adverse.
Changes observed in biomarkers of liver function (including total bilirubin and bile acid levels and alkaline phosphatase, aspartate amino transferase and alanine amino transferase activities) were considered to be incidental because of the direction and low magnitude of these changes.
The other statistically significant differences from controls [i.e. sodium, chloride, calcium, total protein, albumin, albumin to globulin ratio, cholesterol, triglycerides (at 25 mg/kg bw/day)] were not attributed to the test item treatment as they were slight/isolated and/or not dose-related and/or remained within the range of historical control data.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONES:
Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean T4 level was significantly lower in Cohort 1A males (-23%). This was associated with statistically significant, higher mean TSH level
(2.6-fold). These changes were attributed to the test item treatment and correlated with the thyroid follicular cell hypertrophy observed at microscopic examination.
No other relevant differences from controls were noted.
At 200 mg/kg bw/day, when compared to controls, the mean TSH level was significantly higher in Cohort 1A females (2.2-fold). This change was attributed to the test item treatment and correlated with the thyroid follicular cell hypertrophy observed at microscopy. No variation in the T4 level was noted.
No other relevant differences from controls were noted.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 1A - Post-Weaning:
No effects were noted on the quantitative or qualitative parameters at any dose level.

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In term of absolute counts (cells/mg of spleen), no statistically significant differences in mean were observed in test item-treated groups when compared to control group in any of the studied lymphocyte populations.
In term of relative counts (%), statistically significant, lower mean cytotoxic T cell frequency was noted in females given 200 mg/kg/day compared to control group (28.2% vs. 34.1%, respectively). This difference was of low magnitude, remained within the HCD and was observed in only one sex, and was therefore not attributed to the test item treatment.
No statistically significant difference was observed in males.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups (in Percentages Versus Controls) are given in Table 5, 6, and 7, for Cohort 1A, Cohort 1B, and Cohort 3, respectively (in the section "Any other information on results incl. tables). 

Cohort 1A - Post-Weaning:
Increased absolute and relative-to-body liver and kidney weights were recorded in females treated at = 25 mg/kg bw/day and in males treated at = 75 mg/kg bw/day. This correlated respectively with hepatocellular hypertrophy (liver) and tubular basophilia/accumulation of hyaline droplets (kidneys) at microscopic examination.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
This included the increased absolute and/or relative-to-body spleen weights in males and females treated at 200 mg/kg bw/day (up to -18% in males; p<0.01 or 0.05). There were also low absolute and/or relative-to-body thymus weights in males treated at = 75 mg/kg bw/day (up to -16% at 200 mg/kg bw/day; p<0.01 or 0.05).
These differences were of low magnitude and had no microscopic correlates. Therefore they were considered not to be related to test item administration.

Cohort 1B - Post-Weaning:
Increased absolute and/or relative-to-body liver and kidney weights were recorded in males and females treated at = 75 mg/kg bw/day. This correlated respectively with tubular basophilia/accumulation of hyaline droplets (kidneys) at microscopic examination.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 3 - Post-Weaning:
Increased absolute and relative-to-body liver weights were recorded in males and females treated at 200 mg/kg bw/day (p<0.01 or 0.05). This correlated with microscopic hepatocellular hypertrophy.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
This included the decreased absolute and relative-to-body thyroid gland weights recorded in females treated at 75 or 200 mg/kg bw/day that had no gross correlates and that were considered to be unrelated to the test item administration since similar findings were not seen in any other groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
The following findings were considered to be test item-related:
• liver: accentuated lobular pattern and granular aspect in males treated at 200 mg/kg bw/day, correlated with hepatocellular hypertrophy,
• kidney: irregular color or surface, green discoloration, and/or enlargement in males treated at = 75 mg/kg bw/day, and in females treated at 200 mg/kg bw/day, correlated with tubular basophilia and accumulation of hyaline droplets,
• forestomach: yellow deposit in females treated at 200 mg/kg bw/day that correlated with squamous cell hyperplasia and/or hyperkeratosis,
• ureters: dilatation in males treated at = 75 mg/kg bw/day and in females treated at 200 mg/kg bw/day that correlated with microscopic lumen dilatation.
In addition, there were isolated brown discoloration and enlargements in the iliac and lumbar lymph nodes from one high-dose female that correlated respectively with pigment and increased cellularity in paracortex related to the severe alterations in the kidneys of this female (mainly degeneration/necrosis of the glomeruli with inflammation at microscopic examination; see below).
There was also dilated pelvis in the kidneys from occasional males treated at = 25 mg/kg bw/day. The relationship to test item was considered to be ambiguous in view of the low incidence and the occurrence of such common finding in untreated rats, as in parents.

Cohort 1B - Post-Weaning:
The following findings were considered to be test item-related:
• liver: accentuated lobular pattern, thickening and/or granular aspect in males treated at 75 mg/kg bw/day,
• kidney: irregular color or surface and/or green discoloration in males treated at = 75 mg/kg bw/day, correlated with tubular basophilia and accumulation of hyaline droplets,
• forestomach: white mass in 1/20 females treated at 200 mg/kg bw/day that correlated with ulcer, squamous cell hyperplasia and hyperkeratosis.
There was also dilated pelvis in the kidneys from occasional males treated at = 25 mg/kg bw/day, in one female treated at 25 mg/kg bw/day and in 1/20 control males. The incidence was minimally increased at 25 and 200 mg/kg bw/day. The relationship to test item was considered to be ambiguous in view of the low increase in incidence and of the occurrence of such common finding in untreated rats.
The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the brown content seen in the vagina from one high-dose female that correlated with granulocyte aggregate. This was considered to be part of the spontaneous background lesions in view of the low incidence of this change.

Cohort 3 - Post-Weaning:
Regarding Macroscopic Post-Mortem Examination, the following findings were considered to be test item-related:
• kidney: irregular color in males treated at 200 mg/kg bw/day, correlated with tubular basophilia and accumulation of hyaline droplets,
• forestomach: white discoloration in 1/10 males treated at 200 mg/kg bw/day that correlated with squamous cell hyperplasia and hyperkeratosis,
• ureters: dilatation in occasional males and females treated at 25 or 200 mg/kg bw/day.
There was also dilated pelvis in the kidneys from occasional males treated at = 25 mg/kg bw/day, in 1/10 control males and in 1/10 female treated at 25 mg/kg bw/day. The incidence was minimally increased at 200 mg/kg bw/day. The relationship to test item was considered to be ambiguous in view of the low increase in incidence and of the occurrence of such common finding in untreated rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
Test item-related changes were noted in the kidneys (and ureters), liver, thyroid glands, forestomach and esophagus.

Kidneys and ureters
Hematoxylin-eosin-stained slides
There was a spectrum of dose-related degenerative and regenerative renal lesions in males and, at a lower extent, in females treated at = 25 mg/kg bw/day. These lesions included:
• minimal to moderate degeneration/necrosis of tubular cells in males treated at = 75 mg/kg bw/day and in one female treated at 200 mg/kg bw/day,
• minimal to moderate granular casts located in the outer medulla/inner cortex in males treated at = 75 mg/kg bw/day,
• increased severity and incidence of tubular basophilia, graded up to marked and consistent with regeneration following cellular alterations in males and females treated at = 75 mg/kg bw/day,
• minima to moderate tubular dilatation in females treated at 25 mg/kg bw/day, and in males and females treated at = 75 mg/kg bw/day,
• minimal to marked hyaline droplet accumulation in the cortical tubules from males treated at = 25 mg/kg bw/day,
• increased severity and incidence of pelvic dilatation, in males treated at = 25 mg/kg bw/day only, graded up to slight, and probably secondary to renal dysfunction,
• minimal tubular vacuolation seen in 2/20 males treated at 200 mg/kg bw/day and in 1/20 females treated at 25 or 200 mg/kg bw/day that were seen with very low occurrence and thus were of equivocal significance,
• severe degeneration/necrosis of glomeruli accompanied by glomerular inflammation and hemorrhage in one high-dose female, in addition to the various test item-related findings as tubular degeneration/necrosis, basophilia, etc. This was accompanied by intrasinusoidal erythrocytes, pigment and increased cellularity in paracortex in the iliac and lumbar lymph nodes that were considered to be secondary to these severe alterations in the kidneys (draining process in loco-regional lymph nodes).

Alpha-2 microglobulin-stained slides
When compared to controls, there were increased severity of granular staining of alpha-2 microglobulin in the tubules of kidneys from males treated at 200 mg/kg bw/day, associated with presence of staining in dilated tubules (i.e. those containing granular casts) and in the medulla (cytoplasm of collecting ducts and/or granular material in lumen). At a lower extent, the severity of staining was lower in males treated at 200 mg/kg bw/day than controls in the large aggregates. Altogether, these stainings suggested that there was an increase in the amount of alpha-2 microglobulin deposit in the kidneys of males treated at 200 mg/kg bw/day, similarly to the distribution noted in the parents.
The ureters from 1 male treated at 75 mg/kg bw/day, of 3 males and 1 female treated at 200 mg/kg bw/day were dilated. This correlated with the macroscopic findings and was considered to be secondary to the renal/pelvic dysfunction.

Liver
Dose-related minimal hepatocellular hypertrophy was noted in males treated at = 25 mg/kg bw/day and females treated at = 75 mg/kg bw/day.

Thyroid glands
Dose-related minimal follicular cell hypertrophy was noted in males treated at = 25 mg/kg bw/day and females treated at = 200 mg/kg bw/day.

Forestomach
Dose-related minimal to slight squamous cell hyperplasia, hyperkeratosis and/or edema were noted in males and/or females treated at = 25 mg/kg bw/day. This was associated focally with aggregates of granulocytes in the epithelium and/or mixed cell infiltrates in occasional high-dose animals.

Esophagus
Minimal hyperkeratosis of the esophagus was noted in two males treated at 200 mg/kg bw/day.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
There were test item-related changes neither in the male or female reproductive organs.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.

The main microscopic lesions (incidences and severity) observed in the Cohort 1A are presented in Table 9 of the section "Any other information on results incl. tables".

Enumeration of corpora lutea in HE-stained slides
At enumeration of corpora lutea in the ovary of control and high-dose group, there were no differences, with a mean number of corpora lutea of 21.55 (± 7.61) and 19.00 (± 5.57) per animal respectively, reaching no statistical significance.

Enumeration of the number of primordial follicles on PCNA-stained slides
There were no differences between the control and the high-dose groups, with a mean number of 6.76 (± 5.40) and 7.78 (± 3.67) primordial follicles per animal on PCNA-stained slides respectively, reaching no statistical significance.

Cohort 1B - Post-Weaning:
Test item-related changes were noted in the kidneys (and ureters) from males and in the forestomach of one high-dose female.

Kidneys and ureters
There was a spectrum of dose-related degenerative and regenerative renal lesions in males treated at = 25 mg/kg bw/day. These lesions included:
• minimal to slight degeneration/necrosis of tubular cells in males treated at 200 mg/kg bw/day,
• minimal to moderate granular casts located in the outer medulla/inner cortex in males treated at = 75 mg/kg bw/day,
• increased severity and incidence of tubular basophilia, graded up to marked and consistent with regeneration following cellular alterations in males treated at = 25 mg/kg bw/day,
• minimal to moderate tubular dilatation in males treated at = 75 mg/kg bw/day,
• minimal to marked hyaline droplet accumulation in the cortical tubules in males treated at = 25 mg/kg bw/day (increased severity and incidence when compared to controls),
• increased severity and incidence of pelvic dilatation, in males treated at = 25 mg/kg bw/day, graded up to slight, and probably secondary to renal dysfunction,
• minimal tubular vacuolation with low occurrence of one male treated at 75 mg/kg bw/day that was considered to be of low toxicological significance in view of this isolated occurrence.
There was minimal increase of severity and incidence of ureter dilatation in males treated at 200 mg/kg bw/day when compared to controls.
Forestomach
Moderate ulcer and hyperplasia of squamous cells together with slight hyperkeratosis were noted in one female treated at 200 mg/kg bw/day.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included a mammary gland adenocarcinoma in one female treated at 75 mg/kg bw/day or a marked pulmonary abscess in one male treated at 75 mg/kg bw/day that were considered to be part of the spontaneous background

The main microscopic lesions (incidences and severity) observed in the Cohort 1B (Macroscopic Lesions Only) are presented in Table 10 of the section "Any other information on results incl. tables".

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Cohort 3:
Microscopic examination (gross abnormalities only except for liver and thyroid glands).
There were test item-related microscopic changes in kidneys, ureters, liver, thyroid glands and forestomach.
Kidneys and ureters
There was a spectrum of dose-related degenerative and regenerative renal lesions in 1/10 males treated at 200 mg/kg bw/day. These lesions included: minimal degeneration/necrosis of tubular cells, minimal tubular basophilia and moderate hyaline droplet accumulation in the cortical tubules.
Ureter dilatation in males and/or females treated at 25 or 200 mg/kg bw/day were noted. In view of the low occurrence of this change, the toxicological significance remained equivocal.
Forestomach
Moderate hyperplasia of squamous cells together with moderate hyperkeratosis and minimal edema were noted in one male treated at 200 mg/kg bw/day, accompanied by aggregate of granulocytes.
Liver
Dose-related minimal hepatocellular hypertrophy was noted in males and females treated at = 75 mg/kg bw/day.
Thyroid glands (females only)
Minimal follicular cell hypertrophy was noted in 2/10 females treated at 200 mg/kg bw/day.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Cohort 1A:
No effects on mean estrous cycle parameters were noted in Cohort 1A females.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
At 200 mg/kg bw/day, when compared to controls, the mean number of testicular sperm heads and the daily sperm production rate were lower (-16%). While a relationship to the test item treatment cannot be excluded, these findings were considered to be non-adverse given the absence of significant effects on the mean number of epididymal sperm.
No effects on mean number of testicular sperm heads and the daily sperm production rate were observed at 25 or 75 mg/kg bw/day.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

MATING AND FERTILITY DATA (Cohort 1B):
No effects were noted on mating (including the mean number of days taken to mate) or fertility at any dose level.
No test item-related effects were observed on the gestation index, duration of gestation, number of implantation sites, percentage of post-implantation loss, live birth index or pup sex ratio at any dose level.
The higher mean percentage of post implantation loss noted at 75 and 200 mg/kg bw/day was not attributed to the test item treatment as the differences were neither statistically significant nor dose-related and they did not affect the live birth index.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/ developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Non-adverse lower body weight gain was observed in male and female F1 pups at 200 mg/kg bw/day throughout the lactation period, but with recovery after weaning. Absence of effects on pup development, on estrous cycle, mating, fertility, duration of gestation, number of implantation sites or live birth index.

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Adverse effects on kidneys in only one female at 200 mg/kg bw/day in the cohort 1A

3

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Severe kidney toxicity was recorded in the kidneys at 75 and 200 mg/kg bw/day in males, associated with the accumulation of hyaline droplets. This accumulation is a toxic effect specific of male rat with no relevance for human risk assessment.

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All findings recorded in pups during the lactation period were considered to be unrelated to the test item treatment as they were reported in isolated animals and/or are common observations in rat pups of this strain and age.

Cohort 1A, Cohort 1B, Cohort 3 - Post-Weaning
Ptyalism was noted after treatment in all groups (including controls) (Cohort 1A and 1B), and after
treatment from 75 mg/kg bw/day (Cohort 3) with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect. On rare occasions, ptyalism was also noted before treatment.
The other findings, i.e. thin appearance, piloerection, hypoactivity, loud/abdominal breathing, chromodacryorrhea, chromorhynorrhea, half-closed eyes, alopecia/thinning of hair, scabs/wounds,
shortened tail, abnormal growth of teeth and tail bent, were considered to be unrelated to the test
item treatment as they were present with a similar incidence in controls and/or were reported sporadically in only very few animals and/or are commonly observed in untreated laboratory rats.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No relevant effects were noted on mean litter size at birth or on the incidence of pups found dead, missing or cannibalized on Days 1-4 p.p.
The lower mean litter size at birth (not statistically significant) recorded in the 200 mg/kg bw/day group (10.7 vs. 11.8 in controls) was considered to be incidental and not attributed to the test item treatment as the difference was mainly due to the contribution of one female for which only one live pup was recorded on Day 1 p.p. (mean value of 11.3% when this female is excluded). This female accounted for a large part of the higher number of dead, missing and/or cannibalized pups on Days 1-4 p.p. (in total 16 dead pups when this female is excluded, i.e. 8.8%).
The higher mean number/percentage of dead, missing and/or cannibalized pups on Days 1-4 p.p. at 25 (22 i.e. 8.6%) or 75 (20 i.e. 10.2%) mg/kg bw/day was not attributed to the test item treatment as these differences were mainly due to the contribution of isolated females .
Also, no test item-related effects were observed on the viability index at any dose level during lactation of the pups.
The lower mean viability index (not statistically significant) recorded in the 200 mg/kg bw/day group (86.0 vs. 91.9% in controls) was considered to be incidental and not attributed to the test item treatment as the difference was mainly due to the contribution of one female for which 13 pups were found dead on Day 1 p.p.
After culling on Day 4 p.p., no effects were noted on mean litter size or lactation indexes at any dose level.

There was no mortality of the non selected F1 pups.

Cohort 1A - Post-Weaning:
No test item-related deaths occurred in Cohort 1A males or females.
One male given 25 mg/kg bw/day died after episodes of convulsions on Day 50 (after observation to
check for preputial separation). Scabs on the neck were recorded from Day 37, corresponding to the microscopic finding of a slight ulcer with crusts. The cause of death was not determined, but as this death occurred at the lowest dose level, and no unscheduled deaths were recorded in the other gro
ups, a relationship to the test item treatment was excluded.

Cohort 1B - Post-Weaning:
None of the deaths were attributed to the test item treatment.
One male given 75 mg/kg bw/day was prematurely euthanized for humane reasons on Day 38. Si gns of poor clinical condition (hunched posture, thin appearance, dyspnea, abdominal breathing
and piloerection) were observed from Days 37/38, associated with body weight loss (-3%, when co mpared to its last body weight) and reduced food consumption (19 g/day on Days 29-36 vs. 31 g/day
in controls). The cause of morbidity was not determined at microscopic examination.
One female (control group) given 0 mg/kg bw/day was prematurely euthanized for human reasons
on Day 25 p.c. Signs of poor clinical condition (piloerection, hunched posture, pallor of extremities, red discolored urogenital region and cold to the touch) were noted from Days 24/25 p.c., together with difficulties to deliver. Eleven live pups and one dead and one cannibalized pup were found in the bedding while at hysterectomy, one dead fetus and eight placentas were found in the horns. The cause of death/morbidity was attributed to severe tubular degeneration/necrosis in the kidneys and moderate degeneration/necrosis in the adrenal glands, as well as the dead fetus in the uterus.
One female given 25 mg/kg bw/day was prematurely euthanized due to the death of its litter on Day 1 p.p. In addition, signs of poor clinical condition (piloerection and generalized pallor) were observed on the day of death. The cause of death was reproduction problems associated with focal necrosis in the uterus along with thrombus in an adjacent vessel.

Cohort 3 - Post-Weaning:
No unscheduled deaths occurred in Cohort 3 animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, when compared with controls, mean body weight gain was lower in male and female pups throughout the lactation period (-7 and -8%, respectively), reaching statistical significance on Days 4-7 p.p. (-16% in both sexes), and leading to slightly lower mean body weight on Day 21 p.p. (-6 and -7%, respectively, but without statistical significance). These effects were attributed to the test item treatment, but considered as non-adverse in view of the magnitude.
At 25 or 75 mg/kg bw/day, no effects were noted on mean body weight or mean body weight gain.

Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean body weight was slightly lower in males
and females on Day 1 (-5 and -7%, respectively). This was considered to have resulted from the
lower mean body weight recorded before weaning. This difference from controls was no longer note d in females from Day 15 until sacrifice due to mean body weight gains similar or higher to those of controls, reaching statistical significance on Days 15-22 and 29-36 (+5% for the whole period), ind icating the reversibility of this finding. While in males, the mean body weight was continuously lower
between Days 1 and 71 (-4% for the whole period) due to mean body weight gains generally lower th an those of controls. These effects on body weight and body weight change in males were attributed t o the test item treatment,but considered as non-adverse given their low magnitude.
At 25 or 75 mg/kg bw/day, no test item-related effects were noted on mean body weight or mean body
weight change in males or females.

Cohort 1B - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean body weight was minimally lower in males
and females on Day 1 (-6 and-3%, respectively). This was considered to have resulted from the lower mean body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 until sacrifice due to mean boy weight gains generally similar to those of controls or higher (+4% for the whole period), reaching statistical significance on
Days 22-29 and 57-64 (+4% for the whole period), indicating the reversibility of this finding. The statist ically significant, lower mean body weight gain recorded on Days 71-78 was considered to be unrelate d to the test item treatment as this difference was of low magnitude and isolated.
In males, the mean body weight was continuously lower between Days 1 and 113, but particularly be tween Days 8 and 29 (-18 to -7%, vs. controls), mainly due to statistically significant, lower mean body weight gain on Days 1-8 (-34%), while thereafter, the difference from controls was minimal (-2% in terms of body weight on Day 113 and body weight gain on Days 1-113). These effects on body weig ht and body weight change in males were attributed to the test item treatment, but considered as non- adverse given their low magnitude.
At 25 or 75 mg/kg bw/day, no test item-related effects were noted on mean body weight or mean body weight change in males or females. The few statistically significant differences observed in mean body weight gain were not attributed to the test item treatment as they were occasional and/or not dose-related.
No effects were noted on mean body weight or mean body weight change at any dose level during the pregnancy period.
No effects were noted on mean body weight or mean body weight change between Days 1 and 4 p.p. of the lactation period.

Cohort 3 - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean body weight was slightly lower in males and females on Day 1 (-6 and -9%, respectively). This was considered to have resulted from the
lower mean body weight recorded before weaning. This difference from controls was no longer not ed in females from Day 15 until sacrifice due to mean body weight gains similar or higher to those o f controls, reaching statistical significance on Days 22-29 (+7% for the whole period), indicating the
reversibility of this finding.
In males, the mean body weight was continuously lower between Days 1 and 36, but the difference
from controls was minimal from Day 29 due to higher mean body weight gain from Day 22 (-1% for the whole period). These effects on body weight and body weight change in males were attributed to the test item treatment, but considered as non-adverse given their low magnitude
At 75 mg/kg bw/day, when compared to controls, mean body weight was slightly lower in males a nd females from Day 1 (-8 and-6%, respectively, not statistically significant) until Day 8 in females
(-6%; statistically significant) or Day 15 in males (-5%, not statistically significant). Thereafter, mean body weights were similar to those of controls, due to similar or higher mean body weight gains. A relationship to the test item treatment was considered to be unlikely as these effects on body weight
were of low magnitude, not dose-related (males only), and were not reported in Cohort 1A or 1B.
At 25 mg/kg bw/day, no effects were noted on mean body weight or mean body weight change in ma les or females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
Food consumption was unaffected by the test item treatment in males or females at all dose levels. Some differences were reported in test item-treated males when compared to controls (not statistically
significant), but as they were occasional and/or not dose-related and/or noted with an opposite trend during the course of the treatment period, they were considered to be unrelated to the test item tre atment.
Cohort 1B - Post-Weaning:
At 200 m/kg/day, when compared to controls, mean food consumption was significant lower in male
s on Days 1-8 (-15%) and 8-15 (-17%). This effect was attributed to the test item treatment and corr elated with the lower body weight gain recorded during this period (see above). The higher mean food
consumption recorded in females on days 22-29 was not attributed to the test item treatment as this
difference was isolated and of low magnitude.
At 75 mg/kg bw/day, mean food consumption was significantly lower in males on Days 8-15. Although
a relationship to the test item treatment could not be excluded, this finding was isolated and noted in
only one sex, and was therefore considered to be non-adverse.
The higher mean food consumption recorded in males during the postmating period was not attributed
to the test item treatment as this difference was of low magnitude and not dose-related.
At 25 mg/kg bw/day, no relevant effects were noted on food consumption.
No test item-related effects were noted on mean food consumption at any dose level during the pre gnancy period.
The statistically significant higher mean food consumption recorded at 75 or 200 mg/kg bw/day on
Days 14-17 p.c. was not attributed to the test item treatment as these differences were isolated and/ or not dose-related and rather considered to be the result of individual episodes of spillage.
No effects were noted on mean food consumption at any dose level during the lactation period.

Cohort 3 - Post-Weaning:
Food consumption was unaffected by the test item treatment in males or females at all dose levels. Some differences were reported in test item-treated males and females when compared to controls,
but as they were occasional and/or not dose-related and/or noted with an opposite trend during the course of the treatment period, they were considered to be unrelated to the test item treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean red blood cell parameters (including hemo globin concentration, mean cell hemoglobin concentration and reticulocyte count) were minimally to s lightly lower in males (-6, -5% and -15%, respectively). While a relationship to the test item treatment
could not be excluded, these differences were of low magnitude and observed in only one sex, and
were therefore considered to be non-adverse.
The other statistically significant differences from controls (i.e. higher thrombocyte count and fibrin ogen level in females given 200 mg/kg bw/day) were considered to be incidental as they were of low
magnitude and/or remained within the range of historical control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean glucose and creatinine levels were lower in females and males, respectively, and mean triglyceride level was higher in females. While a relat ionship to the test item treatment could not be excluded, these differences were within/close to our
HCD and observed in only one sex, and were therefore considered to be non-adverse.
Changes observed in biomarkers of liver function (including total bilirubin and bile acid levels and alkal ine phosphatase, aspartate amino transferase and alanine amino transferase activities) were consider ed to be incidental because of the direction and low magnitude of these changes.
The other statistically significant differences from controls [i.e. sodium, chloride, calcium, total protein,
albumin, albumin to globulin ratio, cholesterol, triglycerides (at 25 mg/kg bw/day)] were not attributed to the test item treatment as they were slight/isolated and/or not dose-related and/or remained within the range of historical control data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 1A - Post-Weaning:
No effects were noted on the quantitative or qualitative parameters at any dose level.

Sexual maturation:

no effects observed

Description (incidence and severity):

Cohort 1A:
No effects were noted on the mean age of balanopreputial separation or on mean body weight on the day of occurrence in cohort 1A males.
One male in each of the groups receiving 0, 25 or 75 mg/kg bw/day had no or incomplete balanopreputial separation until termination.
No test item-related effects were noted on the mean age at vaginal opening or on mean body weight on the day of occurrence in cohort 1A females.
At 200 mg/kg bw/day, when compared to controls, there was an apparent delay in the mean age at vaginal opening. This difference was not attributed to the test item treatment as all individual values (34 to 44 days) remained within the control range (32 to 43 days), except for one female (44 days).
The mean time to first estrous after vaginal opening in Cohort 1A females was unaffected by the test item treatment.
The differences observed between controls and females given 25 or 200 mg/kg bw/day were not attributed to the test item treatment as they were noted with no real dose-response relationship and the mean values remained within or close to our HCD.

Cohort 1B:
No effects were noted on the mean age of balanopreputial separation or on mean body weight on the day of occurrence in cohort 1B males.
One male in each of the groups receiving 0, 25 or 75 mg/kg bw/day and two males in the 200 mg/kg bw/day group had no or incomplete balanopreputial separation until termination.
At 25, 75 or 200 mg/kg bw/day, when compared to controls, there was an apparent delay in the mean age at which balanopreputial separation occurred. These differences were not attributed to the test item treatment as they were mainly due to the contribution of one male in each group for which the age at balanopreputial separation was much greater than the highest control age (76, 75 and 86 vs. 51 days old).
No effects were noted on mean age at vaginal opening or on mean body weight on the day of occurrence in cohort 1B females.

Cohort 3:
No test item-related effects were noted on the mean age of balanopreputial separation or on mean body weight on the day of occurrence in cohort 3 males.
One male in each of the groups receiving 0 or 75 mg/kg bw/day had no or incomplete balanopreputial separation until termination.
At 200 mg/kg bw/day, when compared to controls, there was an apparent delay in the mean age at which balanopreputial separation occurred. This difference was not attributed to the test item treatment as it was mainly due to the contribution of one male for which the age at balanopreputial separation was far greater than the highest control age (57 vs. 49 days old), while all other individual values remained within or close to the control range (42-49 days old).
No effects were noted on mean age at vaginal opening or on mean body weight on the day of occurrence in cohort 3 females.
For one 200 mg/kg bw/day female, vaginal opening did not occur during the observation period (between the ages of 28 and 48 days).

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No effects were observed on the anogenital distance (AGD) in F1 pups (day 1 p.p.) at any dose level.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples or areolae were observed in any male pup on Day 12 p.p.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were not test item-related organ weight differences in the non selected F1 pups.
The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.
This included the high absolute and/or relative-to-body thymus weights recorded in males treated at 75mg/kg bw/day and in females treated at 25 mg/kg bw/day (p<0.05) that were non-dose-related and due to individual low values in controls.

Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups (in Percentages Versus Controls) for Cohort 1A, Cohort 1B, and Cohort 3, are given in Table 5, 6, and 7, respectively (in the section "Any other information on results incl. tables). 

Cohort 1A - Post-Weaning:
Increased absolute and relative-to-body liver and kidney weights were recorded in females treated
at = 25 mg/kg bw/day and in males treated at = 75 mg/kg bw/day. This correlated respectively with hepatocellular hypertrophy (liver) and tubular basophilia/accumulation of hyaline droplets (kidneys) at microscopic examination.
The other organ weight changes were not considered to be test item related because they were of in sufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
This included the increased absolute and/or relative-to-body spleen weights in males and females
treated at 200 mg/kg bw/day (up to -18% in males; p<0.01 or 0.05). There were also low absolute
and/or relative-to-body thymus weights in males treated at = 75 mg/kg bw/day (up to -16% at 200 mg/ kg bw/day; p<0.01 or 0.05).
These differences were of low magnitude and had no microscopic correlates. Therefore they were c onsidered not to be related to test item administration.

Cohort 1B - Post-Weaning:
Increased absolute and/or relative-to-body liver and kidney weights were recorded in males and
females treated at = 75 mg/kg bw/day. This correlated respectively with tubular basophilia/accumula tion of hyaline droplets (kidneys) at microscopic examination.
The other organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.

Cohort 3 - Post-Weaning:
Increased absolute and relative-to-body liver weights were recorded in males and females treated at
200 mg/kg bw/day (p<0.01 or 0.05). This correlated with microscopic hepatoce llular hypertrophy.
The other organ weight changes were not considered to be test item related because they were of ins ufficient magnitude, were not dose-related and/or did not correlate to microscopic findings.
This included the decreased absolute and relative-to-body thyroid gland weights recorded in females treated at 75 or 200 mg/kg bw/day that had no gross correlates and that were considered to be unr
elated to the test item administration since similar findings were not seen in any other groups. Regarding Macroscopic Post-Mortem Examination, the following findings were considered to be test
item-related:
• kidney: irregular color in males treated at 200 mg/kg bw/day, correlated with tubular basophilia and
accumulation of hyaline droplets,
• forestomach: white discoloration in 1/10 males treated at 200 mg/kg bw/day that correlated with
squamous cell hyperplasia and hyperkeratosis,
• ureters: dilatation in occasional males and females treated at 25 or 200 mg/kg bw/day.
There was also dilated pelvis in the kidneys from occasional males treated at = 25 mg/kg bw/day,
in 1/10 control males and in 1/10 female treated at 25 mg/kg bw/day. The incidence was minimally
increased at 200 mg/kg bw/day. The relationship to test item was considered to be ambiguous in vie w of the low increase in incidence and of the occurrence of such common finding in untreated rats.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related findings were observed at any dose level in pups culled on Day 4 p.p. or euthanized after weaning.
There were no test item-related gross changes in the non selected F1 pups.
The enlarged left kidney associated with white mass in 1/10 females treated at 25 mg/kg bw/day correlated with marked subacute suppurative inflammation.

Cohort 1A - Post-Weaning:
The following findings were considered to be test item-related:
• liver: accentuated lobular pattern and granular aspect in males treated at 200 mg/kg bw/day, corre lated with hepatocellular hypertrophy,
• kidney: irregular color or surface, green discoloration, and/or enlargement in males treated at = 75
mg/kg bw/day, and in females treated at 200 mg/kg bw/day, correlated with tubular basophilia and a ccumulation of hyaline droplets,
• forestomach: yellow deposit in females treated at 200 mg/kg bw/day that correlated with squamous
cell hyperplasia and/or hyperkeratosis,
• ureters: dilatation in males treated at = 75 mg/kg bw/day and in females treated at 200 mg/kg bw/ day that correlated with microscopic lumen dilatation.
In addition, there were isolated brown discoloration and enlargements in the iliac and lumbar lymph
nodes from one high-dose female that correlated respectively with pigment and increased cel lularity in paracortex related to the severe alterations in the kidneys of this female (mainly degen eration/necrosis of the glomeruli with inflammation at microscopic examination; see below).
There was also dilated pelvis in the kidneys from occasional males treated at = 25 mg/kg bw/day. T he relationship to test item was considered to be ambiguous in view of the low incidence and the occ urrence of such common finding in untreated rats, as in parents.

Cohort 1B - Post-Weaning:
The following findings were considered to be test item-related:
• liver: accentuated lobular pattern, thickening and/or granular aspect in males treated at 75 mg/kg
bw/day,
kidney: irregular color or surface and/or green discoloration in males treated at = 75 mg/kg bw/day, c orrelated with tubular basophilia and accumulation of hyaline droplets,
• forestomach: white mass in 1/20 females treated at 200 mg/kg bw/day that correlated with ulcer, squamous cell hyperplasia and hyperkeratosis.
There was also dilated pelvis in the kidneys from occasional males treated at = 25 mg/kg bw/day, in one female treated at 25 mg/kg bw/day and in 1/20 control males. The incidence was minimally i
ncreased at 25 and 200 mg/kg bw/day. The relationship to test item was considered to be ambiguous in view of the low increase in incidence and of the occurrence of such common finding in untreated rats.
The few other isolated gross observations were considered to be consistent with spontaneous find ings encountered in the rats of these strain and age. This included the brown content seen in the va gina from one high-dose female that correlated with granulocyte aggregate. This was considered to
be part of the spontaneous background lesions in view of the low incidence of this change.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the non selected F1 pups, no examination was performed except for the macroscopic abnormality of the left kidney seen in 1/10 females treated at 25 mg/kg bw/day consisting in marked subacute suppurative inflammation. This change was considered to be unrelated to the test item administration.

Cohort 1A - Post-Weaning:
Test item-related changes were noted in the kidneys (and ureters), liver, thyroid glands, forestomach
and esophagus.
Kidneys and ureters
Hematoxylin-eosin-stained slides
There was a spectrum of dose-related degenerative and regenerative renal lesions in males and, at a
lower extent, in females treated at = 25 mg/kg bw/day. These lesions included:
• minimal to moderate degeneration/necrosis of tubular cells in males treated at = 75 mg/kg bw/day
and in one female treated at 200 mg/kg bw/day,
• minimal to moderate granular casts located in the outer medulla/inner cortex in males treated at = 75
mg/kg bw/day,
• increased severity and incidence of tubular basophilia, graded up to marked and consistent with r egeneration following cellular alterations in males and females treated at = 75 mg/kg bw/day,
• minima to moderate tubular dilatation in females treated at 25 mg/kg bw/day, and in males and females treated at = 75 mg/kg bw/day,
• minimal to marked hyaline droplet accumulation in the cortical tubules from males treated at = 25 mg /kg bw/day,
• increased severity and incidence of pelvic dilatation, in males treated at = 25 mg/kg bw/day only,
graded up to slight, and probably secondary to renal dysfunction,
• minimal tubular vacuolation seen in 2/20 males treated at 200 mg/kg bw/day and in 1/20 females
treated at 25 or 200 mg/kg bw/day that were seen with very low occurrence and thus were of equ ivocal significance,
• severe degeneration/necrosis of glomeruli accompanied by glomerular inflammation and he morrhage in one high-dose female, in addition to the various test item-related findings as tubular
degeneration/necrosis, basophilia, etc. This was accompanied by intrasinusoidal erythrocytes, pigm ent and increased cellularity in paracortex in the iliac and lumbar lymph nodes that were considered
to be secondary to these severe alterations in the kidneys (draining process in loco-regional lymph nodes).
Alpha-2 microglobulin-stained slides
When compared to controls, there were increased severity of granular staining of alpha-2 micr oglobulin in the tubules of kidneys from males treated at 200 mg/kg bw/day, associated with presence
of staining in dilated tubules (i.e. those containing granular casts) and in the medulla (cytoplasm of collecting ducts and/or granular material in lumen). At a lower extent, the severity of staining was lower in males treated at 200 mg/kg bw/day than controls in the large aggregates. Altogether, these stainings suggested that there was an increase in the amount of alpha-2 microglobulin deposit in the kidneys of males treated at 200 mg/kg bw/day, similarly to the distribution noted in the parents.
The ureters from 1 male treated at 75 mg/kg bw/day, of 3 males and 1 female treated at 200 mg/kg bw/day were dilated. This correlated with the macroscopic findings and was considered to be second
ary to the renal/pelvic dysfunction.
Liver
Dose-related minimal hepatocellular hypertrophy was noted in males treated at = 25 mg/kg bw/day and females treated at = 75 mg/kg bw/day.
Thyroid glands
Dose-related minimal follicular cell hypertrophy was noted in males treated at = 25 mg/kg bw/day and females treated at = 200 mg/kg bw/day.
Forestomach
Dose-related minimal to slight squamous cell hyperplasia, hyperkeratosis and/or edema were noted in
males and/or females treated at = 25 mg/kg bw/day. This was associated focally with aggregates of granulocytes in the epithelium and/or mixed cell infiltrates in occasional high-dose animals.
Esophagus
Minimal hyperkeratosis of the esophagus was noted in two males treated at 200 mg/kg bw/day. The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings
described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
There were test item-related changes neither in the male or female reproductive organs. There was a good correspondence between the vaginal smears and the histopathological exa mination of estrus cycle.
The main microscopic lesions (incidences and severity) observed in the Cohort 1A are presented in Table 9 of the section "Any other information on results incl. tables".
Enumeration of corpora lutea in HE-stained slides
At enumeration of corpora lutea in the ovary of control and high-dose group, there were no differences, with a mean number of corpora lutea of 21.55 (± 7.61) and 19.00 (± 5.57) per animal respectively,
reaching no statistical significance.
Enumeration of the number of primordial follicles on PCNA-stained slides
There were no differences between the control and the high-dose groups, with a mean number of 6. 76 (± 5.40) and 7.78 (± 3.67) primordial follicles per animal on PCNA-stained slides respectively, r eaching no statistical significance.

Cohort 1B - Post-Weaning:
Test item-related changes were noted in the kidneys (and ureters) from males and in the forestomach
of one high-dose female.
Kidneys and ureters
There was a spectrum of dose-related degenerative and regenerative renal lesions in males treated at
= 25 mg/kg bw/day. These lesions included:
• minimal to slight degeneration/necrosis of tubular cells in males treated at 200 mg/kg bw/day,
• minimal to moderate granular casts located in the outer medulla/inner cortex in males treated at = 75
mg/kg bw/day,
• increased severity and incidence of tubular basophilia, graded up to marked and consistent with re generation following cellular alterations in males treated at = 25 mg/kg bw/day,
• minimal to moderate tubular dilatation in males treated at = 75 mg/kg bw/day,
• minimal to marked hyaline droplet accumulation in the cortical tubules in males treated at = 25 mg/ kg bw/day (increased severity and incidence when compared to controls),
• increased severity and incidence of pelvic dilatation, in males treated at = 25 mg/kg bw/day, graded
up to slight, and probably secondary to renal dysfunction,
• minimal tubular vacuolation with low occurrence of one male treated at 75 mg/kg bw/day that was
considered to be of low toxicological significance in view of this isolated occurrence.
There was minimal increase of severity and incidence of ureter dilatation in males treated at 200 mg/ kg bw/day when compared to controls.
Forestomach
Moderate ulcer and hyperplasia of squamous cells together with slight hyperkeratosis were noted in
one female treated at 200 mg/kg bw/day.
The remaining microscopic findings were not considered to be associated with the test item admini stration because these findings were consistent with spontaneous and background findings described
in the literature, the findings were distributed randomly among groups, and/or their appearance was s imilar to changes found in controls.
This included a mammary gland adenocarcinoma in one female treated at 75 mg/kg bw/day or a ma rked pulmonary abscess in one male treated at 75 mg/kg bw/day that were considered to be part of
the spontaneous background
The main microscopic lesions (incidences and severity) observed in the Cohort 1B (Macroscopic Lesions Only) are presented in Table 10 of the section "Any other information on results incl. tables".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SEX RATIO: No effects were observed on the sex ratio (percentage of males) at any dose level (day 21 p.p.).

LYMPHOCYTE SUBTYPING
Cohort 1A:
In term of absolute counts (cells/mg of spleen), no statistically significant differences in mean were observed in test item-treated groups when compared to control group in any of the studied lymphocyte populations.
In term of relative counts (%), statistically significant, lower mean cytotoxic T cell frequency was noted in females given 200 mg/kg bw/day compared to control group (28.2% vs. 34.1%, respectively) This difference was of low magnitude, remained within our HCD and was observed in only one sex, and was therefore not attributed to the test item treatment.
No statistically significant difference was observed in males.

THYROID HORMONES:
Cohort 1A - Post-Weaning:
At 200 mg/kg bw/day, when compared to controls, mean T4 level was significantly lower in Cohort 1A
males (-23%). This was associated with statistically significant, higher mean TSH level
(2.6-fold). These changes were attributed to the test item treatment and correlated with the thyroid
follicular cell hypertrophy observed at microscopic examination.
No other relevant differences from controls were noted.
At 200 mg/kg bw/day, when compared to controls, the mean TSH level was significantly higher in C ohort 1A females (2.2-fold). This change was attributed to the test item treatment and correlated with
the thyroid follicular cell hypertrophy observed at microscopy. No variation in the T4 level was noted. No other relevant differences from controls were noted.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Description (incidence and severity):

QUANTIFICATION OF ANTI-KLH IGM RESPONSE
Cohort 3:
No conclusion could be drawn as the number of animals showing an anti-KLH IgM response was too low.
Before KLH immunization, all samples had quantifiable anti-KLH IgM concentrations as rat serum is expected to contain IgMs directed against some glycosylated moieties similar to those of KLH. As animals were not exposed to KLH before immunization, it is unlikely that this observation corresponds to anti-KLH IgM.
Before KLH immunization, statistically significant differences were found for mean serum
anti-KLH IgM concentrations between males treated at 200 mg/kg bw/day and females treated at
75 mg/kg bw/day versus corresponding control groups. As mentioned above, since those quantifications unlikely correspond to anti-KLH IgM, this finding was considered to be no relevant.
Five days after the KLH injection, only 1/79 animals showed an anti-KLH IgM response with a % Difference (Day 56 -61) above 50%. The other animals did not show any significant anti-KLH IgM response (% Difference (Day 56-61) < 50%). As the number of animals showing an adequate anti-KLH IgM response was too low including in the control group, it was not possible to draw a conclusion on the impact of the test item on the primary antibody response using this TDAR assay.
In view of these results, another analysis was performed at Charles River Laboratories Montreal
to determine if an anti-KLH response could be detected in the study serum samples using the Test Site procedure.
All animals were negative for the presence of anti-KLH IgM antibodies before the administration of KLH. Post immunization a high number of non-responders (< LLOQ) to KLH (IgM) were observed in all groups, including the control group. Even though the number of non-responder obtained was lower than with the Test Facility procedure, it was still too high to be able to determine any test item related effects on the T-cell dependent antibody responses to KLH.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental immunotoxicity testing

Generation:

F1 (cohort 3)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Remarks:

No conclusion could be drawn on the possible effect of the test item on the development of a humoral immune response. However, no changes in parameters that may reflect immunotoxicity /adverse effects on the immune system were observed.

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/developmental toxicity

Generation:

F1 (cohort 1B)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Non-adverse lower body weight gain was observed in male and female F1 pups at 200 mg/kg bw/day throughout the lactation period, but with recovery after weaning. Absence of effects on pup development (including sexual maturation) and no effects were observed on estrous cycle, mating, fertility, duration of gestation, number of implantation sites or live birth index

3

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/developmental toxicity

Generation:

F1 (cohort 1A)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Non-adverse lower body weight gain was observed in male and female F1 pups at 200 mg/kg bw/day throughout the lactation period, but with recovery after weaning. Absence of effects on pup development (including sexual maturation) and no effects were observed on estrous cycle.

4

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Generation:

F1 (cohort 1A)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Adverse effects on kidneys in only one female at 200 mg/kg bw/day

5

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Severe kidney toxicity was recorded in the kidneys at 75 and 200 mg/kg bw/day in males, associated with the accumulation of hyaline droplets. This accumulation is a toxic effect specific of male rat with no relevance for human risk assessment.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 75 or 200 mg/kg bw/day, when compared to controls, the number of pups (and litters) with findings at gross external examination (head hematoma, scab on tail) was higher on Day 1 p.p. These are common observations after birth.
No relevant differences were noted between the control group and the 25 mg/kg bw/day group.
No relevant findings were recorded in pups on Day 1-4 p.p. at any dose level.
The higher number of pups with hematoma on the head at 200 mg/kg bw/day, was not considered to be adverse, as this is a common observation in rat pups of this strain and age and because the hematoma disappeared on Day 4 p.p.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effects were noted on mean litter size at birth or on the incidence of pups found dead, missing or cannibalized on Days 1-4 p.p.
No effects were observed on the viability index at any dose level during lactation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences between control and test item-treated animals were noted in body weight or body weight change.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were observed in F2 pups. All findings were those commonly observed in pups of this strain and age.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2 (cohort 1B)

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Chemical analysis of the Dose Formulation

The test item concentrations in the administered dose formulations analyzed from the beginning to the end of treatment remained within an acceptable range of variation (-12.3% to +9.2%) when compared to the nominal values (± 15% of the nominal concentrations), except on three occasions (see Study Plan Adherence): one during the P-lactation (group 4),  F1-postweaning (group 2) and F1-premating (group 4) periods. No test item was detected in the control dose formulation.

 

 

Text Table 1

Absolute Splenic Lymphocyte Immunophenotyping in Cohort 1A Males (Mean, Cells/mg Spleen)

Dose level (mg/kg/day)	0	25	75	200	HCD
Number of animals	10	10	10	10	40
Splenocytes	196337	256255	263664	196122	[73602-605531]
		(+31)	(+34)	(0)
B cells	76531	102335	106705	76371	[23368-260668]
		(+34)	(+39)	(0)
NK cells	5812	7887	7020	6017	[2706-20443]
		(+36)	(+21)	(+4)
NKT cells	6583	8648	9029	6522	[2080-33213]
		(+31)	(+37)	(-1)
T cells	76928	99808	98744	74697	[29602-229860]
		(+30)	(+28)	(-3)
Helper T cells	48311	60985	61499	48517	[18796-156037]
		(+26)	(+27)	(0)
Cytotoxic T cells	25047	34153	32530	23010	[10089-73297]
		(+36)	(+30)	(-8)

(): in brackets, percentage (%) difference vs. controls. No statistically significant difference vs. controls.

HCD: Historical Control Data, (n = 5 studies) [min-max].

 

 

Text Table 2

Absolute Splenic Lymphocyte Immunophenotyping in Cohort 1A Females (Mean, Cells/mg Spleen)

Dose level (mg/kg/day)	0	25	75	200	HCD
Number of animals	10	10	10	10	40
Splenocytes	178144	210719	165994	251055	[75530-682243]
		(+18)	(-7)	(+41)
B cells	60853	75024	60007	95170	[22539-268451]
		(+23)	(-1)	(+56)
NK cells	5210	6373	5422	7651	[1904-36929]
		(+22)	(+4)	(+47)
NKT cells	7065	7804	6329	9397	[3215-21816]
		(+10)	(-10)	(+33)
T cells	78761	91974	69768	103098	[30789-266390]
		(+17)	(-11)	(+31)
Helper T cells	49473	58952	44173	70196	[19523-174401]
		(+19)	(-11)	(+42)
Cytotoxic T cells	26307	29452	22921	28704	[9729-95415]
		(+12)	(-13)	(+9)

(): in brackets, percentage (%) difference vs. controls. No statistically significant difference vs. controls. HCD: Historical Control Data, (n = 5 studies) [min-max].

 

Text Table 3

Relative Splenic Lymphocyte Immunophenotyping in Cohort 1A Males (Mean Percent vs. Splenocytes, %)

Dose level (mg/kg/day)	0	25	75	200	HCD
Number of animals	10	10	10	10	50
B cells	38.8	39.1	40.8	39.4	[29.8-46.8]
NK cells	2.9	3.1	2.7	3.1	[1.8-6.1]
NKT cells	3.3	3.4	3.3	3.3	[1.8-8.0]
T cells	39.7	39.5	37.3	37.5	[24.7-47.8]
Helper T cells	63.1	61.3	62.8	65.2	[32.7-77.5]
Cytotoxic T cells	32.5	34.0	32.6	30.2	[20.2-60.6]

No statistically significant difference vs. controls. HCD: Historical Control Data, (n = 5 studies) [min-max].

 

Text Table 4

Relative Splenic Lymphocyte Immunophenotyping in Cohort 1A Females (Mean Percent vs. Splenocytes, %)

Dose level (mg/kg/day)	0	25	75	200	HCD
Number of animals	10	10	10	10	50
B cells	33.0	35.3	35.4	37.9	[27.8-44.0]
NK cells	3.0	2.9	3.3	3.0	[2.1-6.6]
NKT cells	4.0	3.6	3.8	3.7	[1.6-7.5]
T cells	45.0	44.1	42.2	41.2	[22.3-52.4]
Helper T cells	62.4	63.6	63.5	67.7	[48.5-75.8]
Cytotoxic T cells	34.1	32.7	32.6	28.2**	[21.0-49.7]

Statistical significance: **: p<0.01. HCD: Historical Control Data, (n = 5 studies) [min-max].

 

 

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) was considered to be 25 mg/kg bw/day in males and 75 mg/kg bw/day in females based on the following changes in kidneys in P and F1 generations.
The No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity were considered to be 200 mg/kg bw/day in males and females based the absence of adverse effects observed in P, F1 and F2 generations.

Executive summary:

The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, Tert-dodecanethiol, that may occur as a result of pre- and postnatal exposure and to provide an evaluation of systemic toxicity in pregnant and lactating females and in young and adult offspring.

The test item, Tert-dodecanethiol, was administered daily by the oral route (gavage) at the dose level of 0, 25, 75 or 200 mg/kg/day in 0.5% Carboxymethylcellulose/0.5% Tween 80, at a constant dosage volume of 5 ml/kg/day, to sexually-mature male and female rats [parental (P) generation], continuously from 10 weeks before mating and through mating, gestation and weaning of the pups (F1 generation). At weaning, pups were also treated daily by oral gavage at the dose level of 0, 25, 75 or 200 mg/kg/day. Pups were assigned to cohorts for: reproductive/developmental toxicity testing (Cohorts 1A and 1B, including the production of a F2 generation), and developmental immunotoxicity testing (Cohort 3).

P generation

Mortality: no test item-related unscheduled deaths occurred.

Clinical signs: ptyalism was noted after treatment in males and females from all groups (including controls) with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect.

Body weight, body weight change and food consumption: no test item-related effects were noted during the premating, mating, gestation or lactation periods.

Estrous cycle, mating and fertility: no test item-related effects were noted on the estrous cycle, mating (including the mean number of days taken to mate) or fertility.

Delivery data: no test item-related effects were observed on the gestation index, duration of gestation, number of implantation sites, percentage of post-implantation loss, live birth index or pup sex ratio at any dose level.

P generation offspring (pre-weaning F1 pups): no test item-related effects were noted on litter size at birth, incidence of pups found dead, missing or cannibalized on Days 1-4 p.p., or lactation index. No test item-related clinical signs were observed in pups during the lactation period. Body weight gain was lower in male and female pups at 200 mg/kg/day throughout the lactation period (-7 and -8%, respectively), reaching statistical significance on Days 4-7 p.p. (-16% in both sexes), and leading to slightly lower mean body weight on Day 21 p.p. (-6 and -7%, respectively). These effects were attributed to the test item treatment, but considered as non-adverse in view of the magnitude. No effects were noted on the anogenital distance (AGD) or on sex ratio. No nipples or areolae were observed in any male pups on Day 12 p.p.

No test item-related findings were observed at macroscopic post-mortem examination of pups culled on Day 4 p.p. or euthanized after weaning.

Cohort 1A (after weaning)

Mortality: no test item-related unscheduled deaths occurred.

Clinical signs: ptyalism was noted after treatment in males and females in all groups (including controls) with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect.

Body weight, body weight change and food consumption: body weight was slightly lower in males and females at 200 mg/kg/day on Day 1 (-5 and -7%, respectively). This was considered to have resulted from the lower body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 due to body weight gains similar to those of controls or higher, reaching statistical significance on Days 15-22 and 29-36 (+5% for the whole period). In conclusion, no change in bodyweight in females were observed to be test item-related after the weaning. In males, the body weight was continuously lower between Days 1 and 71 due to generally lower body weight gains than those of controls (-4% for the whole period, no statistical differences), indicating the reversibility of this finding. These effects on body weight and body weight change in males were attributed to the test item treatment, but considered as non-adverse given their low magnitude.

No relevant effects were noted on food consumption in males or females at any dose level.

Sexual development: no effects were noted.

Estrous cycles: no effects were noted.

Cohort 1B (after weaning)

Mortality: no test item-related unscheduled deaths occurred.

Clinical signs: ptyalism was noted after treatment in males and females in all groups (including controls) with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect.

Body weight, body weight change and food consumption: body weight was minimally lower in males and females at 200 mg/kg/day on Day 1 (-6 and -3%, respectively). This was considered to have resulted from the lower body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 due to body weight gains generally similar to those of controls or higher, reaching statistical significance on Days 22-29 and 57-64 (+4% for the whole period), indicating the reversibility of this finding. No effects were noted during the pregnancy or lactation period. In conclusion, no change in bodyweight in females were observed to be test item-related after the weaning. In males, the body weight was continuously lower between Days 1 and 113, mainly between Days 8 and 29 (-18 to -7%, vs. controls) due in particular to statistically significant lower body weight gain on Days 1-8 (-34%), while thereafter, the difference from controls was minimal (-2% in terms of body weight on Day 113 and body weight gain on Days 1-13). These effects on body weight and body weight change in males were attributed to the test item treatment, but considered as non-adverse given their low magnitude.

No relevant effects were noted on food consumption in females at any dose level. At
200 mg/kg/day, food consumption was significantly lower in males on Days 1-8 (-15%) and
8-15 (-17%). This effect correlated with the lower body weight gain recorded during this period.

At 75 mg/kg/day, non-adverse and isolated lower body weight gain was recorded in males on Days 8-15 (-17%).

Sexual development: no effects were noted.

Mating and fertility: no effects were noted on the estrous cycle, mating (including the number of days taken to mate) or fertility.

Delivery data: no test item-related effects were observed on the gestation index, duration of gestation, number of implantation sites, percentage of post-implantation loss, live birth index or pup sex ratio at any dose level.

F1 generation offspring (pre-weaning F2 pups): no test item-related effects were noted on litter size at birth, incidence of pups found dead, missing or cannibalized on Days 1-4 p.p., viability index, clinical observations, body weight, body weight gain or macroscopic observations.

Cohort 3 (after weaning)

Mortality: no unscheduled deaths occurred.

Clinical signs: ptyalism was noted after treatment in males and females from 75 mg/kg/day with a dose-related incidence and frequency. This sign, commonly observed when a test item is administered by gavage, did not represent an adverse effect.

Body weight, body weight change and food consumption: body weight was slightly lower in males and females at 200 mg/kg/day on Day 1 (-6 and -9%, respectively). This was considered to have resulted from the lower mean body weight recorded before weaning. This difference from controls was no longer noted in females from Day 15 due to body weight gains similar or higher to those of controls, reaching statistical significance on Days 22-29 (+7% for the whole period), indicating the reversibility of this finding.  In conclusion, no change in bodyweight in females were observed to be test item-related after the weaning.

In males, the body weight was continuously lower on Days 1-36, but the difference from controls was minimal from Day 29 due to higher body weight gain from Day 22 (-1% for the whole period). These effects on body weight and body weight change in males were attributed to the test item treatment, but considered as non-adverse given their low magnitude.

No relevant effects were noted on food consumption in males or females at any dose level.

Sexual development: no effects were .

Quantification of anti-KLM IgM response: no conclusion could be drawn as the number of animals showing an anti-KLH IgM response was too low in both experiments.

Laboratory investigations

Hematology The only non-adverse findings that could be attributed to the test item treatment consisted of lower red blood cell parameters in P generation females at 200 mg/kg/day (including erythrocyte count, hemoglobin concentration and packed cell volume, i.e. -13, -10% and -12%, respectively, vs. controls) and in Cohort 1A males at 200 mg/kg/day (including hemoglobin concentration, mean cell hemoglobin concentration and reticulocyte count, i.e. -6%, -5% and -15%, vs. controls).

Coagulation: no relevant effects were noted in P generation or Cohort 1A animals.

Blood biochemistry: no relevant effects were noted in P generation or Cohort 1A animals. The only non-adverse findings that could be attributed to the test item treatment consisted of lower glucose and triglyceride levels in P generation males at 200 mg/kg/day (same tendency at 75 mg/kg/day), lower creatinine level in Cohort 1A males at 200 mg/kg/day and lower glucose and higher triglyceride levels in Cohort 1A females at 200 mg/kg/day.

Urinalysis: No relevant effects were noted in P generation or Cohort 1A animals.

Thyroid hormones:

• P generation: T4 level was significantly lower in P generation males at 200 mg/kg/day (-22%). This change correlated with the thyroid follicular cell hypertrophy observed at microscopy. No relevant changes in females or in non-selected F1 offspring,
• Cohort 1A: in males, T4 level was significantly lower in males at 200 mg/kg/day (-23%). This was associated with statistically significant, higher TSH level (2.6-fold). In females, TSH level was significantly higher at 200 mg/kg/day (2.2-fold) although no variations of T4 level were noted. These changes correlated with the thyroid follicular cell hypertrophy observed at microscopy in both sexes.

Lymphocyte subtyping (Cohort 1A): there were no test item treatment-related findings.

Sperm analysis (P generation and Cohort 1A males):

• P generation: no effects on sperm analysis parameters,
• Cohort 1A: non-adverse lower number of testicular sperm heads and daily sperm production rate at 200 mg/kg/day (-16%) were noted.

 

Pathology

P generation

Increased liver and kidney weights were recorded in females at = 25 mg/kg/day and in males at = 75 mg/kg/day and correlated respectively with hepatocellular hypertrophy (liver) and tubular basophilia/accumulation of hyaline droplets (kidneys).

At gross examination, there were test item-related accentuated lobular pattern in the liver from males at = 75 mg/kg/day, and enlargement in males at 200 mg/kg/day, that correlated with hepatocellular hypertrophy; irregular color in the kidneys from males at = 75 mg/kg/day, and enlargement in the kidneys from males at 200 mg/kg/day, correlated with tubular basophilia and accumulation of hyaline droplets; thickening and yellow deposit in the forestomach from males and females at 200 mg/kg/day that correlated with squamous cell hyperplasia and/or hyperkeratosis; dilatation in the ureters from males at 200 mg/kg/day that correlated with microscopic lumen dilatation. The dilated pelvis seen in the kidneys from occasional males at = 25 mg/kg/day was considered to be ambiguous.

At microscopic examination, test item-related changes were noted in the kidneys generally in males and females at = 25 mg/kg/day and were characterized by adverse tubular degeneration/necrosis at 75 and 200 mg/kg/day; granular casts, tubular basophilia, dilatation, hyaline droplet accumulation and vacuolation, pelvic dilatation, seen generally from 25 mg/kg/day and considered to be non-adverse; and increased amount of tubular alpha-2uglobulin as demonstrated by IHC. There were also test item-related lesions in the ureters
(non-adverse secondary dilatation in two males at 200 mg/kg/day), liver (non-adverse hepatocellular hypertrophy in males and females at = 25 mg/kg/day), thyroid glands
(non-adverse follicular cell hypertrophy in males at = 75 mg/kg/day and females at = 200 mg/kg/day), forestomach (mainly non-adverse squamous cell hyperplasia, hyperkeratosis and edema in males and females at = 25 mg/kg/day) and adrenal glands (non-adverse increased incidence and severity of cortical hypertrophy in females at 200 mg/kg/day).

At quantitative evaluation of primordium follicles, there were no differences between the high-dose and the control groups while corpora lutea were statistically significantly decreased in females treated at 200 mg/kg/day when compared to controls. This difference was considered to be of no toxicological significance in the light of other results of the study regarding the female genital system and reproduction function.

Cohort 1A

Increased liver and kidney weights were recorded in females at = 25 mg/kg/day and in males at = 75 mg/kg/day. These correlated respectively with hepatocellular hypertrophy (liver) and tubular basophilia/accumulation of hyaline droplets (kidneys).

At gross examination, there were accentuated lobular pattern and granular aspect in the liver from males at 200 mg/kg/day (correlated with hepatocellular hypertrophy); irregular color or surface, green discoloration, and/or enlargement in the kidneys from males at = 75 mg/kg/day, and in females at 200 mg/kg/day (correlated with tubular basophilia and accumulation of hyaline droplets); yellow deposit in the forestomach from females at 200 mg/kg/day (correlated with squamous cell hyperplasia and/or hyperkeratosis); dilatation in the ureters from males at = 75 mg/kg/day and in females at 200 mg/kg/day (correlated with microscopic lumen dilatation).

At microscopic examination, test item-related changes were noted in the kidneys (adverse tubular degeneration/necrosis in males at = 75 mg/kg/day and in 1 female at 200 mg/kg/day; granular casts, basophilia, dilatation, hyaline droplet accumulation, vacuolation and pelvic dilatation seen from 25 mg/kg/day and considered to be non-adverse at 25 mg/kg/day in males and at 25 and 75 mg/kg/day in females; a severe degeneration/necrosis of glomeruli accompanied by glomerular inflammation in one high-dose female accompanied by reactive lesions in the draining lymph nodes; increased amount of tubular alpha2uglobulin as demonstrated by IHC), ureters (non-adverse secondary dilatation), liver (non-adverse hepatocellular hypertrophy in males and/or females at = 25 mg/kg/day), thyroid glands
(non-adverse follicular cell hypertrophy in males and/or females at = 25 mg/kg/day), forestomach (non-adverse squamous cell hyperplasia, hyperkeratosis and edema at = 25 mg/kg/day) and esophagus (non-adverse hyperkeratosis in two males at 200 mg/kg/day).

At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1B

There was no test item-related mortality.

Increased liver and kidney weights were recorded in males and females at = 75 mg/kg/day and correlated respectively with tubular basophilia/accumulation of hyaline droplets (kidneys).

At gross examination, there were test item-related accentuated lobular pattern, thickening and/or granular aspect in the liver from males at 75 mg/kg/day; irregular color or surface and/or green discoloration in the kidneys from males at = 75 mg/kg/day, correlated with tubular basophilia and accumulation of hyaline droplets; and white mass in the forestomach from 1/20 females at 200 mg/kg/day that correlated with ulcer, squamous cell hyperplasia and hyperkeratosis.

There was also ambiguous dilated pelvis in the kidneys from occasional males at = 25 mg/kg/day.

At microscopic examination, test item-related changes were noted in kidneys (adverse tubular degeneration/necrosis in males at 200 mg/kg/day; granular casts, tubular basophilia, dilatation, hyaline droplet accumulation, vacuolation and pelvic dilatation seen from 25 mg/kg/day and considered to be non-adverse at 25 and 75 mg/kg/day), ureters (non-adverse dilatation in males treated at 200 mg/kg/day) and forestomach (adverse ulcer, hyperplasia of squamous cells and hyperkeratosis in one high-dose female).

Cohort 3

Increased liver weights were recorded in males and females at 200 mg/kg/day.

At gross examination, there were test item-related irregular color in the kidney from males at 200 mg/kg/day, correlated with tubular basophilia and accumulation of hyaline droplets; white discoloration in the forestomach from1/10 males at 200 mg/kg/day that correlated with squamous cell hyperplasia and hyperkeratosis; and dilatation in the ureters from occasional males and females at 25 or 200 mg/kg/day.

There was also equivocal dilated pelvis in the kidneys of occasional males from 25 mg/kg/day. 

At microscopic examination, there were test item-related changes in kidneys (adverse tubular degeneration/necrosis in one male at 200 mg/kg/day accompanied by tubular basophilia, dilatation, hyaline droplet accumulation), ureters (non-adverse dilatation in males at 25 or 200 mg/kg/day) and forestomach (non-adverse hyperplasia of squamous cells together with hyperkeratosis and edema at 200 mg/kg/day in males).

Non selected pups : There was no test item-related mortality, organ weight differences or gross changes.

The test item, Tert-dodecanethiol, was administered daily by oral gavage, at the dose level of 0, 25, 75 or 200 mg/kg/day, to sexually-mature male and female rats [parental (P) generation] continuously from 10 weeks before mating and through mating, gestation and weaning of the pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity or developmental immunotoxicity testing.

Systemic toxicity evaluation: The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) was considered to be 25 mg/kg/day in males and 75 mg/kg/day in females based on the following changes in kidneys in P and F1 generations: 

• in males, adverse effects on kidneys in a lot of males at the doses of 75 and 200 mg/kg/day in the P and F1 generation. Severe kidney toxicity was associated with the accumulation of hyaline droplets from 25 mg/kg/day; this accumulation is a toxic effect specific of male rat with no relevance for human risk assessment.
• in females, adverse effects on kidneys in only one female at 200 mg/kg/day in the cohort 1A

Reproductive/developmental toxicity testing:

• in P generation animals: no effects were noted on estrous cycle, mating, fertility, duration of gestation, number of implantation sites or live birth index,
• In Cohort 1A and/or 1B animals: no effects were noted on pup development (including sexual maturation) and no effects were observed on estrous cycle, mating, fertility, duration of gestation, number of implantation sites or live birth index. Non-adverse lower body weight gain was observed in male and female F1 pups at 200 mg/kg/day throughout the lactation period, but with recovery after weaning,
• the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity were considered to be 200 mg/kg/day in males and females based the absence of adverse effects observed in P, F1 and F2 generations.

 

Developmental immunotoxicity testing:

Based on the available data, there was no evidence of effects on the development of the immune system [no impact on the lymphocyte subtyping and no changes in parameters that may reflect adverse effects on the immune system, i.e. hematological changes (e.g. leucocyte count), changes in immune system organ weights or histology (e.g. changes in thymus, spleen, lymph nodes or bone marrow)].