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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 November 2018 to 19 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
22 November 2018 to 19 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
RjHan:SD (CD®)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: appr. 10 weeks (males) and appr. 12 weeks (females)
- Weight at study initiation: 396 g to 441 g (males), 257 g to 311 g (females)
- Fasting period before study: No (except for at least 14 hours prior to blood sampling and during urine collection)
- Housing:Animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust.
- Diet: SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 6 days (males), 13 days (females)

DETAILS OF FOOD AND WATER QUALITY:
The batches of diet, sawdust and wood shavings were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 05 December 2018 (males) or 19 December 2018 (females) To: 18 February 2019 (males) or 19 February 2019 (females)
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) Carboxymethylcellulose (400-800 cps) / 0.5% (v/v) Tween 80 in drinking water tr eated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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. The test item dose forulations were prepared on the days of treatment, stored at room temperature and protected from light and used within 4 hours after the end of their preparation.

VEHICLE
- Concentration in vehicle:0, 10, 20 and 40 mg/mL for the control, low, mid and high dose groups, respectively
- Amount of vehicle: 5 mL/kg bw/day
- Batch no. carboxymethylcellulose (400-800 cps): SLBQ8545V
- Batch no. Tween 80: BCBT5201
- Drinking water treated by reverse osmosis using ELIX 5 (Millipore S.A.)
Details on mating procedure:
Females were paired with males from the same dose level group. One female was placed with one male, in the latter's cage, during the night.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated Day 0 p.c. Each female was placed with the same male until mating occurred or 14 days had elapsed. One pair (low dose group) with no evidence of mating after 14 days was separated and the female was placed for a further 9 days with a different male from the same dose level group who had already mated. The pre-coital time was calculated for each female.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed using an analytical method developed and validated at Charles River Laboratories Evreux (Charles River Laboratories Evreux/Study No. 46438 VAA) prior to dose formulation analysis using High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS).
Dose formulations were analysed three times during the study: Weeks 1, 5 and 9 (referring to the Week 1 of males).
A sample was taken from control and test item dose formulations and analyzed using the validated method.
Acceptance criterion: Measured concentration = nominal concentration ± 15%
Duration of treatment / exposure:
The males were treated starting 4 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed) until euthanasia (11 weeks in total).
The females were treated starting 2 weeks before mating, during the mating period (until evidence of
mating or 2 weeks had elapsed), during gestation, during lactation until Day 21 p.p. inclusive until
euthanasia for females with no delivery.
Frequency of treatment:
Once daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected on the basis of the results of a previous study (Charles River Laboratories Evreux/Study No. 46746 TSR) performed in the same species, in which 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.
In this study, 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 pathology, the test item administration at = 100 mg/kg bw/day induced squamous cell hyperplasia and
hyperkeratosis in the forestomach and centrilobular hepatocellular hypertrophy in the liver in both sexes, and tubular hyaline droplets accumulation and degeneration/regeneration 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 and the maximal tolerated dose level to be lower than 300 mg/kg bw/day.
Positive control:
No.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period (including weekends and public holidays)
- Cage side observations included observations for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day
- 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 males:
once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia;
- Time schedule for examinations females: once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8, 13, 17 and 21 p.p. Females euthanized prematurely were weighed before euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption of males: measured once a week from the first day of treatment until the start of the mating period and after the mating period until euthanasia;
- Food consumption of females: measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8, 8-13, 13-17 and 17-21 p.p. During the mating period, food consumption was not measured for males or females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 14 hours o/n)
- How many animals: All
- All parameters according to guideline were included

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 14 hours o/n)
- How many animals: All
- All parameters according to guideline were included
- Coagulation parameters (Prothrombin time, fibrinogen and activated partial thromboplastin time) were determined from the first five males and lactating females from each group on the day of euthanasia
- Thyroid hormones (T4 and TSH) were determined for pups sampled on Day 21 p.p. and for F0 males sampled at termination.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (at least 14 hours) before necropsy)
- Metabolism cages used for collection of urine: Yes, urine was collected onto thymol crystals
- Animals fasted: Yes
- All parameters according to guideline were included.

FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule for examinations: once at the end of the treatment period (for males: during the last week of treatment; for females: on
Day 21 p.p. after euthanasia of the pups)
- Dose groups that were examined: The first five males and lactating females from each group
- Battery of functions tested:
- Detailed clinical examination (including in the cage "touch escape" or ease of removal from the cage, in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis), in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia;
- The assessment of reactivity to manipulation and to different stimuli (including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature);
- Motor activity (measured once by automated infra-red sensor equipment over a 60 minute period).
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 a.m.):
- during the 2 last weeks of the pre-treatment period (including for the two supplementary females per group, whose data are not presented in the study report),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on the day of sacrifice before euthanasia, to allow correlation with reproductive organs histopathology.
Sperm parameters (parental animals):
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The pups were observed daily for clinical signs and abnormal behavior. The body weight of each pup was recorded on Days 1, 4, 8, 13, 17 and 21 p.p. The following physical development measurements were performed in live pups of each litter:
- anogenital distance (AGD): on Day 1 p.p.,
- number of nipples and of areolae in male pups: on Day 12 p.p.
The AGD was normalized to the cube root of body weight recorded on Day 1 p.p.
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
males: after the end of the mating period (at least 10 weeks of treatment in total),
females: on Day 22 p.p. Females which did not deliver were euthanized on Day 26 p.c. (after a body weight recording to check for a possible un-noticed delivery) by inhalation of carbon dioxide gas followed by cervical dislocation since gestation was suspected.

A complete macroscopic post-mortem examination was performed on all F0 animals including those that were euthanized prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, 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 corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 22 p.p. and for one female in the low dose group that was euthanized on Day 26 p.c. due to no delivery. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

ORGAN WEIGHTS: Yes
Organ weights of the main organs of the first five euthanized as scheduled males and the first five females euthanized on Day 22 p.p. of each group were determined. A table in which all tissue procedures are summarized is attached below.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table (attached below) from the first five euthanized as scheduled males and lactating females of the control and high-dose groups,
- all macroscopic lesions of all groups, including kidneys of the prematurely euthanized low dose group female and the subcutis mass of the prematurely euthanized high dose group female,
- reproductive organs from females that did not conceive, or from pregnant female that did not deliver, to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Based upon the results of the microscopic examination of the high-dose group, the following tissues of the low- and intermediate-dose groups were examined on the first five euthanized as scheduled males and/or lactating females: kidneys (males), liver (males and females), thyroids (males and females), stomach with forestomach (males), forestomach (females), vagina (females).
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS:
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood was sampled), followed by exsanguination when the thyroids were sampled:
pups not selected: on Day 4 p.p.,
surviving pups: on Day 21 p.p.

Pups prematurely euthanized because of dying mother were euthanized by an intraperitoneal injection of sodium pentobarbital.
Found dead and prematurely euthanized pups were submitted to a detailed external examination (including orifices and buccal cavity), after euthanasia when applicable. Particular attention was paid to the external genital organs and to whether the pup had been fed (e.g. presence of milk in the stomach) when possible. Then, they were discarded without any further examination.

The body weight of 1 selected pup/sex/litter (or of two pups from the same sex when there was only one sex in the litter) euthanized on Day 21 p.p. was recorded before euthanasia.

Pups euthanized on Day 21 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.

Blood samples were taken at termination on Day 21 p.p. from at least two pups/litter (chosen by manual randomization; approximately 0.25 mL per pup (to obtain approximately 0.5 mL of blood in total) was collected from the vena cava immediately after euthanasia and then pooled per litter) to determine thyroid hormones.
Statistics:
Body weight, food consumption and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions). The statistical analysis sequence for hematology, blood biochemistry, urinalysis, hormones, motor activity, anogenital distance, nipples/areolae, live birth index, sex-ratio and post-implantation loss parameters and for organ weights (level of significance of 0.05 or 0.01) are attached in Section 7.5.
Data are expressed as group mean values ± standard deviation (body weight, body weight change, food consumption, number ofcorpora lutea, number of implantation sites, number of pups and pup body weight, gestation length) or as proportions (pre-implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index). Whenever appropriate, the experimental unit of comparison was the litter.
Data of non-pregnant females are not included in group mean calculations (body weight, body weight change, food consumption).
Data of pregnant female with no evidence of mating (i.e.not assigned a Day 0p.c.date) are not included in the group mean calculations such as body weight and food consumption for thep.c.period, but all data from the dams and pups are included in the group mean calculations for the p.p.period.
Reproductive indices:
- pre-implantation loss: ((Number of corpora lutea - Number of implantation sites)/Number of corpora lutea)*100;
- post-implantation loss: ((Number of implantation sites - Number of live pups)/ Number of implantation sites)*100;
- mating index: ((Number of mated animals/ Number of paired animals)* 100;
- fertility index: (Number of pregnant female partners/ Number of mated pairs)*100;
- gestation index: (Number of females with live born pups/ Number of pregnant females)*100.
Offspring viability indices:
- live birth index: (Number of live born pups on Day 1 p.p./ Number of delivered pups)* 100;
- viability index on Day 4 p.p.: (Number of surviving pups on Day 4 p.p. (before culling)/ Number of delivered pups)*100;
- lactation index on Day 21 p.p.: (Number of surviving pups on Day 21 p.p./ Number of surviving pups on Day 4 p.p. (after culling))*100;
- AGD/cube root of body weight ratio: (AGD/ ³vBody weight).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in males and females given 100 or 200 mg/kg bw/day during the premating, pregnancy and lactation periods. It was also noted in one isolated female given 50 mg/kg bw/day during the pregnancy period. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.
The other clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (area of hair loss, cutaneous lesions, scabs), chromodacryorrhea, chromorhinorrhea, reddish vaginal discharge, short/broken teeth, mass on thorax area and/or reflux at dosing, were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.
Description (incidence and severity):
n/a
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No unscheduled deaths occurred in males at any dose level during the study. No mortality of females occurred in the control group and the group dosed at 100 mg/kg bw/day.
At 50 mg/kg bw/day, one female was prematurely euthanized on Day 23 p.c. due to difficulties to deliver. Signs of poor clinical condition (pallor of eyes and extremities, cold to the touch, round back, piloerect ion and dyspnea) were noted on the day of sacrifice. At necropsy, there were six alive and two dead fetuses in the uterine horns, enlarged and hemorrhagic placenta, red left adrenal gland, red content in vagina and irregular color in kidneys. This latter finding correlated microscopically with a severe acute degeneration/necrosis of tubules and glomeruli. These changes were considered to be unrelated to test item administration in view of their isolated occurrence and the absence of dose-relationship. They could be secondary to uterine/placental infection and sepsis in spite no bacteria were observed at microscopic examination of the kidney.
At 200 mg/kg bw/day, one female was prematurely euthanized on Day 2 p.p. because of the presence of a mass on the mammary gland. This mass of 3.5 x 3 cm, white and heterogeneous in the subcutis (mammary gland region), was a mammary adenocarcinoma and was considered to be unrelated to the test item treatment.
Due to no delivery, three females were euthanized on Day 26 p.c. without clinical signs prior to death (except for ptyalism in the high dose females):
- one female at 50 mg/kg bw/day. At necropsy, one dead fetus and one placenta were found in the left horn;
- two females at 200 mg/kg bw/day. At necropsy, the females were found to be non pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No relevant effects were observed on mean body weights or mean body weight changes in males at any dose level. The lower mean body weight gain recorded in males given 200 mg/kg bw/day throughout the treatment period (-10% vs. controls) was not attributed to the test item treatment as the difference was slight, due to isolated body weight losses and with no impact on the terminal body weight (-3% vs. controls).
No relevant effects were observed on mean body weights or mean body weight changes in females at any dose level. A lower mean body weight gain recorded in females given 200 mg/kg bw/day during the gestation period (16% vs. controls) was not attributed to the test item treatment as the difference was mainly due to the contribution of one female which also consumed less food (body weight gain of +20 g on Days 14-20 vs. +94 g in controls).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on mean food consumption were observed in males at any dose level. The mean food consumption was considered to be unaffected by the test item treatment at any dose level.
The lower mean food consumption recorded in females given 200 mg/kg bw/day during the lactation period (-10% vs. controls) was not attributed to the test item treatment as the difference was mainly due to the contribution of one female (food consumption values between 22 and 44 g/day; without these values the difference vs. controls felt to -3%).
Description (incidence and severity):
n/a
Description (incidence and severity):
n/a
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted on the hematology parameters at the end of the treatment period. The only statistically significant differences between control and test item-treated animals (i.e. lower mean cell hemoglobin concentration in females at 50 mg/kg bw/day and males at 100 mg/kg bw/day, lower relative reticulocyte count in males at 50 mg/kg bw/day, higher neutrophil counts in males at
200 mg/kg bw/day, lower eosinophil counts in males at 100 mg/kg bw/day, higher large unstained cell counts in males at 50 and 100 mg/kg bw/day), were not attributed to the test item treatment as they were of low magnitude and/or isolated and/or not dose-related.

No test item-related effects were noted on the clotting parameters at the end of the treatment period. The only statistically significant difference between control and test item-treated animals (i.e. longer activated partial thromboplastin time in males at 100 mg/kg/day) was not attributed to the test item treatment as it was not dose-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted on the blood biochemistry parameters at the end of the treatment period.
The only statistically significant differences between control and test item-treated animals were not attributed to the test item treatment as they were of low magnitude and/or isolated (calcium, glucose, creatinine, protein and albumin levels) or were noted with an opposite trend in males and females (triglyceride levels) or because of the direction and magnitude of the changes (total bilirubin and bile
acid levels, alanine aminotransferase activity).
No effects were noted on the mean thyroid hormone levels (T4 and TSH plasma levels) in males at termination.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted on the quantitative or qualitative urinary parameters at the end of the treatment period. The only statistically significant difference between control and test item-treated animals (i.e. lower urinary volume in females given 200 mg/kg bw/day) was not attributed to the test item treatment and it was of low magnitude and isolated.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No behavioral or neurological abnormalities were observed during the tests in any group. The motor activity (60-minute recording period) was unaffected by the test item treatment.
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):
An overview of the microscopic findings is included in Section 7.5.
Test item-related microscopic observations included mainly adverse renal changes in males (degeneration/necrosis of tubules, accumulation of hyaline droplets, casts, tubular dilatation and basophilia) and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid gland, hyperkeratosis and squamous cell hyperplasia in forestomach, stomach (atrophy of glands) and vagina (increased mucification).
Kidneys: Minimal degeneration/necrosis of proximal tubules, associated with minimal to marked accumulation of hyaline droplets in proximal tubules, casts (granular and, to a lesser extent, hyaline), tubular dilatation in cortex and medulla and cortical basophilia (increased severity/incidence) were noted in males treated at = 50 mg/kg bw/day when compared to controls.
Liver: Minimal to slight hepatocellular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day. It was mostly in centrilobular areas but also sometimes diffuse.
Thyroid: Minimal follicular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day, in association with heterogeneous colloid in 2/5 high-dose males.
Forestomach: Minimal to slight hyperkeratosis often associated with squamous cell hyperplasia were noted in males and females treated at = 50 mg/kg bw/day.
Stomach: Minimal atrophy of gastric gland was noted in 4/5 males treated at 200 mg/kg bw/day. This affected mostly the parietal cells.
Vagina: Increased incidence and severity of vaginal mucification was noted in females treated at 100 or 200 mg/kg bw/day.
Additional examination of males/females that did not conceive: The males had no lesions in the reproductive organs.
For one female, the left horn contained one dead fetus and the placenta was partially present. There was a moderate acute granulocytic inflammation in the uterus with edema and bacteria in lumen that were probably secondary to the presence of a dead fetus. Two females had functional estrus cycle (one was in proestrus and the other was in estrus). There were no microscopic abnormalities in their reproductive tract.
The other changes were considered not to be related to the test item administration since they were not dose-related, of low incidence and part of the spontaneous background in the rat kept under laboratory conditions.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
n/a
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No test item-related effects were observed on the mating or fertility data. All animals mated between 1 and 4 days.
All females were pregnant, except two females given 200 mg/kg bw/day, which accounts for the slightly lower mean fertility index noted at this dose level. These isolated cases were considered as incidental. A summary of mating and fertility data is included in tabular form below. All pregnant females delivered, except two females given 50 mg/kg bw/day. No delivery was observed for one of the two females; at necropsy, one dead fetus and one placenta were found in the left horn. Difficulty to deliver was noted for the other female; at necropsy, six alive and two dead fetuses were found in the uterine horns. These occurrences were considered to be unrelated to the test item treatment as they were noted with no dose-relationship.

No test item-related effects were observed on the number of corpora lutea, pre- and post-implantation losses or pups delivered, or on the duration of gestation at any dose level.

The higher mean pre-implantation loss associated with the lower mean number of implantation sites and pups delivered recorded in females given 200 mg/kg bw/day was not attributed to the test item treatment as the difference was due to the contribution of one female (15 corpora lutea but only three pups delivered).
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction performance
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
Dose descriptor:
NOAEL
Remarks:
general 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
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related clinical signs, external observations or abnormal behaviour were observed at any dose level in pups. The results are summarized in a table below.
Description (incidence and severity):
n/a
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test item-related effects on the incidence of pups found dead/sacrificed moribund or cannibalized were noted at any dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight and mean body weight changes per litter recorded in control and test item-treated pups during the lactation period are summarized in tabular format below. At 100 and 200 mg/kg bw/day, when compared to controls, mean body weight was lower at birth in males and females (statistically significant in female pups). Throughout the lactation period mean body weight gain was lower in male and female pups at these doses, resulting in a statistically significant lower mean body weight on Day 21 p.p. (around -10%, dose-related in females).
While a relationship to the test item treatment cannot be excluded, the differences were of low magnitude (all values remained within the range of our historical control data, at least until Day 13 p.p.) and were therefore considered to be non-adverse.
Description (incidence and severity):
n/a
Description (incidence and severity):
n/a
Description (incidence and severity):
n/a
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects were noted on the mean thyroid hormone levels (T4 and TSH plasma levels) in pups on Day 21 p.p. at any dose level. The results are included below.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related effects on the anogenital distance were observed in males or females at any dose level. The mean AGD for males was 4.28, 4.52, 4.35 and 4.36 mm for the control group and the low, mid and high dose group, respectively (HCD: 4.14-6.55). The mean AGD/BW*E1/3 for males was 2.11, 2.23, 2.19 and 2.21 mm for the control group and the low, mid and high dose group, respectively (HCD: 2.16 - 3.26).
The mean AGD for females was 2.42, 2.65, 2.59 and 2.51 mm for the control group and the low, mid and high dose group, respectively (HCD: 1.85 - 4.71). The mean AGD/BW*E1/3 for females was 1.22, 1.33, 1.33* and 1.31 mm for the control group and the low, mid and high dose group, respectively (HCD: 0.92 - 2.37).
* The statistically significant (p<0.05) higher mean anogenital distance corrected for body weight measured in female pups at 100 mg/kg bw/day was not attributed to the test item treatment as the difference from controls was not dose-related and was of low magnitude (+9%).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples or areolae were observed in any male pups on Day 12 p.p.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic changes.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No effects were noted on the sex ratio (percentage of males) at any dose level, which was 45.2, 53.6, 53.1 and 49.1 for the control group and the low, mid and high dose group respectively.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS

The test item concentrations in the administered dose formulations analyzed in Weeks 1 (two preparations), 5 and 9 (referring to Week 1 for males) remained within an acceptable range of variations of -8.9% to +1.8% when compared to the nominal values (± 15% of the nominal concentrations), except for the first formulation of group 3 male in Week 1 (-41.6%). No test item was detected in the control dose formulation.

Conclusions:
Based on the results of a repeated dose toxicity study, combined with screening study for reproduction and development toxicity, performed according to OECD guideline 422 and GLP principles, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/ day based on the absence of adverse findings at this dose level. The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 200 mg/kg bw/day based on the absence of effects on mating or fertility at this dose level. The NOAEL for toxic effects on progeny was considered to be 200 mg/kg bw/day.
Executive summary:

A repeated dose toxicity study combined with screening study for reproduction and development toxicity was performed with Tert-dodecanethiol according to OECD guideline 422 and GLP principles. Three groups of ten male and ten female Sprague-Dawley rats received the test item, Tertdodecanethiol, daily by the oral route (gavage) at dose levels of 50, 100 or 200 mg/kg bw/day, under a constant dosage volume of 5 mL/kg bw/day. A control group of ten male and ten female rats received the vehicle only (0.5% Carboxymethylcellulose / 0.5% Tween 80) under the same experimental conditions. Males were treated for an overall period of approximately 10 weeks: 4 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 8 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks), gestation (3 weeks) and lactation (3 weeks). Males were euthanized after completion of the mating period. Dams and their pups were euthanized on Day 22 p.p. The actual test item concentrations in the dose formulations were confirmed in Weeks 1, 5 and 9 using a validated LC/MS-MS analytical method.

Animals were checked daily for clinical signs and mortality. Detailed clinical examinations were performed weekly. Functional observation battery and motor activity were performed on the first five males and five lactating females during the last days of treatment. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before mating until the females had mated and on the day of sacrifice, before euthanasia. The animals were paired for mating after two (females) or four (males) weeks of treatment and the dams were allowed to litter and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible five males and five females per litter. Pups not selected on Day 4 p.p. were sampled, euthanized and discarded without further examination. The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8, 13, 17 and 21 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p. Hematology, coagulation, blood biochemistry and urinary investigations were performed on the first five males and five lactating females in each group at scheduled euthanasia. Thyroid hormone levels (TSH and T4) were determined in males at sacrifice and in at least two pups/litter euthanized on Day 21 p.p. Males were euthanized after completion of the mating period. Dams were euthanized on Day 22 p.p. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five euthanized males and lactating females in all groups. Pups were euthanized on Day 21 p.p. and submitted to a detailed external examination with a particular attention to the external genital organs. No test item-related deaths occurred in males or females during the study. Ptyalism was observed mainly at 100 and 200 mg/kg bw/day throughout the study. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect. The functional observation battery and motor activity were unaffected by the test item treatment. Body weight and food consumption were not impaired by the test item treatment. The estrous cycle was not impacted by the test item treatment. The mating, fertility and delivery data were unaffected by the test item treatment. At hematology, coagulation, blood biochemistry and urinary investigations, no changes were noted. Thyroid hormone analysis did not reveal any disturbances in F0 males at sacrifice.

At pathology investigations, test item-related increased liver weights were noted in males and/or females at = 50 mg/kg bw/day, and increased kidney weights 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), and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid glands, hyperkeratosis and squamous cell hyperplasia in forestomach at = 50 mg/kg bw/day in males and at = 100 mg/kg bw/day in females, atrophy of glands in stomach in males treated at 200 mg/kg bw/day and increased mucification in vagina at = 100 mg/kg bw/day in females. Observations of the pups from birth to Day 21 p.p. did not show any effects on mortality, viability, clinical signs, sex ratio or anogenital distance. 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 disturbance in pups on Day 21 p.p.

In conclusion:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/day in females based on the absence of adverse findings at this dose level. The adverse renal changes noted in male rats at = 50 mg/kg bw/day (accumulation of hyaline droplets associated with tubular vacuolation and degeneration/necrosis) are specific to male rats with no relevance for human risk assessment. No other adverse effects were observed in males,

- the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 200 mg/kg bw/day based on the absence of effects on mating or fertility at this dose level,

- the NOAEL for toxic effects on progeny was considered to be 200 mg/kg bw/day given the lower pup weight at 100 or 200 mg/kg bw/day, recorded at birth and throughout the lactation period, considered as non-adverse.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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
Remarks:
RjHan:SD (CD®)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: appr. 10 weeks (males) and appr. 12 weeks (females)
- Weight at study initiation: 396 g to 441 g (males), 257 g to 311 g (females)
- Fasting period before study: No (except for at least 14 hours prior to blood sampling and during urine collection)
- Housing: Animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust.
- Diet: SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 6 days (males), 13 days (females)

DETAILS OF FOOD AND WATER QUALITY: The batches of diet, sawdust and wood shavings were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 December 2018 (males) or 19 December 2018 (females) To: 18 February 2019 (males) or 19 February 2019 (females)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.
The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
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 oral exposure:
PREPARATION OF DOSING SOLUTIONS:
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.
The test item dose forulations were prepared on the days of treatment, stored at room temperature and protected from light and used within 4 hours after the end of their preparation.

VEHICLE
- Concentration in vehicle: 0, 10, 20 and 40 mg/mL for the control, low, mid and high dose groups, respectively
- Amount of vehicle: 5 mL/kg bw/day
- Batch no. carboxymethylcellulose (400-800 cps): SLBQ8545V
- Batch no. Tween 80: BCBT5201
- Drinking water treated by reverse osmosis using ELIX 5 (Millipore S.A.)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed using an analytical method developed and validated at Charles River Laboratories Evreux (Charles River Laboratories Evreux/Study No. 46438 VAA) prior to dose formulation analysis using High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS).
Dose formulations were analysed three times during the study: Weeks 1, 5 and 9 (referring to the Week 1 of males).

A sample was taken from control and test item dose formulations and analyzed using the validated method.
Acceptance criterion: Measured concentration = nominal concentration ± 15%
Duration of treatment / exposure:
The males were treated starting 4 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed) until euthanasia (11 weeks in total).
The females were treated starting 2 weeks before mating, during the mating period (until evidence of mating or 2 weeks had elapsed), during gestation, during lactation until Day 21 p.p. inclusive until euthanasia for females with no delivery.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected on the basis of the results of a previous study (Charles River Laboratories Evreux/Study No. 46746 TSR) performed in the same species, in which 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.
In this study, 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 pathology, the test item administration at = 100 mg/kg bw/day induced squamous cell hyperplasia and hyperkeratosis in the forestomach and centrilobular hepatocellular hypertrophy in the liver in both sexes, and tubular hyaline droplets accumulation and degeneration/regeneration 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 and the maximal tolerated dose level to be lower than 300 mg/kg bw/day.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period (including weekends and public holidays)
- Cage side observations included observations for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day
- 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 males: once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia;
- Time schedule for examinations females: once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8, 13, 17 and 21 p.p. Females euthanized prematurely were weighed before euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption of males: measured once a week from the first day of treatment until the start of the mating period and after the mating period until euthanasia;
- Food consumption of females: measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8, 8-13, 13-17 and 17-21 p.p.
During the mating period, food consumption was not measured for males or females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 14 hours o/n)
- How many animals: All
- All parameters according to guideline were included
- Coagulation parameters (Prothrombin time, fibrinogen and activated partial thromboplastin time) were determined from the first five males and lactating females from each group on the day of euthanasia

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 14 hours o/n)
- How many animals: All
- All parameters according to guideline were included
- Thyroid hormones (T4 and TSH) were determined for pups sampled on Day 21 p.p. and for F0 males sampled at termination.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (at least 14 hours) before necropsy)
- Metabolism cages used for collection of urine: Yes, urine was collected onto thymol crystals
- Animals fasted: Yes
- All parameters according to guideline were included.

FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule for examinations: once at the end of the treatment period (for males: during the last week of treatment; for females: on Day 21 p.p. after euthanasia of the pups)
- Dose groups that were examined: The first five males and lactating females from each group
- Battery of functions tested:
- Detailed clinical examination (including in the cage "touch escape" or ease of removal from the cage, in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis), in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia;
- The assessment of reactivity to manipulation and to different stimuli (including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature);
- Motor activity (measured once by automated infra-red sensor equipment over a 60 minute period).
Sacrifice and pathology:
GROSS PATHOLOGY:
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
Males: after the end of the mating period (at least 10 weeks of treatment in total),
Females: on Day 22 p.p. Females which did not deliver were euthanized on Day 26 p.c. (after a body weight recording to check for a possible un-noticed delivery) by inhalation of carbon dioxide gas followed by cervical dislocation since gestation was suspected.

A complete macroscopic post-mortem examination was performed on all F0 animals including those that were euthanized prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, 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 corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 22 p.p. and for one low dose female euthanized on Day 26 p.c. due to no delivery. For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

ORGAN WEIGHTS:
Organ weights of the main organs of the first five euthanized as scheduled males and the first five females euthanized on Day 22 p.p. of each group were determined. A table in which all tissue procedures are summarized is attached below.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table (attached below) from the first five euthanized as scheduled males and lactating females of the control and high-dose groups,
- all macroscopic lesions of all groups, including kidneys of the prematurely euthanized low dose group female and the subcutis mass of the prematurely euthanized high dose group female,
- reproductive organs from females that did not conceive, or from pregnant female that did not deliver, to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Based upon the results of the microscopic examination of the high-dose group, the following tissues of the low- and intermediate-dose groups were examined on the first five euthanized as scheduled males and/or lactating females: kidneys (males), liver (males and females), thyroids (males and females), stomach with forestomach (males), forestomach (females), vagina (females).
Other examinations:
Monitoring of estrous cycle
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 a.m.):
- during the 2 last weeks of the pre-treatment period (including for the two supplementary females per group, whose data are not presented in the study report),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on the day of sacrifice before euthanasia, to allow correlation with reproductive organs histopathology.
Statistics:
Body weight, food consumption and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
The statistical analysis sequence for hematology, blood biochemistry, urinalysis, hormones, motor activity, anogenital distance, nipples/areolae, live birth index, sex-ratio and post-implantation loss parameters and for organ weights (level of significance of 0.05 or 0.01) are attached below.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in males and females given 100 or 200 mg/kg bw/day during the premating, pregnancy and lactation periods. It was also noted in one isolated female given 50 mg/kg bw/day during the pregnancy period. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

The other clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (area of hair loss, cutaneous lesions, scabs), chromodacryorrhea, chromorhinorrhea, reddish vaginal discharge, short/broken teeth, mass on thorax area and/or reflux at dosing, were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.

Mortality:
mortality observed, non-treatment-related
Description (incidence):
No unscheduled deaths occurred in males at any dose level during the study. No mortality of females occurred in the control group and the group dosed at 100 mg/kg bw/day.
At 50 mg/kg bw/day, one female was prematurely euthanized on Day 23 p.c. due to difficulties to deliver. Signs of poor clinical condition (pallor of eyes and extremities, cold to the touch, round back, piloerection and dyspnea) were noted on the day of sacrifice. At necropsy, there were six alive and two dead fetuses in the uterine horns, enlarged and hemorrhagic placenta, red left adrenal gland, red content in vagina and irregular color in kidneys. This latter finding correlated microscopically with a severe acute degeneration/necrosis of tubules and glomeruli. These changes were considered to be unrelated to test item administration in view of their isolated occurrence and the absence of dose-relationship. They could be secondary to uterine/placental infection and sepsis in spite no bacteria were observed at microscopic examination of the kidney.

At 200 mg/kg bw/day, one female was prematurely euthanized on Day 2 p.p. because of the presence of a mass on the mammary gland. This mass of 3.5 x 3 cm, white and heterogeneous in the subcutis (mammary gland region), was a mammary adenocarcinoma and was considered to be unrelated to the test item treatment.

Due to no delivery, three females were euthanized on Day 26 p.c. without clinical signs prior to death (except for ptyalism in the high dose females):
- one female at 50 mg/kg bw/day. At necropsy, one dead fetus and one placenta were found in the left horn;
- two females at 200 mg/kg bw/day. At necropsy, the females were found to be non pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No relevant effects were observed on mean body weights or mean body weight changes in males at any dose level. The lower mean body weight gain recorded in males given 200 mg/kg bw/day throughout the treatment period (-10% vs. controls) was not attributed to the test item treatment as the difference was slight, due to isolated body weight losses and with no impact on the terminal body weight (-3% vs. controls). No relevant effects were observed on mean body weights or mean body weight changes in females at any dose level. A lower mean body weight gain recorded in females given 200 mg/kg bw/day during the gestation period (16% vs. controls) was not attributed to the test item treatment as the difference was mainly due to the contribution of one female which also consumed less food (body weight gain of +20 g on Days 14-20 vs. +94 g in controls).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on mean food consumption were observed in males at any dose level. The mean food consumption was considered to be unaffected by the test item treatment at any dose level.

The lower mean food consumption recorded in females given 200 mg/kg bw/day during the lactation period (-10% vs. controls) was not attributed to the test item treatment as the difference was mainly due to the contribution of one female (food consumption values between 22 and 44 g/day; without these values the difference vs. controls felt to -3%).
Description (incidence and severity):
n/a
Description (incidence and severity):
n/a
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted on the hematology parameters at the end of the treatment period.

The only statistically significant differences between control and test item-treated animals (i.e. lower mean cell hemoglobin concentration in females at 50 mg/kg bw/day and males at 100 mg/kg bw/day, lower relative reticulocyte count in males at 50 mg/kg bw/day, higher neutrophil counts in males at 200 mg/kg bw/day, lower eosinophil counts in males at 100 mg/kg bw/day, higher large unstained cell counts in males at 50 and 100 mg/kg bw/day), were not attributed to the test item treatment as they were of low magnitude and/or isolated and/or not dose-related.

No test item-related effects were noted on the clotting parameters at the end of the treatment period. The only statistically significant difference between control and test item-treated animals (i.e. longer activated partial thromboplastin time in males at 100 mg/kg/day) was not attributed to the test item treatment as it was not dose-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted on the blood biochemistry parameters at the end of the treatment period.

The only statistically significant differences between control and test item-treated animals were not attributed to the test item treatment as they were of low magnitude and/or isolated (calcium, glucose, creatinine, protein and albumin levels) or were noted with an opposite trend in males and females (triglyceride levels) or because of the direction and magnitude of the changes (total bilirubin and bile acid levels, alanine aminotransferase activity).

No effects were noted on the mean thyroid hormone levels (T4 and TSH plasma levels) in males at termination.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted on the quantitative or qualitative urinary parameters at the end of the treatment period.

The only statistically significant difference between control and test item-treated animals (i.e. lower urinary volume in females given 200 mg/kg bw/day) was not attributed to the test item treatment and it was of low magnitude and isolated.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No behavioral or neurological abnormalities were observed during the tests in any group.
The motor activity (60-minute recording period) was unaffected by the test item treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dose-related increased absolute and/or relative-to-body liver weights were noted in males treated at = 50 mg/kg bw/day (p<0.01 for both absolute and relative-to-body weights at 200 mg/kg bw/day, p<0.01 or 0.05 in relative weights only at 50 or 100 mg/kg bw/day; statistical significance was not reached for the absolute weights at 50 or 100 mg/kg bw/day). This difference was also noted in females treated at 100 mg/kg bw/day (p<0.05) and did not reach statistical significance at 200 mg/kg bw/day. These differences correlated with hepatocellular hypertrophy at microscopic examination.

Dose-related increased absolute and relative-to-body kidney weights were noted in males at = 100 mg/kg bw/day (p<0.01). These differences correlated with tubular hyaline droplet accumulation and dilatation at microscopic examination.

An overview of these results is included below.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic changes.
The few gross changes were considered to be part of the spontaneous background seen in the rat kept under laboratory conditions. This included the yellow mass in the epididymal adipose tissue from one high-dose male that correlated with microscopic granulomatous inflammation (probably secondary to fat torsion). There was also a unilateral small and tan kidney in one high dose female that correlated with a microscopic malformation (congenital).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
An overview of the microscopic findings is included below in tabular form below.
Test item-related microscopic observations included mainly adverse renal changes in males (degeneration/necrosis of tubules, accumulation of hyaline droplets, casts, tubular dilatation and basophilia) and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid gland, hyperkeratosis and squamous cell hyperplasia in forestomach, stomach (atrophy of glands) and vagina (increased mucification).

Kidneys: Minimal degeneration/necrosis of proximal tubules, associated with minimal to marked accumulation of hyaline droplets in proximal tubules, casts (granular and, to a lesser extent, hyaline), tubular dilatation in cortex and medulla and cortical basophilia (increased severity/incidence) were noted in males treated at = 50 mg/kg bw/day when compared to controls.

Liver: Minimal to slight hepatocellular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day. It was mostly in centrilobular areas but also sometimes diffuse.

Thyroid: Minimal follicular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day, in association with heterogeneous colloid in 2/5 high-dose males.

Forestomach: Minimal to slight hyperkeratosis often associated with squamous cell hyperplasia were noted in males and females treated at = 50 mg/kg bw/day.

Stomach: Minimal atrophy of gastric gland was noted in 4/5 males treated at 200 mg/kg bw/day. This affected mostly the parietal cells.
Vagina: Increased incidence and severity of vaginal mucification was noted in females treated at 100 or 200 mg/kg bw/day.

Additional examination of males/females that did not conceive:
The males had no lesions in the reproductive organs.
For one female, the left horn contained one dead fetus and the placenta was partially present. There was a moderate acute granulocytic inflammation in the uterus with edema and bacteria in lumen that were probably secondary to the presence of a dead fetus. Two females had functional estrus cycle (one was in proestrus and the other was in estrus). There were no microscopic abnormalities in their reproductive tract.
The other changes were considered not to be related to the test item administration since they were not dose-related, of low incidence and part of the spontaneous background in the rat kept under laboratory conditions.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No effects were observed on the estrous cycle at any dose level during the treatment period.
Details on results:
There was no test item-related intercurrent death during the course of the study. Both unscheduled deaths were related to spontaneous affections (mammary gland adenocarcinoma and renal necrosis probably secondary to uterine infection).

In kidneys, increased severity and incidence of accumulation of hyaline droplets (consistent with a2µglobulin) were noted in males treated at = 50 mg/kg bw/day and correlated with increased weights. In view of the presence of degeneration/necrosis and of the magnitude of the hyaline droplets accumulation (up to marked), these changes were considered to be adverse at all dose-levels. It is noteworthy that the a2µglobulin droplets are specific of the male rat and are not present in humans.

In the liver, hepatocellular hypertrophy was noted in males at 50 mg/kg bw/day and in males and females treated at = 100 mg/kg bw/day, correlated with increased weights. The test item was considered to induce hepatocellular enzymatic induction with secondary increased hepatic clearance of thyroid hormones, and thus hypertrophy of thyroid glands, as described in Histopathology of preclinical toxicity studies (Greaves P., 2012, p. 771) This was considered to be of low magnitude, of the absence of associated deleterious changes as degeneration/necrosis, and since there were no clinical pathology correlates, and thus this was considered to be non-adverse.

Follicular hypertrophy was noted in the thyroid glands from males at 50 mg/kg bw/day and from males and females treated at = 100 mg/kg bw/day. This correlated with the increased weights and was linked to the liver hypertrophy (see above). This was considered to be of low magnitude and in the absence of changes in T4 and TSH plasma levels, was considered to be non-adverse.

In the forestomach, the hyperkeratosis and hyperplasia seen at = 50 mg/kg bw/day were considered to be non adverse in view of the low magnitude of these changes and of the absence of associated deleterious lesions (no associated ulceration or inflammation).

In the stomach, the atrophy seen in 4 males at 200 mg/kg bw/day was considered to be non-adverse in view of the low magnitude of this change and of the absence of associated deleterious lesions (no ulceration).

It was considered that the mucification in the vagina at 100 or 200 mg/kg bw/day was non-adverse in view of the low magnitude of this change (up to moderate) and of the presence of this change in 1/5 controls.

Effect levels

Key result
Dose descriptor:
NOAEL
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

Key result
Critical effects observed:
no

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS

The test item concentrations in the administered dose formulations analyzed in Weeks 1 (two preparations), 5 and 9 (referring to Week 1 for males) remained within an acceptable range of variations of -8.9% to +1.8% when compared to the nominal values (± 15% of the nominal concentrations), except for the first formulation of group 3 male in Week 1 (-41.6%). No test item was detected in the control dose formulation.

Table: Relevant changes in mean final body weights and organ weights in treated groups (% changes from controls)

Sex

Male

Male

Male

Female

Female

Female

Dose level (mg/kg bw/day)

50

100

200

50

100

200

Final body weight

+2

0

-5

-2

-1

+1

Kidneys, absolute

+9

+24**

+26**

0

+4

-2

Kidneys, relative

+7

+25**

+31**

-2

+5

-2

Liver, absolute

+16

+19

+30**

+5

+18*

+16

Liver, relative

+13*

+20**

+35**

+3

+19*

+16

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Applicant's summary and conclusion

Conclusions:
Based on the results of a repeated dose toxicity study, combined with screening study for reproduction and development toxicity, performed according to OECD guideline 422 and GLP principles, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/day in females based on the absence of adverse findings at this dose level. The adverse renal changes noted in male rats at = 50 mg/kg bw/day (accumulation of hyaline droplets associated with tubular vacuolation and degeneration/necrosis) are specific to male rats with no relevance for human risk assessment. No other adverse effects were observed in males.
Executive summary:

A repeated dose toxicity study combined with screening study for reporduction and development toxicity, was performed with Tert-dodecanethiol according to OECD guideline 422 and GLP principles.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Tert-dodecanethiol, daily by the oral route (gavage) at dose levels of 50, 100 or 200 mg/kg bw/day, under a constant dosage volume of 5 mL/kg/day. A control group of ten male and ten female rats received the vehicle only (0.5% Carboxymethylcellulose / 0.5% Tween 80) under the same experimental conditions. Males were treated for an overall period of approximately 10 weeks: 4 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 8 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks), gestation (3 weeks) and lactation (3 weeks). Males were euthanized after completion of the mating period. Dams were euthanized on Day 22 p.p. The actual test item concentrations in the dose formulations were confirmed in Weeks 1, 5 and 9 using a validated LC/MS-MS analytical method.

Animals were checked daily for clinical signs and mortality. Detailed clinical examinations were performed weekly. Functional observation battery and motor activity were performed on the first five males and five lactating females during the last days of treatment.

Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before mating until the females had mated and on the day of sacrifice, before euthanasia.

The animals were paired for mating after two (females) or four (males) weeks of treatment and the dams were allowed to litter and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible five males and five females per litter. Pups not selected on Day 4 p.p. were sampled, euthanized and discarded without further examination.

Hematology, coagulation, blood biochemistry and urinary investigations were performed on the first five males and five lactating females in each group at scheduled euthanasia. Thyroid hormone levels (TSH and T4) were determined in males at sacrifice. A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five euthanized males and lactating females in all groups.

No test item-related deaths occurred in males or females during the study. Ptyalism was observed mainly at 100 and 200 mg/kg bw/day throughout the study. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect. The functional observation battery and motor activity were unaffected by the test item treatment. Body weight and food consumption were not impaired by the test item treatment. The estrous cycle was not impacted by the test item treatment. Mating, fertility and delivery data were unaffected by the test item treatment.

At hematology, coagulation, blood biochemistry and urinary investigations, no changes were noted. Thyroid hormone analysis did not reveal any disturbances in males at sacrifice.

At pathology investigations, test item-related increased liver weights were noted in males and/or females at = 50 mg/kg bw/day, and increased kidney weights 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), and non-adverse hepatocellular hypertrophy in liver, follicular hypertrophy in thyroid glands, hyperkeratosis and squamous cell hyperplasia in forestomach at = 50 mg/kg bw/day in males and at = 100 mg/kg bw/day in females, atrophy of glands in stomach in males treated at 200 mg/kg bw/day and increased mucification in vagina at = 100 mg/kg bw/day in females.

At the dose level of 50 mg/kg bw/day, treatment-related changes were limited to increased liver weight in males associated in males with minimal hepatocellular and thyroid follicular hypertrophies and minimal to moderate hyaline droplet nephropathy. Treatment-related changes consisted of increased liver and kidney weights in males and females associated with minimal to slight (at 200 mg/kg bw/day only) hepatocellular hypertrophy and minimal thyroid follicular hypertrophy in both sexes and moderate to marked hyaline droplet nephropathy in males.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg bw/day in females based on the absence of adverse findings at this dose level. The adverse renal changes noted in male rats at = 50 mg/kg bw/day (accumulation of hyaline droplets associated with tubular vacuolation and degeneration/necrosis) are specific to male rats with no relevance for human risk assessment. No other adverse effects were observed in males.