tert-dodecanethiol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In the extended one-generation reproduction toxicity study (CRL 2021), the 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 adverse changes in kidneys in P and F1 generations. And the NOAEL for reproduction/developmental toxicity was considered to be 200 mg/kg/day in males and females based the absence of adverse effects observed in P, F1 and F2 generations.

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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

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.

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

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.

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

75 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:

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

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

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: 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).

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

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

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.

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

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: 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.

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.

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

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.

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

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.

Behaviour (functional findings):

not examined

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.

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.

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

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.

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

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.

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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].

 

 

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)].

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Both OECD 442 and 443 are considered to be reliable with a klimisch score of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Extended one generation reproduction toxicity study (OECD 443, CRL 2021)

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:

• in Cohort 1A: no relevant effects on lymphocyte subtyping,


• in Cohort 3: 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 adverse effects on the immune system were observed, e. hematological changes, changes in immune system organ weights or histology (e.g. changes in thymus, spleen, lymph nodes or bone marrow).

 

Combined 28 -day repeated toxicity study and reproduction screening (OECD 422, CRL 2020):

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.

Effects on developmental toxicity

Description of key information

Treatment with t-dodecyl mercaptan did not produce a teratogenic effect in mice or rats after inhalation exposure, or in rabbits after oral exposure.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

only two concentration tested

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: 12 weeks old
- Weight at study initiation: 205 and 264 grams on gestation day 0
- Fasting period before study: no
- Housing: individually in hanging wire-mesh cages, nn gestation day 6 animals were transferred to stainless steel and glass exposure chambers
- Diet (ad libitum, except during the exposure period ): Purina® Certified Rodent Chow® #5002
- Water (ad libitum, except during the exposure period): tap water
- Acclimation period: 14 day

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 76-85 (exposure chambers)
- Humidity (%): 35-41 (exposure chambers)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass exposure chambers with a volume of one cubic meter
- Method of holding animals in test chamber: individually in hanging wire-mesh cages
- System of generating vapor: Vapor atmospheres of t-dodecyl mercaptan (TDM) were generated by one of two methods. The first method was used to generate the 25 ppm group atmosphere. With this system a syringe-drive pump (Sage model 355) delivered the test materiai at a known, constant rate to the top of a 19.6 cm high column filled with 5 mm diameter glass beads. Air heated by a 400 watt cartridge heater passed up the bead column counter-current to the liquid flow. Vaporization occurred within the bead column. The vapors were then piped to the air inlet of the exposure chamber where dilution with chamber ventilation air (approximately 250 L/min) reduced the concentration to the desired level. Heat tape was wrapped around the trap to prevent condensation. The current supplied to both the cartridge heater and the heat tape was controlled individually by variable autotransformers.
The TDM concentration for the 100 ppm group was generated with a second method of vapor generation. With this system all of the chamber ventilation air was passed through an atomization chamber which had been charged with a known amount of test material. An FMI laboratory pump was then used to deliver the test material to an atomizer (Spraying Systems, No. 1650 liquid nozzle, No. 64 air nozzle). The atomization step served to greatly increase the surface area of the test material which increased, in turn, the vaporization rate to obtain a relatively high vapor concentration of test material. Glass wool was placed in the exit pipe of the atomization chamber to prevent aerosol from passing into the exposure chamber. To overcome the resistance of the glass wool filter, the exposure chamber was operated at a negative pressure of approximately 0.2 - 0.4 inches of vater.
- Temperature, humidity, pressure in air chamber: 76-85 °F, 35-41%
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: Since the generation method for the 100 ppm group vapor level involved an atomization step, samples were collected to determine the amount of aerosol in the chamber atmosphere. Samples were collected on 25 mm glass fiber filters at a rate of 1 L/min for 30 minutes. The filters were placed in 2 ml of n-hexane to extract the collected TDM and 2 pl of the resultant solution were injected into the GC. Two injections were made of each of 4 filter samples. Three concurrent vapor samples were collected for each filter sample. During the experiment the vapor phase concentration averaged 53 ppm while the samples of recovered aerosol averaged 0.18 ppm. From these data we concluded that aerosol would not constitute an appreciable fraction of the total TDM concentration in the vapor exposure atmosphere.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: The actual vapor concentration of TDM in the chamber atmosphere was determined by a gas chromatograph fitted with a gas sampling valve and sample loop. The GC was connected to a Hewlett-Packard Model 3388A integrator for data collection and reduction and was calibrated with liquid-phase standards of TDM in n-hexane.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see above

Details on mating procedure:

At the end of the acclimation period, all animals were weighed and subjected to a detailed physical examination. At this time, animals considered suitable for study were cohabitated with stock males utilized exclusively for this purpose.
One female and one male animal of the same species, strain and source were placed together for mating. The occurrence of copulation was determined by daily inspection for a copulatory plug or vaginal smear for sperm. The day evidence of mating was detected was
designated day 0 of gestation and the female was returned to an individual cage, assigned a permanent animal number and properly identified by ear-tag.

Duration of treatment / exposure:

G6 to G19

Frequency of treatment:

6 hours/day

Duration of test:

up to gestation day 20

Dose / conc.:

22.7 ppm (analytical)

Remarks:

Target conc.: 25 ppm

Dose / conc.:

88.6 ppm (analytical)

Remarks:

Target conc.: 100 ppm

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Mated females were consecutively assigned in a block design to one control group and two t-dodecyl mercaptan treated groups of 25 rats. The order in which the mated females were assigned corresponded to the day the copulatory plug was observed and the order in which the animal appeared on the breeding record. The first mated female on the breeding record was assigned to the first group. The second mated female was assigned to the next group. Animals were assigned in this manner until the required number of mated females had been placed into each group.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to treatment, the females were observed twice daily for mortality and overt changes in appearance and behavior. They were observed twice daily for mortality and once daily for clinical signs of toxicity from gestation day 6 through sacrifice. Females not surviving to the scheduled sacrifice were necropsied in an attempt to determine the cause of death.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 16 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: On gestation day 20 all surviving dams were sacrificed by carbon dioxine inhalation. The abdominal and thoracic cavities and organs of the dams were examined for grossly evident morphological changes and the carcasses discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and location of viable and nonviable fetuses

Fetal examinations:

All fetuses were individually weighed and examined for external malformations and variations, including the palate and eyes. Each fetus was externally sexed and individually numbered and tagged for identification. Approximately one-third of the fetuses were placed in Bouin's fixative for subsequent visceral examination by razor-blade sectioning as described by Wilson. The remaining two-thirds of the fetuses were fixed in alcohol, macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson for subsequent skeletal examination.

Statistics:

The male to female fetal sex distribution and the number of litters with malformations were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.
The number of early and late resorptions, nonviable fetuses and postimplantation loss were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
The mean number of viable fetuses, total implantations, corpora lutes and mean fetal body weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equai or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance ot differences.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no biologically meaningful differences in the appearance and behavior of rats in the t-dodecyl mercaptan treated groups when compared to those of the rats in the control group. Incidental observations occurring in a few control and/or treated rats included hair loss, soft stool, reddened conjunctiva, wet red matter or red liquid in the vagins or toes red, swollen and/or scabbed. A circumscribed areas in the inguinal area was noted in a female in the 100 ppm group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One rat in the 100 ppm group died on gestation day 14. Antemortem observations included thinness, an unkempt haircoat and reddened conjunctiva. Mottled kidneys were noted at necropsy; the cause of death could not be determined. The normally developing implants in this female were inadvertently not examined further. Survival was 100% in the control group and the 25 ppm group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related decrease in mean maternal body weight gain of the rats in the treated groups when compared to the control value over the entire exposure period (gestation days 6-20) and the entire gestation period (gestation days 0-20).

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The occurrence of necropsy findings in the 25 ppm and 100 ppm groups was similar to that of the control group. Incidental observations included hydronephrosis, pitted kidneys, calculi in the kidneys, ureters and urinary bladder and distended ureters.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

There were no biologically meaningful or statistically significant differences in the mean number of postimplantation loss, total implantations and corpora lutea in the treated groups.

Key result

Dose descriptor:

LOAEC

Effect level:

22.7 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

decreases in mean body weight gain

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slight (less than 10%) but statistically significant increase in mean fetal body weight in comparison to the control value occurred in the 100 ppm group. The 100 ppm group mean fetal body weight was comparable to the mean value in the historical control data. The mean fetal body weight in the 25 ppm group was similar to that of the control group and the historical control data; no statistically significant differences from the control group were observed.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The incidence of fetal malformations in litters of the rats in the t-dodecyl mercaptan treated groups was comparable to that of the control group; no statistically significant differences were observed.
There were no biologically meaningful differences in the occurrence of genetic and developmental variations between the control and treated groups.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 88.6 ppm (analytical)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Treatment with t-dodecyl mercaptan by whole body inhalation at an actual exposure level of 88.6 ppm or less did not produce a teratogenic effect in rats.

Executive summary:

In a study comparable to OECD 414 guideline, pregnant Charles River COBS®CD® rats, randomly assigned to one control group and two t-dodecyl mercaptan treated groups of 25 animals, were used to determine the teratogenic potential of t-dodecyl mercaptan. Mean actual exposure levels of 22.7 and 88.6 ppm, which corresponded to desired exposure levels of 25 and 100 ppm, were administered by whole body inhalation exposure on gestation days 6 through 19 on a 6-hour daily exposure schedule. The control group was exposed to filtered air only on a comparable regimen. Cesarean sections were performed on all surviving rats on gestation day 20 and fetuses removed for teratologic evaluation.

One rat died at 100 ppm. The cause of death could not be determined. Survival was 100% in the control rats and the 25 ppm group. There were no biologically meaningful or relevant statistically significant differences in the mean number of total implantations, corpora lutes and fetal body weight or in the fetal sex distribution in all Cesarean section observations when compared to the control values. No adverse treatment-related or statistically significant differences in the incidence of fetal malformations when compared to those of the control group. Dose-related decreases in mean maternal body weight gain occurred in the t-dodecyl mercaptan treated rats. There were no meaningful differences in the appearance, behavior and necropsy when compared to those of the control group. Treatment with t-dodecyl mercaptan by whole body inhalation at an actual exposure level of 88.6 ppm or less did not produce a teratogenic effect in rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

only two concentration tested

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: 12 weeks old
- Weight at study initiation: 24-33 grams on gestation day 0
- Fasting period before study: no
- Housing: individually in hanging wire-mesh cages, nn gestation day 6 animals were transferred to stainless steel and glass exposure chambers
- Diet (ad libitum, except during the exposure period ): Purina® Certified Rodent Chow® #5002
- Water (ad libitum, except during the exposure period): tap water
- Acclimation period: 14 day

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 76-85 (exposure chambers)
- Humidity (%): 35-41 (exposure chambers)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass exposure chambers with a volume of one cubic meter
- Method of holding animals in test chamber: individually in hanging wire-mesh cages
- System of generating vapor: Vapor atmospheres of t-dodecyl mercaptan (TDM) were generated by one of two methods. The first method was used to generate the 25 ppm group atmosphere. With this system a syringe-drive pump (Sage model 355) delivered the test materiai at a known, constant rate to the top of a 19.6 cm high column filled with 5 mm diameter glass beads. Air heated by a 400 watt cartridge heater passed up the bead column counter-current to the liquid flow. Vaporization occurred within the bead column. The vapors were then piped to the air inlet of the exposure chamber where dilution with chamber ventilation air (approximately 250 L/min) reduced the concentration to the desired level. Heat tape was wrapped around the trap to prevent condensation. The current supplied to both the cartridge heater and the heat tape was controlled individually by variable autotransformers.
The TDM concentration for the 100 ppm group was generated with a second method of vapor generation. With this system all of the chamber ventilation air was passed through an atomization chamber which had been charged with a known amount of test material. An FMI laboratory pump was then used to deliver the test material to an atomizer (Spraying Systems, No. 1650 liquid nozzle, No. 64 air nozzle). The atomization step served to greatly increase the surface area of the test material which increased, in turn, the vaporization rate to obtain a relatively high vapor concentration of test material. Glass wool was placed in the exit pipe of the atomization chamber to prevent aerosol from passing into the exposure chamber. To overcome the resistance of the glass wool filter, the exposure chamber was operated at a negative pressure of approximately 0.2 - 0.4 inches of vater.
- Temperature, humidity, pressure in air chamber: 76-85 °F, 35-41%
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: Since the generation method for the 100 ppm group vapor level involved an atomization step, samples were collected to determine the amount of aerosol in the chamber atmosphere. Samples were collected on 25 mm glass fiber filters at a rate of 1 L/min for 30 minutes. The filters were placed in 2 ml of n-hexane to extract the collected TDM and 2 pl of the resultant solution were injected into the GC. Two injections were made of each of 4 filter samples. Three concurrent vapor samples were collected for each filter sample. During the experiment the vapor phase concentration averaged 53 ppm while the samples of recovered aerosol averaged 0.18 ppm. From these data we concluded that aerosol would not constitute an appreciable fraction of the total TDM concentration in the vapor exposure atmosphere.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: The actual vapor concentration of TDM in the chamber atmosphere was determined by a gas chromatograph fitted with a gas sampling valve and sample loop. The GC was connected to a Hewlett-Packard Model 3388A integrator for data collection and reduction and was calibrated with liquid-phase standards of TDM in n-hexane.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see above

Details on mating procedure:

At the end of the acclimation period, all animals were weighed and subjected to a detailed physical examination. At this time, animals considered suitable for study were cohabitated with stock males utilized exclusively for this purpose.
One female and one male animal were placed together for mating. The occurrence of copulation was determined by daily inspection for a copulatory plug or vaginal smear for sperm. The day evidence of mating was detected was
designated day 0 of gestation and the female was returned to an individual cage, assigned a permanent animal number and properly identified by ear-tag.

Duration of treatment / exposure:

G6 to G19

Frequency of treatment:

6 hours/day

Duration of test:

up to gestation day 17

Dose / conc.:

22.7 ppm (analytical)

Remarks:

Target conc.: 25 ppm

Dose / conc.:

88.6 ppm (analytical)

Remarks:

Target conc.: 100 ppm

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): Mated females were consecutively assigned in a block design to one control group and two t-dodecyl mercaptan treated groups of 25 mice each. The order in which the mated females were assigned corresponded to the day the copulatory plug was observed and the order in which the animal appeared on the breeding record. The first mated female on the breeding record was assigned to the first group. The second mated female was assigned to the next group. Animals were assigned in this manner until the required number of mated females had been placed into each group.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to treatment, the females were observed twice daily for mortality and overt changes in appearance and behavior. They were observed twice daily for mortality and once daily for clinical signs of toxicity from gestation day 6 through sacrifice. Females not surviving to the scheduled sacrifice were necropsied in an attempt to determine the cause of death.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 15, and 17

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: On gestation day 20 all surviving dams were sacrificed by carbon dioxine inhalation. The abdominal and thoracic cavities and organs of the dams were examined for grossly evident morphological changes and the carcasses discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and location of viable and nonviable fetuses

Fetal examinations:

All fetuses were individually weighed and examined for external malformations and variations, including the palate and eyes. Each fetus was externally sexed and individually numbered and tagged for identification. Approximately one-third of the fetuses were placed in Bouin's fixative for subsequent visceral examination by razor-blade sectioning as described by Wilson. The remaining two-thirds of the fetuses were fixed in alcohol, macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson for subsequent skeletal examination.

Statistics:

The male to female fetal sex distribution and the number of litters with malformations were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.
The number of early and late resorptions, nonviable fetuses and postimplantation loss were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
The mean number of viable fetuses, total implantations, corpora lutes and mean fetal body weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equai or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance ot differences.

Clinical signs:

no effects observed

Description (incidence and severity):

The surviving mice in the control and t-dodecyl mercaptan treated group were similar in appearance, behavior, and necropsy findings.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival was 100% in the control group and the 100 ppm group. In the 25 ppm group, one mouse died on gestation day 13. Prior to death thinness, an unkempt haircoat and ptosis of the eyelid were observed in this female. At necropsy no gross lesions were evident and the cause of death could not be determined; there was also whole litter resorption.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a decrease in the mean maternal body weight gain of the 25 ppm group mice when compared to that of mice in the control group over the entire exposure period (gestation days 6-17) and over the entire gestation period (gestation days 0-17). No comparable decrease occurred in the 100 ppm group mice at these intervals (Table 2).

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the 100 ppm group delivered twelve fetuses on gestation day 16. Two additional fetuses were retained in the uterus. Antemortem findings included unkempt haircoat; red matter on the limbs, abdomen, anogenital area and tail; and a moribund appearance. No gross lesions were noted at necropsy and all fetuses were externally normal.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There was a slight increase in the mean postimplantation loss and a corresponding decrease in the mean number of viable fetuses in the treated groups when compared to those of the control group. In addition, the mean postimplantation loss in the treated groups was slightly increased in comparison to the high value in the historical control data (Table 4).

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Five dams each in the 25 and 100 ppm groups and three dams in the control group had whole litter resorption.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In the 25 ppm group there was a statistically significant increase in the mean number of early resorptions when compared to the control value; however, no statistically significant differences in the remaining components of postimplantation loss (the mean number of nonviable fetuses and the number of late resorptions) or in the mean postimplantation loss were observed. Similarly, there were no statistically significant differences in mean postimplantation loss or its composent parameters in the 100 ppm group.

Dead fetuses:

no effects observed

Description (incidence and severity):

The decreases in the mean number of viable fetuses in the treated groups were not statistically significant.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment-related or statistically significant differences in the mean number of corpora lutea or the mean implantations in treated groups when compared to those of the control group.

Key result

Dose descriptor:

LOAEC

Effect level:

22.7 ppm (analytical)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

increase in post-implatation loss

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in mean fetal body weight in the 25 ppm group when compared to that of the control group. The difference from the control group was less than 10%. The mean fetal body weight in the 25 ppm group was similar to the mean value in the historical control data. In the 100 ppm group, the mean fetal body weight was comparable to the control value and the historical control data; no statistically significant difference from the control group was noted.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The incidence of litters with fetal malformations was reduced in the 100 ppm group in comparison to that of the control group, while in the 25 ppm group the occurrence of fetal malformations in litters was comparable to the control value. No statistically significant differences were noted. Forked cervical arches occurred in 10 fetuses distributed among six litters in the control group, and in three fetuses each distributed among two litters each in 25 and 100 ppm group. In the 25 ppm group, four fetuses distributed among two litters had cleft palate; this malformation was present as single instances in the control group and in the 100 ppm group. The remaining malformations observed in the control and/or treated group mice were present in one or two fetuses in one or two litters.
No biologically meaningful differences in the incidence of genetic and developmental variations were observed in the fetuses in the treated groups when compared to the control values.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 88.6 ppm (analytical)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Treatment with t-dodecyl mercaptan by whole body inhalation at an actual exposure level of 88.6 ppm or less did not produce a teratogenic effect in mice.

Executive summary:

In a study comparable to OECD 414 guideline, pregnant Charles River CD®-1 mice, randomly assigned to one control group and two t-dodecyl mercaptan treated groups of 25 animals each, were used to determine the developmental toxicity/teratogenic potential of t-dodecyl mercaptan. Mean actual exposure levels of 22.7 and 88.6 ppm, which corresponded to desired exposure levels of 25 and 100 ppm, were administered by whole body inhalation exposure on gestation days 6 through 16 on a 6-hour daily exposure schedule. The control group was exposed to filtered air only on a comparable regimen. Cesarean sections were performed on all surviving mice on gestation day 17 and fetuses removed for teratologic evaluation.

One mouse died at 25 ppm. The cause of death could not be determined. Survival was 100% in the control and the 100 ppm groups. One mouse delivered prematurely at 100 ppm. There was a slight treatment-related increase in mean postimplantation loss, reflective of a slightly increased occurrence of whole litter resorption, and, correspondingly, a slight reduction in the mean number of viable fetuses in the t-dodecyl mercaptan treated mice when compared to these of the control group mice. There were no biologically meaningful or relevant statistically significant differences in the mean number of total implantations, corpora lutes and fetal body weight or in the fetal sex distribution in the mice in the t-dodecyl mercaptan treated groups when compared to the control values. No adverse treatment-related or statistically significant differences in the incidence of fetal malformations occurred in the t-dodecyl mercaptan treated mice when compared to those of the control group. There were no meaningful differences in maternal weight gain of the t-dodecyl mercaptan treated mice or the appearance, behavior and necropsy findings of the t-dodecyl mercaptan treated mice when compared to those of the control group.

Treatment with t-dodecyl mercaptan by whole body inhalation at an actual exposure level of 88.6 ppm or less did not produce a teratogenic effect in mice.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 January 2019 to 27 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France)
- Age: at the beginning of the treatment period, the animals were 18-20 weeks old
- Mean: at the beginning of the treatment period, the animals had a mean body weight of 3770 g (range: 3165 g to 4485 g)
- Fasting period before study: no
- Housing: individually housed in noryl cages
- Diet: free access to breeding pelleted diet "type 110C", batch No. 18206 (SAFE, Augy, France)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of at least 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8 h/16 h

IN-LIFE DATES: 15 January 2019 to 27 February 2019

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:

PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Suitable formulation in the selected vehicle
- Concentration in vehicle: 16.67, 33.33 and 66.67 mg/mL
- Amount of vehicle: 3 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations were within an acceptable range of variation (-3.6% to +1.4%) when compared to nominal values (+/- 15%) in the first and the last weeks of the treatment period.
Homogeneity: The dose formulations containing the test item and prepared at 2 mg/mL and 333.3 mg/mL in 0.5% (w/v) carboxymethylcellulose (400-800 cPs) / 0.5% (v/v) Tween 80 in drinking water treated by reverse osmosis were found to be homogeneous.
Stability: Diluted analytical samples prepared from 1.6 mg/mL and 400 mg/mL dose formulations in 0.5% (w/v) carboxymethylcellulose (400-800 cPs) / 0.5% (v/v) Tween 80 in drinking water treated by reverse osmosis were found to be stable for 3 days when they were stored at +5°C.

Details on mating procedure:

- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as Day 0 post coitum

Duration of treatment / exposure:

Days 6 to 28 post coitum inclusive

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection::
The dose levels were selected in agreement with the Sponsor, on the basis of the results of the previous study, where 20 pregnant New Zealand White females rabbits were given the test item daily by gavage from Day 6 to Day 28 p.c., at different doses (0 mg/kg/day, 33 mg/kg/day, 100 mg/kg/day and 300 mg/kg/day, 5 females per dose). All animals treated with 300 mg/kg/day had strongly reduced food consumption since the first days of treatment, resulting in mean body weight lower than control (-9% on Day 12 and -14% on Day 15). Emaciated appearance was recorded from Day 14 p.c. as well as soft feces. The 300 mg/kg/day group was prematurely euthanized for humane reason on Day 17 p.c. At lower doses, no significant effect was noticed. The Maximal Tolerated Dose level was between 100 and 300 mg/kg/day.
Therefore, 200 mg/kg/day was selected as the high-dose level. The low- and mid-doses were selected using a ratio representing a two-fold interval (i.e. 50 and 100 mg/kg/day).

- Rationale for animal assignment: stratified procedure.

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during treatment period, including weekends.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, between 1 and 3 hours post dosing, at approximately the same time of day, for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c., and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM EXAMINATION:
- Sacrifice on Day 29 post coitum
- Examined: principal thoracic and abdominal organs.

ORGAN WEIGHTS:
- Liver
- Spleen
- Kidney

Ovaries and uterine content:

The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight
- Number of corpora lutea
- Number of early and late resorptions
- Number of dead and live fetuses
- Number of uterine scars
- Number of implantation sites
- Gross evaluation of placentas

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: all fetuses per litter
- Other : fetal weight, fetal sex

Statistics:

Pathdata software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).
Other 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).

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Average fetal body weight = Sum of individual fetal weights / Number of live fetuses

Historical control data:

Cf attached document

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female was transiently cold to the touch and had an emaciated appearance (up to 12 days,correlating with transient body weight loss and reduced food consumption between Days 6 and 19 p.c.).
These observations were considered related to the test item treatment and adverse.
Other clinical signs were transiently observed (loud/abdominal breathing, absence of urine, brownish/reddish vaginal discharge, blood in the bedding, scabs or short teeth) in one or two animals per group. These observations are common observation in this species and strain, affected the animals before the treatment period, were noted also in control group and/or were not dose related. They were therefore considered incidental and not test item treatment-related.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 200 mg/kg/day, three females were euthanized before the end of the study.
Before euthanasia on Day 23 p.c., one female was cold to the touch and had emaciated appearance, correlating with a body weight loss (from Day 9 p.c. reaching -18% on Day 19 p.c. when compared to Day 6 p.c.) and no food consumption from Day 9 p.c., correlating with transient absence of feces recorded on Day 17 p.c. At hysterectomy, this female had nine corpora lutea and nine implantation sites (eight live fetuses and one late resorptions).
Two other females were prematurely euthanized on Days 25 and 27 p.c., respectively, for abortion (at least one placenta and one fetus in the bedding). Before abortion, both females had clinical signs similar to the first female (cold temperature / emaciated appearance correlating with a body weight loss (from Day 9 p.c., reaching -14% and -10%, on Day 24 p.c. compared to Day 6 p.c., respectively) and no or drastically reduced food consumption from Day 9 p.c.). At hysterectomy, one female had eight corpora lutea and eight implantation sites (six live fetuses, one implantation scar and one late resorption). the other female had ten corpora lutea and ten implantation sites (recorded as scars).
At necropsy, one female had pale liver, one another female also had pale liver accompanied with spleen reduced in size and a colored deposit in the stomach. No gross findings were noticed at examination of female.
Abortion and premature euthanasia were related to poor clinical condition of the animals and attributed to the test item administration.
In the other groups, no unscheduled deaths occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, when compared with controls, lower mean body weight gain was recorded (statistically significant from Day 15 p.c.). At the end of the treatment period, the mean body weight was 7% lower than in controls (p<0.01). This resulted in a body weight loss (in average) from the start of the treatment period, except between Days 12 to 15 p.c., when animals gained in average 22 g (vs. 77 g in controls). These changes were considered to be test item-related and adverse.
At 100 mg/kg/day, when compared with controls, statistically significant lower mean body weight gain was recorded throughout the treatment period (+182 g vs. +328 g, p<0.01). At the end of the treatment period, the mean body weight was considered unaffected. This effect was therefore attributed to the test item administration but not adverse.
There were no effects on mean body weight and mean body weight change in the 50 mg/kg/day group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was reduced in a dose dependent manner.
In case there was almost no food consumption, carrots and additional hay were distributed daily in order to check if this absence of food intake was transient. Animals were closely monitored, in particular for absence of urine/feces in the bedding.
At 200 mg/kg/day and when compared with controls, mean food consumption was markedly lower (in average approximately -44%; p<0.001) from the start of the treatment period. This effect was associated with a lower mean body weight gain and a lower body weight and was therefore considered adverse.
At 100 mg/kg/day and when compared with controls, mean food consumption was lower (in average approximately -18%; p<0.001) from Day 15 p.c. This effect was associated with a lower body weight gain and attributed to the test item administration but not considered adverse in absence of significant impact on body weight.
At 50 mg/kg/day, mean food consumption was slightly lower than in controls between Days 24 and 29 p.c. (-12%, not statistically significant), with no impact on mean body weight. Therefore, this effect was attributed to the test item administration but not adverse.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, when compared to controls, there were increased absolute and relative-to-body liver weights (+33% and +43%, respectively; p<0.01).
The increased relative-to-body kidney weight (+11%; p<0.01) was related to the reduced body weight and therefore considered of no toxicological relevance.
At 100 mg/kg/day, when compared to controls, there were increased absolute and relative-to-body liver weights (+12%; p<0.05 and +16%; p<0.01, respectively).
These changes were considered test item treatment-related.
Other variations were not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Among females treated at 200 mg/kg/day and euthanized at the end of treatment period, one had a dilated intestine (caecum) with feces and another one had a reddish content in the urinary bladder. These observations were made at a low frequency but only in the high-dose group. Therefore, a test item treatment-related effect was considered doubtful.
Other findings (serous content in uterine horn, translucent content in pericardium, irregular liver surface or abnormal lobation, depressed area in stomach, periovarian serous cysts, bilobed or reduced in size gall bladder) were noticed across the groups. As these observations were also reported in control animals, were not reported in the high-dose group or are common necropsy findings in pregnant rabbits, it was considered that none of them are test item treatment-related at any dose.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Uterus weight, carcass weight and net body weight change from Day 6 post coitum:
See Table 6.
At 200 mg/kg/day and when compared with controls, there was a slight to moderate decrease in mean gravid uterus weight (-12%, not statistically significant), mean carcass weight (-6%, p<0.05), and mean net body weight change (-432.6 g vs. -152.0 g in controls, p<0.001). Decrease in mean carcass weight was associated with a lower mean body weight. Decrease in mean gravid uterus weight was associated with a lower fetal body weight. Therefore, these effects were considered test item treatment related and adverse.

At 100 mg/kg/day, mean gravid uterus weight was unaffected. Mean carcass weight and mean net body weight change were slightly reduced when compared to controls. In the absence of significant effect on mean body weight and mean fetal body weight, these findings were considered test item treatment-related but not adverse.
At 50 mg/kg/day, there were no effects on mean gravid uterus weight, mean carcass weight and mean net body weight change.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Abortion and premature euthanasia were related to poor clinical condition of the animals and attributed to the test item administration.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related effects.
Number of dams with pre-implantation loss = 14, 14, 14, 15 at 0, 50, 100 or 200 mg/kg/day, respectively
Number of dams with post-implantation loss = 10, 8, 9, 7 at 0, 50, 100 or 200 mg/kg/day, respectively

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related effects.

Number of dams with total resorption = 1 female at 100 mg/kg/day

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related effects.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses.

Description (incidence and severity):

n/a

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One group had less than 20 females with implantation sites at necropsy (dose level 50 mg/kg/day), as recommended in the guideline. This group and the high-dose group (200 mg/kg/day) contained 19 females with live fetuses at term. None of these groups were considered inappropriate according to the guideline since there were more than 16 females with implantation sites in each group.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

other: decreased net body weight change increase absolute and relative-to-body liver weight

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day and when compared with controls, there was a marked decrease in mean fetal body weight in both sexes (-13%, p<0.05), especially in female fetuses (-14%, p<0.01).
At 100 mg/kg/day and when compared with controls, there was a slight decrease in mean fetal body weight, especially in female fetuses (-9%, not statistically significant, still within the range of Historical Control Data).
These effects were considered to be test item administration-related and adverse at the highest dose (200 mg/kg/day).
There were no effects on fetal body weight at 50 mg/kg/day.

Description (incidence and severity):

n/a

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no effects on fetal sex-ratio (percentage of male fetuses) at any tested dose level.

Description (incidence and severity):

n/a

Description (incidence and severity):

n/a

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related external malformations.
All malformations (open eyes: fetus in low-dose, gastroschisis and acephaly: fetus in mid-dose) were not observed at the highest dose and were therefore considered incidental and not attributed to the test item administration.

Number of fetuses with external malformations = 0 (0.0%), 1 (0.6%), 1 (0.5%), 0 (0.0%) at 0, 50, 100 or 200 mg/kg/day, respectively
Number of litters with external malformations = 0 (0.0%), 1 (5.3%), 1 (4.5%), 0 (0.0%) at 0, 50, 100 or 200 mg/kg/day, respectively

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related skeletal malformations.
One fetus had a misshapen scapula bone. Even rare, this malformation was not considered to be test item treatment-related due to its low incidence and the absence of other similar or associated findings.
The other malformations were not observed (or at lower incidences) in the highest dose level group and/or observed at higher incidence in control group. Thus, they were considered to be unrelated to the test item treatment.

Number of fetuses with skeletal malformations = 9 (4.9%), 6 (3.8%), 9 (4.5%), 3 (1.9%) at 0, 50, 100 or 200 mg/kg/day, respectively
Number of litters with skeletal malformations = 6 (28.6%), 5 (26.3%), 8 (36.4%), 3 (15.8%) at 0, 50, 100 or 200 mg/kg/day, respectively

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related soft tissue malformations.
The "absence of brain" corresponds to the anencephaly in one fetus (mid-dose, considered incidental, not observed at the highest dose) and abnormal liver lobation was only observed in control.

Number of fetuses with visceral malformations = 1 (0.5%), 0 (0.0%), 1 (0.5%), 0 (0.0%) at 0, 50, 100 or 200 mg/kg/day, respectively
Number of litters with visceral malformations = 1 (4.8%), 0 (0.0%), 1 (4.5%), 0 (0.0%) at 0, 50, 100 or 200 mg/kg/day, respectively

Other effects:

effects observed, treatment-related

Description (incidence and severity):

External variations:
There were no test item treatment-related variations at external examination of the fetuses.
The few findings recorded (malrotated paw, paw hyperflexion and enlarged abdomen with blackish color) were observed in controls, at low incidence, with no dose-relationship and/or are common findings in this species and strain. Therefore, they were not attributed to the test item administration.
Number of fetuses with external variations = 1 (0.5%), 1 (0.6%), 4 (2.0%), 5 (3.1%) at 0, 50, 100 or 200 mg/kg/day, respectively
Number of litters with external variations =1 (4.8%), 1 (5.3%), 4 (18.2%), 4 (21.1%) at 0, 50, 100 or 200 mg/kg/day, respectively

Skeletal variations:
In the 200 mg/kg/day group and when compared with controls, there were increases in the litter and fetal incidences (sometimes statistically significant) of unossified bones (pubis, talus, 1st metacarpal bone, 1st to 4th sternebra(e), centrum of caudal vertebra(e), odontoide). There were also increases in the litter and fetal incidences (sometimes statistically significant) of bones with incomplete ossification (1st metacarpal bone, hyoid).
These findings were considered to be associated with a delayed ossification (the corresponding cartilages being present). In association with the low mean fetal body weight these findings were considered test item-related and adverse.
In the 100 mg/kg/day group and when compared with controls, incomplete ossification of hyoid centrum was noticed, with a higher litter incidence than at higher dose level. This finding was therefore considered test item related.

Supernumerary 13th ribs, incomplete ossification of pubis, centrum of vertebra(e) or interparietal bone were also noticed, with litter and fetal incidences within the range of the Historical Control Data.
All other findings were not observed at the high-dose level and/or with a lower incidence than in control groups. Thus, they were considered to be unrelated to the test item treatment.
Overall, it is considered that fetal effects (delayed ossification, decreased fetal body weight) are related to the severe signs of toxicity noticed in dams from 100 mg/kg/day (decreased body weight and/or decreased food consumption).

Cartilage:
In the 200 mg/kg/day group and when compared with controls, there were statistically significant increases in the incidence of the non ossified cartilage (cartilage present) [i.e. metacarpal bone, pelvic girdle and talus]. Increases in the incidences of non-ossified cartilage of rib were also noted but to a lesser extent (not statistically significant). Corresponding incidence were above the upper limit of the Historical Control Data.
These findings were considered to be associated with a delayed ossification and related to unossification or incomplete ossification. They were considered test item-related, due to the related decreased fetal body weight in relation with the maternal toxicity.
Misshapen cartilage of scapula was associated with misshapen scapula bone.
The other findings reported in the summary table [i.e. presence of cartilage of hyoid centrum, cervical vertebra(e), rib, forepaw median phalanx] appeared with an increased incidence when compared to controls but were not dose-related, noted without a statistically significant occurrence and/or were within the range of the Historical Control Data. Therefore, a test item relationship was considered to be unlikely.
At lower dose levels, all other findings were not observed at the high-dose and/or with a lower incidence than in control groups.


Visceral variations:
There were no test item treatment-related soft tissue variations.
When compared with controls, paleness of spleen was observed at a higher incidence in the 200 mg/kg/day group. Thin abdominal wall was observed in two fetuses (male low-dose and female mid-dose), with enlarged abdomen with blackish color, and was not observed at the highest dose.
These variations were therefore considered incidental and not attributed to test item treatment.

All other variations (eye opacity, small spleen, small, bilobed or enlarged gall bladder, dilated renal pelvis, enlarged kidney, ovarian and periovarian serous cysts, short or absent innominate artery, absent brachiocephalic trunk, fluid-filled abdomen) were isolated, also observed in controls, or not observed at the highest dose. Therefore, these findings were considered incidental and not attributed to the test item administration

Number of fetuses with visceral variations = 34 (18.7%), 24 (15.3%), 49 (24.4%), 32 (19.9%) at 0, 50, 100 or 200 mg/kg/day, respectively
Number of litters with visceral variations = 17 (81.0%), 12 (63.2%), 17 (77.3%), 11 (57.9%) at 0, 50, 100 or 200 mg/kg/day, respectively

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: Skeletal examinations

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

The test item was administered to time-mated New Zealand White female rabbits by gavage, once daily, from Day 6 to Day 28 p.c., inclusive, at the dose levels of 50, 100 and 200 mg/kg/day.
The control group received the vehicle, 0.5% carboxymethylcellulose/0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, with a dosage volume of 3 mL/kg/day.
On the basis of the results obtained in this study:
. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day based on adverse findings recorded at 200 mg/kg/day (poor clinical condition, decreased body weight, reduced food intake, decreased net body weight change) accompanied by increase absolute and relative-to-body liver weight,
. the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 100 mg/kg/day based on adverse findings recorded at 200 mg/kg/day (reduced fetal body weight with fetal ossification delays) in a context of maternal toxicity. No test-item treatment related fetal malformations were observed.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rabbits from implantation until the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive].

Methods

Three groups of 24 time-mated female New Zealand White rabbits received the test item, by the oral route (gavage), at dose levels of 50, 100 or 200 mg/kg/day, once daily from Day 6 to Day 28 p.c. inclusive.

Another group of 24 time-mated female rabbits received the vehicle only, 0.5% carboxymethylcellulose/0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions and acted as a control group. A constant dosage volume of 3 mL/kg/day was used.

The test item concentrations in the dose formulations were determined.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). The liver, spleen and kidneys of each dam were weighed.

Results

Chemical analyses

The concentrations of the test item in the dose formulations, assessed in the first and the last weeks of the treatment period remained within an acceptable range of variations (-3.6% to +1.4%) when compared to the nominal value (± 15% of the nominal concentrations). No test item was detected in the control dose formulation.

Pregnancy status

There were 21/24, 19/24, 22/24 and 19/24 pregnant females with live fetuses in the control and 50, 100 and 200 mg/kg/day groups, respectively.

Mortality

At 200 mg/kg/day, three females were euthanized before the end of the treatment period (between Days 23 and 27 p.c.) for poor clinical condition (one female) or abortion (two females). These animals also had low body temperature and emaciated appearance associated with reduced food consumption and body weight loss (down to -18% when compared to the first day of treatment). These effects were considered test item treatment-related and adverse. There were no unscheduled deaths in control, 50 or 100 mg/kg/day groups.

Clinical signs

At 200 mg/kg/day, among surviving females, one was transiently cold to the touch and had an emaciated appearance correlating with transient body weight loss and reduced food consumption. This was considered test item treatment-related and adverse.

Body weight, body weight change and food consumption

At 200 mg/kg/day, when compared with controls, mean food consumption was markedly lower (in average approximately -44%; p<0.001) and lower mean body weight gain was recorded throughout the treatment period (in average 22 g vs. 77 g in controls). At the end of the treatment period, the mean body weight was reduced (-7%, p<0.01) when compared with controls. These changes were considered to be test item-related and adverse.

At 100 mg/kg/day, when compared with controls, lower mean food consumption (in average approximately -18%; p<0.001) from Day 15 p.c. and mean body weight gain (+182 g vs. +328 g, p<0.01) were recorded throughout the treatment period). At the end of the treatment period, the mean body weight was considered unaffected. This effect was therefore attributed to the test item administration but not adverse.

At 50 mg/kg/day, mean food consumption was slightly lower than in controls between Days 24 and 29 p.c. (-12%, not statistically significant), with no significant impact on the body weight and body weight gain. Therefore, this effect was attributed to the test item administration but not adverse.

Net body weight change

At 200 mg/kg/day and when compared with controls, there was a slight to moderate decrease in mean gravid uterus weight (associated with lower fetal body weight), mean carcass weight (associated with lower dam body weight) and mean net body weight change (-432.6 g vs. -152.0 g in controls, p<0.001). These findings were considered test item treatment-related and adverse.

At 100 mg/kg/day, mean gravid uterus weight was unaffected. Mean carcass weight and mean net body weight change were slightly reduced when compared with controls. Considering that there was no associated significant effect on mean body weight and mean fetal body weight, these effects were considered test item treatment-related but not adverse.

At 50 mg/kg/day, there were no effects on mean gravid uterus weight, mean carcass weight and mean net body weight change.

Macroscopic post-mortem examination and organ weight

At macroscopic examination, there were no test item-related findings at any tested dose level.

From 100 mg/kg/day, there was a dose dependent increase in absolute and relative-to-body liver weights. This effect was considered test item treatment-related.

At 50 mg/kg/day, there were no effects on organ weight.

Hysterectomy data

There were no test item treatment-related effects.

Fetal body weight and sex-ratio

At 200 mg/kg/day and when compared with controls, there was a decreased fetal body weight (-10% in males, -14% in females).

At 100 mg/kg/day, this decrease was also observed in a lesser extent in females (-9%) but was not considered adverse.

At 50 mg/kg/day, there were no effects on fetal body weight.

There were no effects on sex-ratio at any tested dose.

Fetal examinations

External examination: there were no test item treatment-related variations or malformations at external examination.

Soft tissue examination: there were no test item treatment-related variations or malformations at soft tissue examination.

Cartilage and skeletal examinations:

.at 200 mg/kg/day and when compared with controls and/or Historical Control Data, there were higher litter and/or fetal incidences of variations [delayed ossification; i.e. unossification of pubis, talus, 1st metacarpal bone, 1st to 4th sternebra(e), centrum of caudal vertebra(e), odontoide; incomplete ossification of 1st metacarpal bone, hyoid]. These findings in association with maternal toxicity and lower fetal body weight at this dose level were considered to be test-tem treatment related and adverse,

. at 100 mg/kg/day, there were incomplete ossification of hyoid centrum (variations). This finding was considered to be test-item treatment related but not adverse,

. at 50 mg/kg/day, there were no effects on cartilage,

. there were no test item-related malformations at skeletal examination.

Conclusion

The test item was administered to time-mated New Zealand White female rabbits by gavage, once daily, from Day 6 to Day 28 p.c., inclusive, at the dose levels of 50, 100 and 200 mg/kg/day.

The control group received the vehicle, 0.5% carboxymethylcellulose/0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, with a dosage volume of 3 mL/kg/day.

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day based on adverse findings recorded at 200 mg/kg/day (poor clinical condition, decreased body weight, reduced food intake, decreased net body weight change) accompanied by increase absolute and relative-to-body liver weight,

. the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 100 mg/kg/day based on adverse findings recorded at 200 mg/kg/day (reduced fetal body weight with fetal ossification delays) in a context of maternal toxicity. No test-item treatment related fetal malformations were observed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The rabbit study is considered to be reliable with a klimisch score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

733 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental study in rats (1983)

In a study similar to OECD TG 414, 25 pregnant Sprague-Dawley rats/concentration were exposed (inhalation; whole body) to t-dodecyl mercaptan at analyzed concentrations of 22.6 (0.19 mg/L) and 88.6 ppm (0.73 mg/L) for 6 hours/day during GD 6-19. One female died at the 88.6 ppm concentration level and the cause of death was undetermined. There were no test substance-related body weight changes. Also, there were no test substance-related fetal malformations. The NOAEC for maternal toxicity developmental toxicity was 88.6 ppm (0.73 mg/L) (highest exposure concentration tested). 

 

Developmental study in rabbits (2020) :

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rabbits from implantation until the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive].

Three groups of 24 time-mated female New Zealand White rabbits received the test item, by the oral route (gavage), at dose levels of 50, 100 or 200 mg/kg/day, once daily from Day 6 to Day 28 p.c. inclusive.

Another group of 24 time-mated female rabbits received the vehicle only, 0.5% carboxymethylcellulose/0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions and acted as a control group. A constant dosage volume of 3 mL/kg/day was used.

There were 21/24, 19/24, 22/24 and 19/24 pregnant females with live fetuses in the control and 50, 100 and 200 mg/kg/day groups, respectively.

At 200 mg/kg/day, three females were euthanized before the end of the treatment period (between Days 23 and 27 p.c.) for poor clinical condition (one female) or abortion (two females). These animals also had low body temperature and emaciated appearance associated with reduced food consumption and body weight loss (down to -18% when compared to the first day of treatment). These effects were considered test item treatment-related and adverse. There were no unscheduled deaths in control, 50 or 100 mg/kg/day groups. At 200 mg/kg/day, among surviving females, one was transiently cold to the touch and had an emaciated appearance correlating with transient body weight loss and reduced food consumption. This was considered test item treatment-related and adverse.

At 200 mg/kg/day, when compared with controls, mean food consumption was markedly lower (in average approximately -44%; p<0.001) and lower mean body weight gain was recorded throughout the treatment period (in average 22 g vs. 77 g in controls). At the end of the treatment period, the mean body weight was reduced (-7%, p<0.01) when compared with controls. These changes were considered to be test item-related and adverse.

At 100 mg/kg/day, when compared with controls, lower mean food consumption (in average approximately -18%; p<0.001) from Day 15 p.c. and mean body weight gain (+182 g vs. +328 g, p<0.01) were recorded throughout the treatment period). At the end of the treatment period, the mean body weight was considered unaffected. This effect was therefore attributed to the test item administration but not adverse.

At 50 mg/kg/day, mean food consumption was slightly lower than in controls between Days 24 and 29 p.c. (-12%, not statistically significant), with no significant impact on the body weight and body weight gain. Therefore, this effect was attributed to the test item administration but not adverse.

At 200 mg/kg/day and when compared with controls, there was a slight to moderate decrease in mean gravid uterus weight (associated with lower fetal body weight), mean carcass weight (associated with lower dam body weight) and mean net body weight change (-432.6 g vs. -152.0 g in controls, p<0.001). These findings were considered test item treatment-related and adverse. At 100 mg/kg/day, mean gravid uterus weight was unaffected. Mean carcass weight and mean net body weight change were slightly reduced when compared with controls. Considering that there was no associated significant effect on mean body weight and mean fetal body weight, these effects were considered test item treatment-related but not adverse. At 50 mg/kg/day, there were no effects on mean gravid uterus weight, mean carcass weight and mean net body weight change.

At macroscopic examination, there were no test item-related findings at any tested dose level. From 100 mg/kg/day, there was a dose dependent increase in absolute and relative-to-body liver weights. This effect was considered test item treatment-related. At 50 mg/kg/day, there were no effects on organ weight.

There were no test item treatment-related effects.

At 200 mg/kg/day and when compared with controls, there was a decreased fetal body weight (-10% in males, -14% in females). At 100 mg/kg/day, this decrease was also observed in a lesser extent in females (-9%) but was not considered adverse. At 50 mg/kg/day, there were no effects on fetal body weight. There were no effects on sex-ratio at any tested dose.

External examination: there were no test item treatment-related variations or malformations at external examination.

Soft tissue examination: there were no test item treatment-related variations or malformations at soft tissue examination.

Cartilage and skeletal examinations:

.at 200 mg/kg/day and when compared with controls and/or Historical Control Data, there were higher litter and/or fetal incidences of variations [delayed ossification; i.e. unossification of pubis, talus, 1st metacarpal bone, 1st to 4th sternebra(e), centrum of caudal vertebra(e), odontoide; incomplete ossification of 1st metacarpal bone, hyoid]. These findings in association with maternal toxicity and lower fetal body weight at this dose level were considered to be test-tem treatment related and adverse,

. at 100 mg/kg/day, there were incomplete ossification of hyoid centrum (variations). This finding was considered to be test-item treatment related but not adverse,

. at 50 mg/kg/day, there were no effects on cartilage,

. there were no test item-related malformations at skeletal examination.

 

The test item was administered to time-mated New Zealand White female rabbits by gavage, once daily, from Day 6 to Day 28 p.c., inclusive, at the dose levels of 50, 100 and 200 mg/kg/day.

The control group received the vehicle, 0.5% carboxymethylcellulose/0.5% Tween 80 in drinking water treated by reverse osmosis, under the same experimental conditions, with a dosage volume of 3 mL/kg/day.

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day based on adverse findings recorded at 200 mg/kg/day (poor clinical condition, decreased body weight, reduced food intake, decreased net body weight change) accompanied by increase absolute and relative-to-body liver weight,

. the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 100 mg/kg/day based on adverse findings recorded at 200 mg/kg/day (reduced fetal body weight with fetal ossification delays) in a context of maternal toxicity. No test-item treatment related fetal malformations were observed.

 

Developmental toxicity study in mice (1983)

A study was conducted with 25 CD-1 female mice/concentration at the same concentrations as the above rat study, with exposure for 6 hours/day during GD 6-16. One mouse died at the 22.6 ppm (0.19 mg/L) level, and the cause of death was unknown. There were no test substance-related changes in body weights. There were no statistically significant test substance-related fetal malformations noted. The mean number of total implants, corpora lutea, fetal body weight and sex ratio were unaffected by exposure to the test substance. It should be noted that 3/25 control mice and 5/25 mice from each exposure concentration exhibited total implant resorption; the incidence of resorptions was not considered to be related to test substance. The NOAEC for maternal and developmental toxicity was 88.6 ppm (0.73 mg/L) (highest exposure concentration tested)

Toxicity to reproduction: other studies

Additional information

In the 90-day and 28-day repeated toxicity studies, no histological effect was observed on the reproductive organs and the sperm parameters.

Justification for classification or non-classification

Based on the available data, no classification for reproductive toxicity is required for Tert-dodecanethiol according to the Regulation EC n°1272/2008.