Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 2nd to November 9th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 2nd to November 9th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
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:
July 29th, 2016
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age of animals when supplied to laboratory: 7 to 8 weeks old.
- Weight at study initiation: 211 - 241 g for males and 187 - 204 g for females.
- Housing: from arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5 × 38 × 20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5 × 26.6 × 18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages (measuring 42.5 × 26.6 × 18.5 cm) for the gestation period, birth and lactation. Nesting material was provided inside suitable bedding bags. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
- Diet (e.g. ad libitum): a commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study, except before sample blood sample.
- Water (e.g. ad libitum): drinking water was supplied ad libitum to each cage via water bottles except before the collection of urine. Before starting urine collection water bottles were removed from each cage and each animal received approximately 10ml/kg of drinking water by gavage,in order to obtain urine samples suitable for analysis.
- Acclimation period: 20 days (main groups) and 41 days (recovery groups) before the start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C.
- Humidity (%): 55 % ± 15 %.
- Air changes (per hr): 15 to 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is a possible route of exposure of the test item in man.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved/suspended in the vehicle. The formulations were prepared weekly (concentrations of 25, 100 and 400 mg/ml). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the substance is slightly soluble in water and corn oil was chosen as vehicle.
- Concentration in vehicle: 0 (group 1), 25 (group 2), 100 (group 3) and 400 (group 4) mg/ml
- Amount of vehicle (if gavage): 2 ml/kg bw based on the most recently recorded body weight for each animal.
#Main groups: for the males dose volumes were adjusted once per week for each animal according to the last recorded body weight. For females: dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating; during the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1,4, 7 and 13 postpartum.
#Recovery groups: for both males and females the amount of test item or control item was adjusted once per week for each animal according to the last recorded body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The correct preparation of dose formulation was performed by the laboratory Innovative Environmental Services(IES) Ltd using HPLC-ELSD based on external standards calibration.

#HPLC-ELSD condition:
- HPLC Column: COSMOSIL 5C18-AR, 100 Å, 250 x 4.6 mm (Nacalai tesque)
- Detector: Detector: Sedere SEDEX 85
- Eluent: eluent A:water and eluent B: tetrahydrofuran
Minutes: 0 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 3 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 8 min; % Eluent A: 25 % ; % Eluent B: 75 %
Minutes: 28 min; % Eluent A: 15 % ; % Eluent B: 85 %
Minutes: 29 min; % Eluent A: 0 % ; % Eluent B: 100 %
Minutes: 32 min; % Eluent A: 0 % ; % Eluent B: 100 %
Minutes: 32.1 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 40 min; % Eluent A: 50 % ; % Eluent B: 50 %
- Injection Volume: 50 μl
- Injection Temperature: 45 °C
- Flow Rate: 1.0 ml/minute
- Column Temperature: 45 °C in a thermostatic oven
- Retention Time: approx. 16.8 min.
- Detector Temperature: 40 °C

#RESULTS: the recoveries of test item in all test samples including homogeneity test samples ranged from 84 % to 106 %. Thus, the correct preparation of dose formulation samples at the test facility (Research Toxicology Centre S.p.A - RTC) and the homogenous distribution of the test item in the corn oil vehicle at concentration 25 g/l , 100 g/l and 400 g/l could be confirmed.
Duration of treatment / exposure:
Main groups: 29 - 30 days for males (i.e. 2 weeks prior to paring, during the paring and until the day before necropsy) and for at least 51 days for females (i.e. 2 weeks before paring, during paring, post coitum and post partum periods until day 13 post partum or the day before sacrifice).
Recovery groups: 4 consecutive weeks (total 28 days) for males and 6 consecutive weeks (total 42 days) for females. No treatment was given during the recovery period (4 weeks both for males and for females).
Frequency of treatment:
Once a day, 7 days per week.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 male and 10 female rats for each main group (group 1 to 4) and 5 male and 5 female rats for each recovery (group 5 to 8).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels of 50, 200 and 800 mg/kg bw/day were selected based on information from a preliminary non-GLP compliant study.
- Rationale for animal assignment (if not random): on the day of allocation (7 days prior to the start of treatment for the main groups and 28 days for the recovery groups), all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed. The rat numbers formed the last digits of a computer generated 8 figure animal number (the remaining digits of the animal number were different for each concurrent study and served to ensure unique animal numbering for any study employing computerised data collection). The computerised system used in this study was the Xybion Path/Tox System, Version 4.2.2. Even numbers were chosen for males and odd numbers for females.
- Rationale for selecting satellite groups: a 4 week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. All dose levels included 5 additional animals per sex to be sacrificed after 4 weeks of recovery (Groups 5 to 8).
- Post-exposure recovery period in satellite groups: 4 weeks.
Observations and examinations performed and frequency:
MORTALITY (all groups)
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CAGE SIDE OBSERVATIONS (all groups)
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes. Data are reported until Week 4 (males of main and recovery groups), Week 6 (females of main and recovery groups),Week 4 of recovery (recovery groups).

DETAILED CLINICAL OBSERVATIONS (all groups)
Once before commencement of treatment (for main groups only) and at least once daily during the study,each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day.

BODY WEIGHT (all groups)
- Main groups: males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 19 post coitum. Dams were also weighed on Days 1, 4, 7, 13 postpartum and just before necropsy.
- Recovery groups: each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION (all groups):
-Main groups: the weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7,14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 postpartum starting from Day1 post partum.
- Recovery groups: the weight of food consumed by each cage of rats was recorded at weekly intervals starting from allocation in males while following allocation starting from the second week of allocation in females.
Note: the test item was administered by oral gavage and not in diet.

HAEMATOLOGY
- Time schedule for collection of blood: just before sacrificial procedure.
- Anaesthetic used for blood collection: isofluorane anesthesia.
- Animals fasted: yes.
- How many animals:5 males and 5 females randomly selected from each main group during sacrificial procedure.
- Parameters checked:
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count (Neutrophils / Lymphocites / Eosinophils / Basophils / Monocytes / Large unstained cells)
Plateletes
Coagulation parameters (Prothrombin time and Activated partial thromboplastin time)
In addition, as part of the necropsy procedure, blood samples were taken from the abdominal vena cava of all parental male and female rats from each main group and from animals of the recovery groups, under isofluorane anaesthesia, to perform the determination of serum T3, T4 and TSH levels.

CLINICAL CHEMISTRY
- Time schedule for collection of blood:just before sacrificial procedure.
- Animals fasted: yes.
- How many animals: 5 males and 5 females randomly selected from each main group during sacrificial procedure.
- Parameters checked:
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Inorganic phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

URINALYSIS:
- Time schedule for collection of urine:collection during the last week of treatment .
- Animals fasted: Yes . The urynalysis was performed on the same 5 males for each main group selected for blood collection in clinical pathology investigations.
- Parameters checked :
Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, were examined microscopically for:
Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

NEUROBEHAVIOURAL EXAMINATION (all groups)
- Time schedule and dose group for examinations: once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) , for assessment of grip strength and for motor activity measurement (for 5 minutes). For the females of the main groups the tests were performed on Day 13 postpartum. Measurements were performed using a computer generated random order (for the main groups). Once during Week 4 of recovery, these evaluations were also performed in all animals.
- Battery of functions tested: sensory activity, grip strength, motor activity.

BIOANALYSIS - Thyroid hormone determination (T3, T4 and TSH)
As a part of the necropsy procedure approximately 0.8 ml of blood samples were taken from all parental males and females of the main groups and from animals of the recovery groups. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and thyroid stimulating hormone (TSH) by a multiplex assay. The determination was restricted as samples from all parental males from all main groups.

OTHER: other analysis such as vaginal smears (main groups), mating (main groups), parturition and gestation length (main groups), data on pups (e.g.anogenital distance and nipple count), blood collection for thyroid hormone determination (T3, T4, TSH) for pups were performed during the study. These data are reported in the specific section for reproductive/developmental toxicity (see section 7.8.1 of this IUCLID dossier).
Sacrifice and pathology:
SACRIFICE: parental and recovery animals were killed by exsanguination under isofluorane anaesthesia.
- Main groups: the males of the main groups were killed after the mating of all females on Days 30 and 31 of the study.The females of the main groups with live pups were killed on Day 14 postpartum and the non pregnant females were killed on Day 27 or 28 post coitum.
-Recovery groups: all males and all females of recovery groups were killed after 4 weeks of recovery.

GROSS PATHOLOGY (all groups)
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed:
Adrenal glands
Brain(cerebrum,cerebellum,medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Parathyroid glands
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus (where present)
Thyroid gland
Uterus – cervix
The ratios of organ weight to body weight were calculated for each animal.

HISTOPATHOLOGY:
The histopathological examination was performed for the following tissues on 5 males and on 5 females of the control and high dose groups (already selected for clinical pathology evaluation) killed at term:
Adrenal glands
Bone marrow (from sternum)
Brain
Clitoral gland
Caecum
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem brionchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary glands (males and females)
Mammary area
Optic nerves
Ovaries
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina
All abnormalities were examinated for all animal of all main groups.

OTHER: Specific analysis on parental females (i.e. number of visible implantation sites and number of corpora lutea) and on pups (i.e. external examination, sex confirmation and nipple count, thyroid weight) were performed during the study. These data are reported in the specific section for reproductive/developmental toxicity (see section 7.8.1 of this IUCLID dossier).
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Clinical signs:
no effects observed
Description (incidence and severity):
No significant clinical signs were observed throughout the study in treated animals of main groups of both sexes. During the postpartum phase, in one female of the low dose group (no. X0730033, 50 mg/kg/day) hairloss in different region of the ventral region (neck, thoracic and abdomen region) was noted generally from Day 11 to the end (Day 14) of the postpartum phase. In addition, in a single female animal of Group 1 (no.X0730011), starting from Day 12 post partum up to the end of the observation period (Day 14 postpartum), a palpable mass was noted in the mammary area (right).
Recovery animals did not show any clinical signs during treatment, as well as during the recovery phase.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No difference in body weights and body weights gain were recorded in animals of both sexes compared to the control group both for main and for recovery groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were observed in either males and females compared to the control group both for main and recovery groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with controls, slight decrease of haemoglobin and haematocrit was recorded in males dosed at 800 mg/kg/day (9 %, both parameters). Reticulocytosis was recorded in the same group animals (53 % above controls). In addition, males dosed at 50 mg/kg/day showed decreases of mean corpuscular volume (4 %), mean corpuscular haemoglobin (4 %) and increase of neutrophils (101 %). Mean corpuscular volume was also decreased in males receiving 800 mg/kg/day (3 %). Due to the absence of dose-relation and the minimal magnitude, these changes were considered unrelated to treatment. No change was recorded in coagulation parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Bilirubin was increased in males dosed at 800 mg/kg/day. Compared with mean control data, the increment was 2.4 fold. Due to the absence of other related changes, this finding was considered of no toxicological relevance. Urea was increased in one female dosed at 800 mg/kg/day (no. X07300063). Compared with mean control data, the increment was 2.1 fold. Due to the low incidence, this finding was considered incidental. Other statistically significant differences were observed between controls and treated animals, such as: increases of albumin in males dosed at 800 mg/kg/day (5 %) and increase of phosphorus in females receiving 50 mg/kg/day (48 %). Due to the absence of dose-relation or the minimal severity, these changes were considered of no toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes were observed in urine analysis.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant changes were observed in absolute and relative organ weight of treated animals that completed the treatment or recovery period, when compared to the controls. The sporadic statistically significant changes observed in some organs (i.e. relative heart weight of high dose female group) of animals sacrificed at the end of treatment or the recovery phase, were not considered toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals that completed the treatment or recovery period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related. The sporadic changes such as small prostate observed respectively in one high dose male (no. X0730068) and one low dose male (no. X0730026), single or multiple dark and/or red area/s depressed in the glandular region of the stomach of one high dose male (no. X0730078), two high dose females (nos. X0730069 and X0730077) and three low dose females (nos. X0730025, X0730027 and X0730031) and one subcutaneous mass observed in one control dose female (no. X0730011) were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item for both main groups and for recovery groups . No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated animals for both main groups and for recovery groups. No toxicological relevance was attributed to the statistically significant increase in motor activity indicated in treated males because it was related the low control value measured (when compared to all other control values in the study).
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The M-Adenocarcinoma in the mammary glands of one control female (no. X0730011), which correlated to the subcutaneous mass observed during necropsy, was considered spontaneous.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone determination for parental males: Thyroid Stimulating Hormone (TSH) was increased in a number of parental males dosed at 200 and 800 mg/kg/day. Compared with controls, the increases were 1.8 and 2.1-fold, respectively. No relevant changes of the thyroid hormones T3 or T4 were recorded. Since no related changes of T3/T4 levels were recorded and no thyroid findings were recorded at histopathological evaluation, the above finding was considered to be of no toxicological relevance.
Details on results:
The results related to reproductive parameters (i.e oestrous cycle and spermatogenic cycle), pairing combination, mating performance, implantation, pre-birth loss, gestation length) and data on pups (e.g. sex ratio, anogenital distance, clinical signs of pups and thyroid weight of pups, thyroid hormone determination) are reported in the specific section for reproductive/developmental toxicity (see section 7.8.1 of this IUCLID dossier).
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect was observed during the study
Key result
Critical effects observed:
no
Conclusions:
Based on results in male and female Sprangue Dawley rats exposed to dose levels of 50, 200, and 800 mg/kg bw/day of test substance for at least 29 days, no observed adverse effect level (NOAEL) is 800 mg/kg bw/day for general toxicity and for reproduction/developmental toxicity for both males and females.
Executive summary:

The toxic effects on Sprague Dawley rats after repeated dosing with test item, as well as any effects of the substance on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated. A 4 week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. The vehicle was corn oil (2 ml/kg).

The test item was administered according to the following doses for the main groups: 0 mg/kg bw/day (group 1), 50 mg/kg bw/day (group 2), 200 mg/kg bw/day (group 3) and 800 mg/kg bw/day (group 4). Each main group was composed by 10 males and by 10 females.

The test item was administered according to the following doses for the recovery groups: 0 mg/kg bw/day (group 5), 50 mg/kg bw/day (group 6), 200 mg/kg bw/day (group 7) and 800 mg/kg bw/day (group 8). Each recovery group was composed by 5 males and by 5 females.

 

Main groups:

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29/30 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and postpartum periods until Day 13 post partum (for at least 51 days). The two not pregnant female were dosed up to the day before necropsy.

The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), body weight, food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis in males), litter data, sex ratios, macroscopic observations and organ weights. Histopathological examination was performed in control and high dose groups (five animals/sex/group randomly selected), as well as on all abnormalities detected during post mortem observation at the end of treatment period. The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups.

Thyroid hormone determination was performed in all males.

Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of 1/pup/sex/litter (where possible) at Day 14 postpartum were determined.

All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 postpartum were subjected to an external examination. Sex was determined by internal gonads inspection.

All live pups sacrificed on Day 14 postpartum were examined for external abnormalities and sex confirmation by gonadal inspection.

 

Recovery groups:

Recovery animals (males) were treated for up to 4 consecutive weeks and killed after 4 weeks of recovery period. Recovery animals (females) were treated for up to 6 weeks, when the majority of the main group females were killed. The females were sacrificed after 4 weeks of recovery period. The following parameters were evaluated in these animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, and macroscopic observations and organ weights.

Results:

- Mortality and fate of females: no mortality occurred throughout the study. Two females of the control group were found not pregnant at necropsy. The number of females with live pups on Day 14 post partum was: 8 in the control, 10 in the low dose, 10 in the mid-dose and 9 in the high dose group.

- Clinical signs, neurotoxicity assessment including motor activity, grip strength, sensory reaction to stimuli for main and recovery groups: no significant clinical signs were observed throughout the study in all treated main animals of both sexes. Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups.Recovery animals did not show any clinical signs during treatment, as well as during the recovery phase. Observation of treated animals at removal from the cage and in an open arena (neurotoxicity assessment), as well as, in motor activity, grip strength and sensory reaction to stimuli did not show differences between control and treated groups.

- Body weight and body weight gain for main and recovery groups: no difference in body weights and body weights gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and recovery groups.

- Food consumption: for main and recovery groups: no effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

- Haematology including coagulation for main groups: the changes observed in some haematological parameters were considered unrelated to treatment, due to the absence of dose-relation and the minimal magnitude. No changes were recorded in coagulation parameters evaluated.

- Clinical chemistry for main groups: fluctuations observed in some bio chemicals parameters were considered of no toxicological relevance due to the absence of dose-relation, low incidence or the minimal severity.

- Urinalysis for main groups: no changes were observed in urine analysis.

- Thyroid hormones: no treatment-related differences were noted in hormones levels in parental males when compared to control group animals.

- Terminal body weight and organ weights for main and recovery groups: no changes were observed in terminal body, absolute and relative organ weight of treated animals, when compared to the controls.

- Macroscopic observations for main and recovery groups: no remarkable differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery period, when compared with controls.

- Microscopic observations: no treatment-related changes were noted in animals sacrificed at the end of the treatment period. In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

Note: the results related to oestrous cycle, reproductive parameters, implantation, pre-birth loss, gestation length, data for pups till Day 13 post partum (i.e.clinical signs, anogenital distance, thyroid weight abnd tyroid hormones) are reported in the section 7.8.1 reproductive toxicity of this IUCLID dossier.

 

Conclusion:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity (and also for reproductive/ developmental toxicity- see section 7.8.1 of this IUCLID dossier) was considered to be 800 mg/kg bw/day for both males and females.

 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
IUPAC Name:
Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age of animals when supplied to laboratory: 7 to 8 weeks old.
- Weight at study initiation: 211 - 241 g for males and 187 - 204 g for females.
- Housing: from arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5 × 38 × 20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5 × 26.6 × 18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages (measuring 42.5 × 26.6 × 18.5 cm) for the gestation period, birth and lactation. Nesting material was provided inside suitable bedding bags. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
- Diet (e.g. ad libitum): a commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study, except before sample blood sample.
- Water (e.g. ad libitum): drinking water was supplied ad libitum to each cage via water bottles except before the collection of urine. Before starting urine collection water bottles were removed from each cage and each animal received approximately 10ml/kg of drinking water by gavage,in order to obtain urine samples suitable for analysis.
- Acclimation period: 20 days (main groups) and 41 days (recovery groups) before the start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C.
- Humidity (%): 55 % ± 15 %.
- Air changes (per hr): 15 to 20 air changes per hour.
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The oral route was selected as it is a possible route of exposure of the test item in man.

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved/suspended in the vehicle. The formulations were prepared weekly (concentrations of 25, 100 and 400 mg/ml). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the substance is slightly soluble in water and corn oil was chosen as vehicle.
- Concentration in vehicle: 0 (group 1), 25 (group 2), 100 (group 3) and 400 (group 4) mg/ml
- Amount of vehicle (if gavage): 2 ml/kg bw based on the most recently recorded body weight for each animal.
#Main groups: for the males dose volumes were adjusted once per week for each animal according to the last recorded body weight. For females: dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating; during the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1,4, 7 and 13 postpartum.
#Recovery groups: for both males and females the amount of test item or control item was adjusted once per week for each animal according to the last recorded body weight.
Details on mating procedure:
- M/F ratio per cage: during mating animals were housed one male to one female (monogamous amting).
- Length of cohabitation: the female was paired with the same male until positive identification of copulation occurred.
- Proof of pregnancy: vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found in the cage tray).
- After successful mating each pregnant female was caged (how): the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The correct preparation of dose formulation was performed by the laboratory Innovative Environmental Services(IES) Ltd using HPLC-ELSD based on external standards calibration.

#HPLC-ELSD condition:
- HPLC Column: COSMOSIL 5C18-AR, 100 Å, 250 x 4.6 mm (Nacalai tesque)
- Detector: Detector: Sedere SEDEX 85
- Eluent: eluent A:water and eluent B: tetrahydrofuran
Minutes: 0 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 3 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 8 min; % Eluent A: 25 % ; % Eluent B: 75 %
Minutes: 28 min; % Eluent A: 15 % ; % Eluent B: 85 %
Minutes: 29 min; % Eluent A: 0 % ; % Eluent B: 100 %
Minutes: 32 min; % Eluent A: 0 % ; % Eluent B: 100 %
Minutes: 32.1 min; % Eluent A: 50 % ; % Eluent B: 50 %
Minutes: 40 min; % Eluent A: 50 % ; % Eluent B: 50 %
- Injection Volume: 50 μl
- Injection Temperature: 45 °C
- Flow Rate: 1.0 ml/minute
- Column Temperature: 45 °C in a thermostatic oven
- Retention Time: approx. 16.8 min.
- Detector Temperature: 40 °C

#RESULTS: the recoveries of test item in all test samples including homogeneity test samples ranged from 84 % to 106 %. Thus, the correct preparation of dose formulation samples at the test facility (Research Toxicology Centre S.p.A - RTC) and the homogenous distribution of the test item in the corn oil vehicle at concentration 25 g/l , 100 g/l and 400 g/l could be confirmed.
Duration of treatment / exposure:
Main groups: 29 - 30 days for males (i.e. 2 weeks prior to paring, during the paring and until the day before necropsy) and for at least 51 days for females (i.e. 2 weeks before paring, during paring, post coitum and post partum periods until day 13 post partum or the day before sacrifice).
Recovery groups: 4 consecutive weeks (total 28 days) for males and 6 consecutive weeks (total 42 days) for females. No treatment was given during the recovery period (4 weeks both for males and for females).
Frequency of treatment:
Once a day, 7 days per week.
Details on study schedule:
A total of 142 Sprague Dawley SD rats (67 males and 75 virgin females), 7 to 8 weeks old were ordered. A health check was then performed by a veterinarian. An acclimatisation period of 20 days (main groups) and 41 days (recovery groups) was allowed before the start of treatment .
The treatment of main groups (10 males and 10 females for each group) was scheduled as follows:
- for males: weeks prior to paring, during the paring and until the day before necropsy.
- for females: 2 weeks before paring, during paring, post coitum and post partum periods until day 13 post partum or the day before sacrifice.
The treatment of recovery groups (5 males and 5 females for each group) was scheduled as follows: 4 consecutive weeks (total 28 days) for males and 6 consecutive weeks (total 42 days) for females. No treatment was given during the recovery period (4 weeks both for males and for females).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 male and 10 female rats for each main group (group 1 to 4) and 5 male and 5 female rats for each recovery (group 5 to 8).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels of 50, 200 and 800 mg/kg bw/day were selected based on information from a preliminary non-GLP compliant study.
- Rationale for animal assignment (if not random): on the day of allocation (7 days prior to the start of treatment for the main groups and 28 days for the recovery groups), all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed. The rat numbers formed the last digits of a computer generated 8 figure animal number (the remaining digits of the animal number were different for each concurrent study and served to ensure unique animal numbering for any study employing computerised data collection). The computerised system used in this study was the Xybion Path/Tox System, Version 4.2.2. Even numbers were chosen for males and odd numbers for females.
- Rationale for selecting satellite groups: a 4 week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. All dose levels included 5 additional animals per sex to be sacrificed after 4 weeks of recovery (Groups 5 to 8).
- Post-exposure recovery period in satellite groups: 4 weeks.

Examinations

Parental animals: Observations and examinations:
MORTALITY (all groups)
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CAGE SIDE OBSERVATIONS (all groups)
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes. Data are reported until Week 4 (males of main and recovery groups), Week 6 (females of main and recovery groups),Week 4 of recovery (recovery groups).

DETAILED CLINICAL OBSERVATIONS (all groups)
Once before commencement of treatment (for main groups only) and at least once daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day.

BODY WEIGHT (all groups)
- Main groups: males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 19 post coitum. Dams were also weighed on Days 1, 4, 7, 13 postpartum and just before necropsy.
- Recovery groups: each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION (all groups):
-Main groups: the weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7,14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 postpartum starting from Day1 post partum.
- Recovery groups: the weight of food consumed by each cage of rats was recorded at weekly intervals starting from allocation in males while following allocation starting from the second week of allocation in females.
Note: the test item was administered by oral gavage and not in diet.

HAEMATOLOGY
- Time schedule for collection of blood: just before sacrificial procedure.
- Anaesthetic used for blood collection: isofluorane anesthesia.
- Animals fasted: yes.
- How many animals:5 males and 5 females randomly selected from each main group during sacrificial procedure.
- Parameters checked:
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count (Neutrophils / Lymphocites / Eosinophils / Basophils / Monocytes / Large unstained cells)
Plateletes
Coagulation parameters (Prothrombin time and Activated partial thromboplastin time)
In addition, as part of the necropsy procedure, blood samples were taken from the abdominal vena cava of all parental male and female rats from each main group and from animals of the recovery groups, under isofluorane anaesthesia, to perform the determination of serum T3, T4 and TSH levels.

CLINICAL CHEMISTRY
- Time schedule for collection of blood:just before sacrificial procedure.
- Animals fasted: yes.
- How many animals: 5 males and 5 females randomly selected from each main group during sacrificial procedure.
- Parameters checked:
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Inorganic phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

URINALYSIS:
- Time schedule for collection of urine: collection during the last week of treatment .
- Animals fasted: Yes . The urynalysis was performed on the same 5 males for each main group selected for blood collection in clinical pathology investigations.
- Parameters checked :
Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, were examined microscopically for:
Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

NEUROBEHAVIOURAL EXAMINATION (all groups)
- Time schedule and dose group for examinations: once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) , for assessment of grip strength and for motor activity measurement (for 5 minutes). For the females of the main groups the tests were performed on Day 13 postpartum. Measurements were performed using a computer generated random order (for the main groups). Once during Week 4 of recovery, these evaluations were also performed in all animals.
- Battery of functions tested: sensory activity, grip strength, motor activity.

BIOANALYSIS - Thyroid hormone determination (T3, T4 and TSH)
As a part of the necropsy procedure approximately 0.8 ml of blood samples were taken from all parental males and females of the main groups and from animals of the recovery groups. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and thyroid stimulating hormone (TSH) by a multiplex assay. The determination was restricted as samples from all parental males from all main groups.
Oestrous cyclicity (parental animals):
For stock females oestrus cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle. For females allocated to groups, vaginal smears were taken daily in the morning starting two weeks before pairing throughout the mating period, until a positive identification of copulation was made. The vaginal smear data were examined to determine not only anomalies of the oestrous cycle but also the pre-coital interval (i.e., the number of nights paired prior to the detection of mating). Vaginal smears were also taken from all females, before dispatch to necropsy.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight and epididymis weight.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On Day 4 postpartum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. At least two pups (males or females, selected for culling) were sacrificed for blood collection. Two male pups were selected for dams X0730007 and X0730019 (Group 1) and X0730071 (Group 4). Two female pups were selected for dams X0730059 (Group 3) and X0730065 (Group 4). For litters with 8 pups (no. X0730043, Group 3), two female pups were sacrificed for blood collection.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number and sex of pups: as soon as possible after parturition was considered complete (Day 0 postpartum), all pups (live and dead) were counted, sexed and only live pups were identified.
- Weight gain: live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
- Behavioural abnormalities: observations were performed once daily for all litters.
- Anogenital distance (AGD): the AGD of each pup was measured on Day 1 postpartum. The AGD was normalized to the cube root of body weight collected on Day 1 postpartum.
- Nipple count: nipple count was performed on day 13 post partum in males pups and again after euthanasia of the pups on day 14 post partum.
- Bioanalysis - Thyroid hormone determination (T3, T4 and TSH): on day 4 post partum, blood sample (approximately 0.2 ml) were collected from two of the non selected pups (1 sample/sex where possible) of the main groups. Blood samples were withdrawn under light ether isofluorane anaesthesia from the heart (by intracardiac puncture). On Day 14 postpartum, blood samples (approximately 0.5 ml) were withdrawn under light ether or isofluorane anaesthesia from the heart (by intracardiac puncture) from at least two pups (1 sample/sex where possible) per litter. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and thyroid stimulating hormone (TSH) by a multiplex assay. The determination was restricted to samples from pups on Day 14 post partum.

GROSS EXAMINATION OF DEAD PUPS: all pups found dead in the cage were examined for external and internal abnormalities.


Postmortem examinations (parental animals):
SACRIFICE: parental and recovery animals were killed by exsanguination under isofluorane anaesthesia.
- Main groups: the males of the main groups were killed after the mating of all females on Days 30 and 31 of the study. The females of the main groups with live pups were killed on Day 14 postpartum and the non pregnant females were killed on Day 27 or 28 post coitum.
-Recovery groups: all males and all females of recovery groups were killed after 4 weeks of recovery.

GROSS PATHOLOGY (all groups)
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed:
Adrenal glands
Brain(cerebrum,cerebellum,medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Parathyroid glands
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus (where present)
Thyroid gland
Uterus – cervix
The ratios of organ weight to body weight were calculated for each animal.

HISTOPATHOLOGY:
The histopathological examination was performed for the following tissues on 5 males and on 5 females of the control and high dose groups (already selected for clinical pathology evaluation) killed at term:
Adrenal glands
Bone marrow (from sternum)
Brain
Clitoral gland
Caecum
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem brionchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary glands (males and females)
Mammary area
Optic nerves
Ovaries
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina
All abnormalities were examinated for all animal of all main groups.
Postmortem examinations (offspring):
SACRIFICE
Pups that had completed the scheduled test period (Day 4 or 14 postpartum) were euthanised by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling by ether or isofluorane anaesthesia.

GROSS NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 postpartum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed on Day 14 postpartum were killed and examined for external abnormalities and sex confirmation by gonadal inspection. The ventral region of pups was checked for presence of nipples/areolae.

ORGAN WEIGHTS
On day 14 post partum thyroid was weighed from one male and one female pups selected for blood collection of hormone determination and preserved in 10 % neutral buffered formalin. The thyroid weights were determined after fixation.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
COPULATION INDEX (%) for Males = [(N. of males mated) / (N. of males paired)] x 100

FERTILITY INDEX (%) for Males = [(N. of males which induced pregnancy) / (N. of males paired)] x 100

COPULATION INDEX (%) for Females = [(N. of females mated) / (N. of females paired)] x 100

FERTILITY INDEX (%) for Females = [(N. of pregnant females) / (N. of females paired)] x 100

PRE-COITAL INTERVAL for Males and Females: Number of days between paring and mating
Note: Positive identification of copulation was performed by vaginal smears analysis (sperm identification, vaginal plug in situ or copulation plug found in the cage tray).

GESTATION LENGHT: gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum). A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. X0730013 and X0730017 (Group 1) which did not give birth after 25 days of post coitum period were sacrificed on Days 28 and 27 post coitum, respectively. These animals were found not pregnant at necropsy.

PRE-IMPLANTATION LOSS: [(N. of corpora lutea - N. of visible implantation) / N. of corpora lutea] x 100

PRE-NATAL LOSS: [(N. of visible implantation- live litter size at birth) / N. of visible implantation] x 100
Offspring viability indices:
PUP LOSS day 0 post partum (%): [(Total litter size at birth) - (live litter size at birth) / Total litter size at birth ] x 100

CUMULATIVE LOSS on day 4 post partum (%): [(Total litter size at birth) - (Live litter size at day 4 before culling) / Total litter size at birth] x 100

POST NATAL LOSS on day 13 post partum (%): [(Live litter size on day 4 after culling) - (Live litter size on day 13) / (Live litter size on day 4 after culling)] x 100

SEX RATIO on days 4 and on days 14 post partum

ANOGENITAL DISTANCE

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No significant clinical signs were observed throughout the study in treated animals of main groups of both sexes. During the postpartum phase, in one female of the low dose group (no. X0730033, 50 mg/kg/day) hairloss in different region of the ventral region (neck, thoracic and abdomen region) was noted generally from Day 11 to the end (Day 14) of the postpartum phase. In addition, in a single female animal of Group 1 (no.X0730011), starting from Day 12 post partum up to the end of the observation period (Day 14 postpartum), a palpable mass was noted in the mammary area (right).
Recovery animals did not show any clinical signs during treatment, as well as during the recovery phase.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No difference in body weights and body weights gain were recorded in animals of both sexes compared to the control group both for main and for recovery groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were observed in either males and females compared to the control group both for main and recovery groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with controls, slight decrease of haemoglobin and haematocrit was recorded in males dosed at 800 mg/kg/day (9 %, both parameters). Reticulocytosis was recorded in the same group animals (53 % above controls). In addition, males dosed at 50 mg/kg/day showed decreases of mean corpuscular volume (4 %), mean corpuscular haemoglobin (4 %) and increase of neutrophils (101 %). Mean corpuscular volume was also decreased in males receiving 800 mg/kg/day (3 %). Due to the absence of dose-relation and the minimal magnitude, these changes were considered unrelated to treatment. No change was recorded in coagulation parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Bilirubin was increased in males dosed at 800 mg/kg/day. Compared with mean control data, the increment was 2.4 fold. Due to the absence of other related changes, this finding was considered of no toxicological relevance. Urea was increased in one female dosed at 800 mg/kg/day (no. X07300063). Compared with mean control data, the increment was 2.1 fold. Due to the low incidence, this finding was considered incidental. Other statistically significant differences were observed between controls and treated animals, such as: increases of albumin in males dosed at 800 mg/kg/day (5 %) and increase of phosphorus in females receiving 50 mg/kg/day (48 %). Due to the absence of dose-relation or the minimal severity, these changes were considered of no toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes were observed in urine analysis.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item for both main groups and for recovery groups . No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated animals for both main groups and for recovery groups. No toxicological relevance was attributed to the statistically significant increase in motor activity indicated in treated males because it was related the low control value measured (when compared to all other control values in the study).
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The M-Adenocarcinoma in the mammary glands of one control female (no. X0730011), which correlated to the subcutaneous mass observed during necropsy, was considered spontaneous.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone determination for parental males: Thyroid Stimulating Hormone (TSH) was increased in a number of parental males dosed at 200 and 800 mg/kg/day. Compared with controls, the increases were 1.8 and 2.1-fold, respectively. No relevant changes of the thyroid hormones T3 or T4 were recorded. Since no related changes of T3/T4 levels were recorded and no thyroid findings were recorded at histopathological evaluation, the above finding was considered to be of no toxicological relevance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cycle was similar between treated and control animals.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted in spermatogenic cycle in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital intervals and copulatory index were similar between treated and control animals. Fertility index both for males and females was 100 % each in all treated groups and 80 % in control group. Two control males (no. X0730014 and X0730018) failed to induce pregnancy. These cases were considered incidental. Gestation periods were similar between treated and control group females. All pregnant dams gave birth on Day 22 post coitum (mean value). Corpora lutea, implantations, total litter size and pre-natal loss (percentage) were similar in control and treated groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect was observed during the study.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Apparently no food intake (milk), small appearance and cold to touch were the main clinical signs noted in control and treated pups. No toxicological significance was attributed to these findings which are commonly observed in the litters during the lactation period.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Found dead or missing pups were also observed both in control and treated groups. No toxicological significance was attributed to these findings which are commonly observed in the litters during the lactation period. No significant differences were observed in litter data at birth, on Days 1 (pup loss %), 4 (cumulative loss %) and 13 post partum (post natal loss %) of treated groups when compared to the control group.
Body weight and weight changes:
no effects observed
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratios at birth showed a statistically significant increase in number of female pups and, consequently, a significant decrease in a percentage of males in the high dose group. In addition, statistically significant decrease in percentage of male pups was also recorded in mid-dose group. This findings was considered incidental.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No differences were noted in thyroid weight between control and treated pups at day 14 post coitum.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Autolysis of all organs were mainly observed in pups dying during the lactation period. No abnormalities were recorded at the external and internal examination in culled pups at Day 4 postpartum or in pups sacrificed with dam on Day 14 post partum.
Other effects:
no effects observed
Description (incidence and severity):
No relevant changes was recorded in Thyroid hormone determination (T3, T4 and TSH) in pups on day 14 post partum.
The slight differences with significance at statistical analysis when compared to the control group, in longer AGD noted in male pups of mid- and high dose groups (200 and 800 mg/kg/day), were not considered relevant since it is a positive trend (more “masculine”) and not associated with adverse effects (i.e: feminisation). No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups in female pups.
Nipple count was performed on day 13 post partum in males pups and no nipple was observed. No nipple was observed also after euthanasia of the pups on day 14 post partum.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect was observed during the study.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on results in male and female Sprangue Dawley rats exposed to dose levels of 50, 200, and 800 mg/kg bw/day of test substance for at least 29 days, no observed adverse effect level (NOAEL) is 800 mg/kg bw/day for general toxicity and for reproduction/developmental toxicity for both males and females.
Executive summary:

The toxic effects on Sprague Dawley rats after repeated dosing with test item, as well as any effects of the substance on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated. A 4 week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. The vehicle was corn oil (2 ml/kg).

The test item was administered according to the following doses for the main groups: 0 mg/kg bw/day (group 1), 50 mg/kg bw/day (group 2), 200 mg/kg bw/day (group 3) and 800 mg/kg bw/day (group 4). Each main group was composed by 10 males and by 10 females.

The test item was administered according to the following doses for the recovery groups: 0 mg/kg bw/day (group 5), 50 mg/kg bw/day (group 6), 200 mg/kg bw/day (group 7) and 800 mg/kg bw/day (group 8). Each recovery group was composed by 5 males and by 5 females.

 

Main groups:

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29/30 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and postpartum periods until Day 13 post partum (for at least 51 days). The two not pregnant female were dosed up to the day before necropsy.

The following investigations were performed: mortality check, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), body weight, food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis in males), litter data, sex ratios, macroscopic observations and organ weights. Histopathological examination was performed in control and high dose groups (five animals/sex/group randomly selected), as well as on all abnormalities detected during post mortem observation at the end of treatment period. The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups.

Thyroid hormone determination was performed in all males.

Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of 1/pup/sex/litter (where possible) at Day 14 postpartum were determined.

All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 postpartum were subjected to an external examination. Sex was determined by internal gonads inspection.

All live pups sacrificed on Day 14 postpartum were examined for external abnormalities and sex confirmation by gonadal inspection.

 

Recovery groups:

Recovery animals (males) were treated for up to 4 consecutive weeks and killed after 4 weeks of recovery period. Recovery animals (females) were treated for up to 6 weeks, when the majority of the main group females were killed. The females were sacrificed after 4 weeks of recovery period. The following parameters were evaluated in these animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, and macroscopic observations and organ weights.

 

Results:

- Mortality and fate of females: no mortality occurred throughout the study. Two females of the control group were found not pregnant at necropsy. The number of females with live pups on Day 14 post partum was: 8 in the control, 10 in the low dose, 10 in the mid-dose and 9 in the high dose group.

- Clinical signs, neurotoxicity assessment including motor activity, grip strength, sensory reaction to stimuli for main and recovery groups: no significant clinical signs were observed throughout the study in all treated main animals of both sexes. Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups.Recovery animals did not show any clinical signs during treatment, as well as during the recovery phase. Observation of treated animals at removal from the cage and in an open arena (neurotoxicity assessment), as well as, in motor activity, grip strength and sensory reaction to stimuli did not show differences between control and treated groups.

- Body weight and body weight gain for main and recovery groups: no difference in body weights and body weights gain were recorded in animals of both sexes compared to the control group, throughout the study, both for main and recovery groups.

- Food consumption: for main and recovery groups: no effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

- Haematology including coagulation for main groups: the changes observed in some haematological parameters were considered unrelated to treatment, due to the absence of dose-relation and the minimal magnitude.No changes were recorded in coagulation parameters evaluated.

- Clinical chemistry for main groups: fluctuations observed in some bio chemicals parameters were considered of no toxicological relevance due to the absence of dose-relation, low incidence or the minimal severity.

- Urinalysis for main groups: no changes were observed in urine analysis.

- Thyroid hormones: no treatment-related differences were noted in hormones levels in parental males and in pups at Day 14 post partum, when compared to control group animals.

- Oestrous cycle, reproductive parameters, pairing combination and mating performance: oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any differences that could be related to treatment.

- Implantation, pre-birth loss data and gestation length of females: gestation periods were similar between treated and control females. All pregnant dams gave birth on Day 22 post coitum (mean value). Corpora lutea, implantations, total litter size and pre-natal loss (percentage) were similar in control and treated groups.

- Litter data at birth, on Day 1 and Day 4 postpartum (before culling) and on Day 13 (after culling) postpartum and sex ratios: litter data and sex ratios were unaffected by treatment.

- Clinical signs of pups: there were no compound-related effects. Pre-weaning clinical signs were comparable between treated and control groups.

- Anogenital distance: no negative effects were seen between the control and treated pups in anogenital distance.

- Thyroid weight of pups at Day 14 postpartum: no treatment related changes were seen between control and treated pups.

- Terminal body weight and organ weights for main and recovery groups: no changes were observed in terminal body, absolute and relative organ weight of treated animals, when compared to the controls.

- Macroscopic observations for main and recovery groups: no remarkable differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery period, when compared with controls.

- Microscopic observations: no treatment-related changes were noted in animals sacrificed at the end of the treatment period. In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

Note: the neurotoxicity assessment on parental animals was reported in this IUCLID dossier section as additional info but it is not relevant for reproductive / developmental toxicity screening.

 

Conclusion:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for reproductive/ developmental toxicity (and also for general toxicity - see section 7.5.1 of this IUCLID dossier) was considered to be 800 mg/kg bw/day for both males and females.

 

Categories Display