Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Apr 2021 to 17 Sep 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetramethylthiuram monosulphide
EC Number:
202-605-7
EC Name:
Tetramethylthiuram monosulphide
Cas Number:
97-74-5
Molecular formula:
C6H12N2S3
IUPAC Name:
N,N-dimethyl[(dimethylcarbamothioyl)sulfanyl]carbothioamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague-Dawley was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: males - 11 weeks; females - 12 weeks
- Weight at study initiation: males - between 271 and 383 g; females - between 207 and 289 g
- Housing: The animals were single housed in polycarbonate cages containing appropriate bedding. The details of animals division between the cages can be found in Table 1 of "Any other information on materials and methods incl. tables" section. Individual housing of F0 animals was chosen as group housing for pregnant animals can adversely affect gestation and lactation, and to avoid aggressive behavior around mating. Toward the end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material.
- Diet: SSNIFF rat/mouse pelleted maintenance diet, ad libitum except during designated procedures
- Water: tap water filtered with a 0.22 µm filter, ad libitum
- Acclimation period: Yes
- Animal enrichment: For psychological/environmental enrichment, animals were provided with rat hut, nylabone and wooden log, except when interrupted by study procedures/activities.

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. Periodic analyses of water were performed by the Testing Facility. It was considered that there were no known contaminants in the feed or water that interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS (targeted)
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes: 8 to 15 filtered, non-recycled air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 July 2021 To: 17 Sep 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared according to stability data and divided into aliquots.

VEHICLE
- Concentration in vehicle: 0, 1, 2 and 6 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples (3 samples per dose level) were collected for analysis on Week 1, 3 and
7 from each dose group. Dose analysis was performed prior to dose administration at each occasion. All samples analyzed were collected by the analytical laboratory for same day analysis, where possible, or stored for analysis within known formulation stability period. Analyses were performed by HPLC/UV using a validated analytical procedure.
Duration of treatment / exposure:
Males: 7 weeks
Females: 7 - 9 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale (please see "Any other information on materials and methods incl. tables" section for details): The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study performed in the same species in which the test item was administered daily by gavage to male and female Sprague-Dawley rats at dose levels of 30, 50, 100 or 250 mg/kg bw/day in corn oil for 14 days. The dosing volume was 10 mL/kg bw/day from 50 mg/kg bw/day and was 5 mL/kg bw/day at 30 mg/kg/day. The dose levels of 50, 100 and 250 mg/kg bw/day were considered to exceed the Maximum Tolerated Dose (MTD) under the experimental conditions of the study based on the severity of findings and/or on the absence of recovery throughout the 14-day study duration. Therefore, 30 mg/kg bw/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 2- or 3-fold interval (i.e. 5 and 10 mg/kg bw/day).
- Rationale for animal assignment: During the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition and allocated to groups (by sex) according to randomization procedure based on body weight, so that the average body weight of each group was similar. In addition, only females with regular estrous cycles were allocated to the groups (regularity of estrous cycles being confirmed 2 days before the beginning of the dosing period). At the beginning of the dosing period, the weight variation of animals did not exceed ± 20% of the mean weight of each sex.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily beginning upon arrival through termination

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once before the beginning of the treatment period and then at least once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: At least 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 of mated females: GD 0, 4, 7, 11,14, 17 and 20, PND 1, 4, 7, 10
and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly; from Day 1 until the start of the mating period for all
animals and after the end of the mating period (males only);
- Time schedule for examinations of mated females: GD0-4, GD4-7, GD7-11, GD11-14 and GD14-20
PND1-4, PND4-7, PND 7-10 and PND10-13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of euthanasia
- Anaesthetic used for blood collection: isofluorane
- Animals fasted: Yes, o/n
- How many animals: First five males and lactating females
- Following parameters were examined: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count, Platelet count,
White blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count
Basophil count, Large unstained cells count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of euthanasia
- Animals fasted: Yes, o/n
- How many animals: First five males and lactating females
- Following parameters were examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, Total bilirubin, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride

PLASMA HORMONES: Yes, thyroid hormone (T4) and thyroid stimulating hormone (TSH)
- Time of blood sample collection: All F0 males and females: PND 14 and at termination; pups: PND 4 and PND 13 (2 pups per litter)
- Animals fasted: F0 animals only
The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for pups sampled on PND 13 and for F0 males sampled at termination. Plasma samples obtained on PND 4 from pups and on PND 14 from F0 females were kept at -80°C pending possible analysis. As no relevant changes were noted in PND 13 pups and F0 males, no further analyses were performed.

URINALYSIS: Yes
- Time schedule for collection of urine: day of euthanasia
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume, Color, Appearance/Clarity, Specific gravity, pH, Protein, Glucose, Bilirubin, Ketones, Blood, Nitrites

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the dosing period (for females this was performed on PND 13 after euthanasia of the pups)
- Dose groups that were examined: The first five males and lactating females from each group euthanized as scheduled
- Battery of functions tested: 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, horizontal and vertical movements

OTHER:
- Post-dose observations: In addition to the daily cage side observation, animals were observed between 1 and 3 hours post-dose on the days of dosing
Oestrous cyclicity (parental animals):
Oestrous cyclicity was determined from a fresh vaginal lavage each morning during the 2 weeks of the pretreatment period, from the beginning of the dosing period during the pre-mating and mating periods until the females were mated and on the day of necropsy just before euthanasia (unscheduled or scheduled), to allow correlation with reproductive organs histopathology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of pups born (live, dead and cannibalized) on PND 1, external abnormalities of the pups, number and sex of pups alive on PND 1, 4, 7, 10 and 13, daily clinical signs and abnormal behavior, individual body weight of live pups on PND 1, 4, 7, 10 and 13, anogenital distance (AGD) on PND 1, number of nipples and of areolae in male pups on PND12, external and necropsy findings of dead pups

The first clinical examination (on Day 1 p.p.) included:
- 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.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals; following completion of the gestation period (a minimum of 28 days of administration)
- Maternal animals: All surviving animals - PND 14 or in case of females with litter loss - after the last pup was found dead or missing

GROSS NECROPSY
- Gross necropsy consisted of macroscopic post-mortem examination of the principal thoracic and abdominal organs (Unscheduled or Scheduled euthanasia). This included examination of the
external surfaces and all orifices; the cranial cavity and 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. Number of Corpora lutea and implantation sites were recorded.

ORGAN WEIGHTS
The organs detailed in Table 1 ("Any other information on materials and methods incl. tables") were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight recorded immediately before euthanasia) was calculated.

HISTOPATHOLOGY
Representative samples of tissues identified in Table 2 ("Any other information on materials and
methods incl. tables") were collected and preserved. Histopathological evaluation was performed
on all tissues listed in Table 2 (except Harderian glands) from the first five euthanized as scheduled males and lactating females of the control and high-dose groups, all macroscopic lesions of all groups, all tissues listed in Table 3 from all animals that died or were euthanized prematurely. Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Postmortem examinations (offspring):
SACRIFICE
- Time schedule: PND13

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs,
comprising also a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs and the internal reproductive organs

HISTOPATHOLOGY / ORGAN WEIGTHS
The body weight of 1 selected pup/sex/litter (or of 2 pups from the same sex when there was only one sex in the litter) euthanized on PND 13 was recorded before euthanasia. Thyroids with parathyroids of the selected pups were weighed after fixation.
Statistics:
Data are expressed as group mean values ± standard deviation (body weight, body weight change,
food consumption, number of corpora 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. 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).
Statistical analysis of hematology, blood biochemistry, coagulation, urinalysis, hormones, anogenital distance, nipples/areolae, live birth, viability and lactation indexes, sex-ratio,
post-implantation loss and motor activity data were performed by Provantis software and PATHDATA software was used to perform the statistical analysis of organ weight data (see also pictures attached).
Reproductive indices:
See "Any other information on materials and methods incl. tables" section
Offspring viability indices:
See "Any other information on materials and methods incl. tables" section

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day, test item-related clinical signs consisted of decreased activity in 4/10 males during the premating period, thin appearance in 3/5 surviving females from the premating period until the beginning of the lactation period, erected fur in all males from the premating period until Week 7 and in all females until the lactation period, hunched posture in 2/10 males from the premating period until Week 5 and in all females until the lactation period, and/or abdominal breathing in 7/10 males and 3/5 surviving females on a few occasions at the beginning of the pre-mating period.
Although these test item-related findings were transient and no longer observed at the end of the study, they were considered to be adverse in both sexes as they were associated with adverse changes in mean body weight, mean body weight changes and/or mean food consumption.
Hypersalivation was also noted from 10 mg/kg bw/day in both sexes with a dose-related incidence. This clinical sign is commonly observed when a test item is administered by gavage and it was therefore not considered to be adverse.
All other clinical signs noted during the study (i.e. chromorhynorrhea, chromodacryorrhea, alopecia, scabs, thin fur, wound, missing teeth) were considered incidental as they were occasional, observed with no dose-relationship and/or are commonly observed in laboratory rats of this strain and age.
Mortality:
mortality observed, treatment-related
Description (incidence):
At the dose level of 30 mg/kg bw/day 5 out of 10 females were euthanized or died prematurely. The cause of death for two of the females was considered to be the ulcers observed in the stomach mucosa in a context of dystocia in one of them. The first females was found dead on GD 24. Erected fur and hunched posture were noted prior to death. At macroscopic post-mortem examination, 12 dead fetuses were observed in the uterine horns, and blackish and discoloration along with depressions was noted in the stomach. As this female was pregnant without delivery on GD24, a relationship between difficulties to deliver (dystocia) and test item related effects could not be ruled out. At microscopic examination slight multifocal erosion/ulcers were observed in the stomach and correlated with the macroscopic findings. The ulcers were considered to be test item related and the cause of the poor condition observed in this animal prior to death.
The second females was prematurely euthanized on PND 2 for ethical reasons (erected fur, hunched posture, pallor of extremities, prostration and thin appearance). At macroscopic post-mortem examination, 16 implantation scars were recorded in the uterine horns, the thymus was reduced in size and blackish discoloration along with depressions was noted in the stomach. At microscopic examination, moderately decreased lymphoid cellularity was observed in the thymus and slight multifocal erosion/ulcers were observed in the stomach. Both findings were consistent with the macroscopic findings observed in the same tissues. The findings in the thymus were considered to be secondary to the poor clinical condition and associated stress. The stomach ulcers were considered to be related to the test item and the cause of the poor condition in this animal and therefore its prematurely euthanasia. Thirteen pups (5 found dead and 8 moribund) were found in the bedding. All showed absence of milk in the stomach, and dilatation of the stomach was noted in one of them. The cause of the death for 3 other females was undetermined. No unscheduled deaths were observed in the other female groups. There were no unscheduled deaths in males at any dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pre-mating (males and females) and post-mating (males):
In males at 30 mg/kg bw/day and when compared with controls, there was an overall moderately low mean body weight gain during the treatment period (+120 g vs. +166 g in controls; p<0.01), mainly due to a markedly lower body weight gain over the first week of the study period (+5g vs. +42 g in control; p<0.001). This correlated with lower mean food consumption and signs of poor clinical condition and was therefore considered to be test item-related and adverse. In females at 30 mg/kg bw/day and when compared with controls, a marked mean body weight loss (-16 g vs. +2 g in controls; p<0.001) was recorded over the first week of the treatment period. Mean body weight gain returned towards control values in the second week of the premating period. This finding correlated with low dose-related mean food consumption and resulted in an overall lower mean body weight gain throughout the premating period (-7 g vs. +4 g in controls; p<0.01) and in a lower mean body weight on Day 8 (-8% vs. controls). This effect was therefore considered to be test item-related and adverse based on the severity of the differences.
In males at 10 mg/kg bw/day and when compared with controls, a slightly lower mean body weight gain was noted over the first week of treatment (+33 g vs. +42 g in controls; not statistically significant). This test item-related effect was not considered to be adverse based on the low magnitude of the difference. No relevant differences from controls were observed in females at 5 and 10 mg/kg bw/day.

Gestation period:
At 30 mg/kg bw/day and when compared with controls, there was an overall statistically significant low mean body weight gain (+93 g vs. +159 g in controls between GD 0 and GD 20) resulting in statistically significant low mean body weights throughout the whole gestation period (+336 g vs. +426 g in controls on GD 20; -21%). At 10 mg/kg/day and when compared with controls, there was statistically significant low mean body weight gains at the beginning (GD 0 to GD 4) and at the end of the gestation period (GD 17 to GD 20) resulting in an overall statistically significant low body weight gain during the whole gestation period (+119 g vs. +159 g in controls) and in a statistically significant low mean body weight at the end of gestation (385 g vs. 423 g in controls; -9%). At 5 mg/kg/day and when compared with controls, there was a statistically significant low mean body weight gain between GD 7 and GD 11 resulting in a statistically significant low body weight gain during the whole gestation period (+133 g vs. +159 g in controls) and in a low body weight at the end of gestation (393 g vs. 423 g in controls; -7%; not statistically significant).
These changes were attributed to the test item and were considered to be adverse at 10 and 30 mg/kg bw/day due to their severity and as they correlated with dose-related effects on mean food consumption.

Lactation period:
At 30 mg/kg bw/day and when compared with controls, there were statistically significant low mean body weights that corresponded to the previous low mean body weights of the gestation period. The same severity persisted on PND 4 with a trend to a return to normal values from PND 7 as females gained more weight than controls over the lactation period. At 10 mg/kg bw/day and when compared with controls, a statistically significant low mean body weight was recorded on PND 1 only. This difference corresponded to the previous low mean body weights recorded during the gestation period and was no longer observed during the lactation period as females gained more weight than controls over the lactation period.
Changes in the mean body weight were attributed to the test item and were considered to be adverse at 30 mg/kg bw/day due to their severity and as they correlated with dose-related effects on mean food consumption
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Pre-mating (males and females) and post-mating (males) period:
In males, at 30 mg/kg bw/day and when compared with controls, markedly low mean food consumption (-31%; p<0.001) was noted in the first week of treatment. This correlated with a low mean body weight gain. This effect was therefore considered to be test item-related and adverse based on the severity and as it also impacted mean body weight and overall mean body weight change.
In females, at 30 mg/kg bw/day and when compared with controls, severely low mean food consumption was recorded in the first week of treatment ( 41%; p<0.001). This correlated with body weight loss. The same trend, but less pronounced, was recorded in the second week of treatment (-13%; p<0.05). These effects were considered to be test item-related and adverse based on their severity and as they also impacted mean body weight and body weight change during the premating period.
At 10 mg/kg bw/day, the same tendency to low mean food consumption was observed in females in the first week of treatment. However, these changes were not considered to be adverse as they were less pronounced and not statistically significant. There were no relevant changes in males at this dose level.
At 5 mg/kg bw/day, there were no changes on mean food consumption in males and females.

Gestation period:
From 10 mg/kg bw/day and when compared with controls, slight to marked, dose-related, low mean food consumption was recorded on some occasions at 10 mg/kg bw/day (down to -23%; p<0.001) and throughout the gestation period at 30 mg/kg bw/day (down to -35%; p<0.001). This correlated with dose-related low mean body weight gains. These effects were considered to be test item-related and adverse based on their severity and as they also impacted mean body weight and body weight change during the gestation period.
At 5 mg/kg bw/day and when compared with controls, slightly low mean food consumption was observed on some occasions but without statistically significance. This change was considered to be test item-related as this correlated with slightly lower mean body weight gain. As this difference was of minimal magnitude and slightly affected the mean body weight, this test item-related effect was not considered as adverse.

Lactation period:
At 30 mg/kg bw/day and when compared with controls, markedly low mean food consumption was recorded throughout the lactation period (down to -39%; p<0.001). These dose-related effects were considered to be test item-related and adverse based on their severity.
At 10 and 5 mg/kg bw/day and when compared with controls, the same tendency to low mean food consumption (from -8% to -19%) was observed during the lactation period. These differences were considered to be test item-related but not adverse as they were less pronounced, not statistically significant and did not impact the mean body weight or mean body weight gain. At 5 mg/kg bw/day, the statistically significance observed from PND 10 to PND 13 (-19%; p<0.05) was mainly due to the contribution of one litter.
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):
At 30 mg/kg bw/day and when compared with controls and/or historical control data, red blood cell count (males: -22.8% and females: -24.9% vs. controls; p<0.001), hemoglobin level (males: -13.9% and females: -17.5% vs. controls; p<0.001) and/or hematocrit (males: -13.9% and females: -16.7% vs. controls with p<0.01 and p<0.001, respectively) were slightly to moderately decreased in males and females. Mean cell volume (males: +10.7% and females: +11.4% vs. controls with p<0.001 and p<0.01, respectively) and mean corpuscular hemoglobin (males: +11.6% and females: +9.8% vs. controls with p<0.001 and p<0.05, respectively) were slightly increased in both sexes. Slight increases in absolute reticulocyte counts (males: +26.2% and females: +17.0%; not statistically significant) were also noted and correlated with the decrease in red blood cell mass.
At 10 mg/kg bw/day and when compared with controls and/or historical control data, the same trend, but less pronounced, was noted in the red blood cell count from males (-11.2% with p<0.01) and in the hemoglobin concentration and hematocrit (-7.6% and -7.7%, respectively with p<0.01) from females. At 5 mg/kg bw/day, there were no test item-related effects in males and females.
These differences were not considered to be adverse due to their slight magnitude and/or as they were not associated with adverse histopathological findings.
Other differences between control and test item-treated groups, including those that were statistically significant, were not dose-related, were of minimal magnitude and/or were consistent with normal biological variations, were not considered to be test item-related.
There were no test item-related effects on coagulation parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day and when compared with controls and/or historical control data, there were a slight increase in total bilirubin levels in males and females (2-fold with p<0.01 and 2.9-fold with p<0.001, respectively) and a moderately increase in cholesterol level in males (+59.4% with p<0.01). Slight, but not statistically significant, increases in mean urea (+22.1% vs. controls) and creatinine (+13.3% vs. controls) levels were noted in males. These differences were not considered to be adverse as they were not associated with adverse histopathological correlates.
At 10 and 5 mg/kg bw/day, there were no test item-related effects in males and females.
All other variations in blood biochemistry parameters, including those that were statistically significant, of low magnitude, of opposite trends, consistent with normal biological variations, observed with no dose relationship, and/or had no biological significance were considered as unrelated to the test item.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on thyroid hormone concentrations. Variations in thyroid hormone levels were of low magnitude (not statistically significant), were not consistent with each other, were observed with no dose relationship, were within the range of the historical control data and/or had no biological significance. They were therefore considered as unrelated to the test item.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day and when compared with controls and/or historical control data, moderate changes were observed in males, consisting of increased mean urinary volume (2.3-fold with p<0.05) associated with decreased gravity (-2% with p<0.01) and high mean pH (+8% with p<0.05). These findings were not considered to be adverse as they were of low magnitude and/or and did not correlate with any clinical signs and/or adverse histopathological correlates.
As the variations observed in mean urine volumes and mean pH of females at all dose levels were poorly dose-related and/or were not statistically significant and did not correlate with any other findings, these differences were not considered to be test item-related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item treatment-related findings at FOB. Low mean landing foot splay and rectal temperature values were recorded in females at 10 and 30 mg/kg bw/day. They were considered to be of no toxicological importance as they were poorly dose-related, were not statistically significant and/or did not correlate with any other findings.
There were no dose-related relevant differences between the test item-treated and control groups for motor activity (horizontal movements and rearing) in males or females.
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):
Erythrophagocytosis was observed in the mesenteric lymph node of 7/10 males and 4/5 females at 30/mg/kg bw/day, 1/10 males at 10 mg/kg bw/day and 1/10 female controls. Pigment was observed in the mesenteric lymph node of 2/10 males at 30 mg/kg bw/day. The erythrophagocytosis was characterized by erythrocytes rosetted around and/or phagocytized by sinus macrophages. The pigment was observed in sinus macrophages of the lymph node with a brown golden color consistent with hemosiderin.
Slightly decreased cellularity was observed as diffusely distributed in the thymus of 3/5 females at 30 mg/kg bw/day.
No cycling activity was observed at microscopic evaluation of the female reproductive tract. This was consistent with the lactating stage of the females at the time of necropsy.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the species.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no test item-related effects on estrous cycle during the pre-mating period. At 30 mg/kg/day and when compared with controls, there was a slightly lower percentage of females with all estrous stages as a consequence of a slightly higher mean number of days of diestrus (+21%, not statistically significant). This was mainly due to the contribution of two females. In the absence of any associated findings on estrous cycle length, time taken to mate and fertility, a relationship to the test item was considered unlikely.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects on mating or fertility indices and all groups had similar pre-coital time intervals (number of days taken to mate).
At 30 mg/kg bw/day and when compared with controls, a slightly lower gestation index (90.0% vs. 100.0% in controls) was noted. This was due to the contribution of one female (found dead on GD 24 with dead fetuses in presence of severe maternal toxicity) for which a test item relationship could not be excluded. As this finding was isolated, it was not considered to be adverse. There were no effects on reproductive data.
At 30 mg/kg bw/day and when compared with controls, higher statistically significant mean duration of gestation was observed (p<0.05). As the difference was of slight magnitude and as individual values remained within the range of control values [21 -22 days] and the historical control data, this test item-related finding was not considered as adverse.

See "Any other information on results incl. tables" section.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day and when compared with controls, an adverse increased number of clinical signs due to lack of maternal care (thin appearance, generalized pallor, abnormal color of the body, blackish color of the abdomen, cold to the touch, abdominal breathing, dehydration, absence of milk in the stomach, stiffness and/or hypoactivity) was observed in pups, along with an increased incidence of litters affected (8/9 vs. 0/10 in controls).
Other findings (thinning of hair, hematoma, scab, wound, missing digit and/or short tail) recorded in pups during the lactation period were not dose-related, were of isolated occurrence and/or were common observations in this species and strain of rats maintained under the experimental condition of this study.
No test item-related external abnormalities were observed at any dose level. The tail was misshapen
in one pup at 30 mg/kg bw/day. As this finding was of isolated occurrence, it was considered to be incidental.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day and when compared with controls and/or historical control data, there were markedly low live birth and PND 4 viability indexes (-21.7% and -21% with p<0.01 and p<0.001, respectively). These findings that could be attributed to abnormal maternal behavior, were considered to be test item related and adverse.
There were no test item-related effects on pup viability at 5 or 10 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 30 and 10 mg/kg bw/day, in both sexes and when compared with controls, there were moderately to severely dose-related low mean pup body weights (down to -15% and -16% in male and female pups at 10 mg/kg bw/day and down to -40% and -38% in male and female pups at 30 mg/kg bw/day, respectively) over the whole lactation period, along with slight to severe, low mean body weight gains (statistically significant up to p<0.05 at 10 mg/kg bw/day and p<0.001 at 30 mg/kg bw/day). Taking into account the magnitude of the effects, these findings were considered to be adverse.
At 5 mg/kg bw/day, there were no effects on mean pup body weight or body weight change. Low mean body weights and mean body weight gains recorded at the end of the lactation period were considered to be incidental and not test item-related.
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):
no effects observed
Description (incidence and severity):
At 30 mg/kg bw/day in males and females, when compared with controls, the higher AGD/BW cube root ratio was considered to be related to the lower mean body weight. As AGD values remained within the range of the historical control data, they were not considered to be test item-related.
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no effects on sex ratio (mean percentage of males) at any dose level.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Generation:
F1
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: based on adverse effects (i.e. dystocia in 1/10 females) at 30 mg/kg/day in presence of severe maternal toxicity,

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Relevant for humans:
presumably yes

Any other information on results incl. tables

Reproductive Data


Pairing, Mating and Fertility Data (Parent Animals)


The summary of mating and fertility data is presented in the following table:


Table 1. Summary of Mating and Fertility Data












































































Dose level (mg/kg/day)



0



5



10



30



Number of animals paired (M+F)



10+10



10+10



10+10



10+10



Number of males mated



10



10



10



10



Number of females mated



10



10



10



10



Mean number of days taken to mate (a)



2.9



3.3



2.7



2.0



Females mating index (%)



100



100



100



100



Number of pregnant females



10



10



10



10



Fertility index (%)



100.0



100.0



100.0



100.0



Females with live pups



10



10



10



9



Gestation index (%)



100.0



100.0



100.0



90.0



M: male; F: female.


(a): no statistically significant difference vs. controls.


 


Delivery Data (Females F0)


The summary of reproductive and litter data is presented in the following table:


Table 2. Summary of Reproductive and Litter Data














































































Dose level


(mg/kg/day)



0



5



10



30



HCD [min.- max.]



Number of pregnant females



10



10



10



10



30



Number of females which delivered



10



10



10



9



30



Mean duration of gestation (days ± SD)



21.4±0.5



21.6±0.5



21.3±0.5



22.0*±0.0



[22.0-22.2]



Mean number of corpora lutea ± SD



16.7±1.5



16.3±2.5



15.6±1.8



15.6±4.0



[14.0-16.6]



Mean number of implantation sites ± SD



16.2±1.8



13.8±5.4



15.4±1.5



14.0±2.4



[13.4-14.4]



Mean pre-implantation loss (% ± SD)



3.0±5.8



16.4±29.5



1.1±2.3



7.9±11.6



[4.0-14.7]



Mean number of pups delivered ± SD



15.0±1.6



12.0±5.2



14.8±1.8



12.8±2.4



[10.7-12.3]



Mean post-implantation loss (% ± SD)



7.1±6.67



14.9±15.91



4.0±4.85



8.8±5.40



[16.7-22.5]



Statistical significance, *: p<0.05.


 


Observations of the Progeny During the Lactation Period


Mortality


The number of pups found dead or cannibalized and the number litters affected are presented in the table below:


Table 3. Incidence of Mortality









































Dose level (mg/kg/day)



0



5



10



30



Number of litters



10



10



10



9



Total number of pups on PND 1



150



120



148



115



Number of pups found dead


(litters affected)



4 (1)



0 (0)



3 (2)



34 (9)



Number of pups cannibalized


(litters affected)



0 (0)



3 (2)



0 (0)



8 (3)



 


Clinical Signs and External Abnormalities


Clinical signs recorded in F1 pups during the lactation period are presented in the following table:


Table 4. Clinical Signs or External Abnormalities in Pups During Lactation
(Number of Pups and Litters Affected per Group).


















































































































































Dose level (mg/kg/day)



0



5



10



30



Thin appearance



 



 



 



10 (1): PND 1-2



Generalized pallor



 



 



1 (1): PND 4



13 (1): PND 1



Cold to the touch



 



 



 



27 (3): PND 1-2



Abdominal breathing



 



 



 



3 (2): PND 1-13



Abnormal color of the body



 



 



 



9 (2): PND 2



Blackish color of abdomen



 



 



 



5 (1): PND 1-2



Dehydration



 



 



 



39 (6): PND 1-13



Absence of milk in the stomach



 



 



 



12 (5): PND 2-3



Stiffness



 



 



 



14 (2): PND 1-2



Hypoactivity



 



 



 



2 (2): PND 1-13



Thinning of hair



 



8 (1): PND 10-13



 



 



Hematoma (head)



4 (3): PND 1-3



1 (1): PND 1-2



2 (2): PND 1-4



 



Scab (eye, nose, mouth, shoulder, hindlimb)



2 (2): PND 2-5



1 (1): PND 1-3



1 (1): PND 4-13



1 (1): PND 4-8



Short tail



1(1): PND 1-4



 



 



 



Misshapen tail



 



 



 



1 (1): PND 1-13



Wound
(hindlimb, thorax, head)



1(1): PND 1-4



 



3 (1): PND 4



 



Missing digit



1(1): PND 3-4



 



 



 



Number of litters



10



10



10



9a



Number of affected litters [%]



4 [40]



3 [30]



3 [30]



8 [89]



(): in brackets, number of litters.


Duration: first day of apparition until last day of presence.


No statistical analysis performed.


a: including 3 females prematurely euthanized for ethical reasons on PND 1 or 2 and 1 female prematurely euthanized for dead litter on PND 3.


 


Pup Viability


The pup viability indexes are presented in the table below:


Table 5. Viability Indexes






































Dose level (mg/kg/day)



0



5



10



30



HCD



Live birth index (%)



97.5



94.3


(-3.3)



98.2


(+0.6)



76.2**


(-21.7)



93.9



PND 4 viability index (%)



97.5



93.0


(-4.6)



98.2


(+0.6)



76.8***


(-21.0)



93.2



PND 13 lactation index (%)



100.0



100.0


(0.0)



100.0


(0.0)



97.5


(-2.4)



96.3



(): in brackets, percentage (%) difference vs. controls.


Statistical significance, **: p<0.01 and ***: p<0.001.


HCD: Historical Control Data (OECD 421/422, 2016 to 2018, n = 3 studies); 


 


Pup Body Weight and Body Weight Change


Mean pup body weight and body weight change are presented in the following tables:


Table 6. Mean Body Weight (g)


































































































































Sex



Males



 



 



 



Females



 



 



 



Dose level
(mg/kg/day)



0



5



10



30a



0



5



10



30a



. PND 1



7.3



7.8


(+7)



6.2**


(-15)



5.1#


(-30)



6.9



7.3


(+6)



5.8#


(-16)



4.8#


(-30)



. PND 4 (preculling)



10.6



11.4


(+8)



9.0*


(-15)



6.9#


(-35)



10.3



10.5


(+2)



8.5*


(-17)



6.7#


(-35)



. PND 7



18.3



18.7


(+2)



16.0**


(-13)



10.9#


(-40)



17.7



17.4


(-2)



15.3*


(-14)



10.8#


(-39)



. PND 10



27.0



27.6


(+2)



23.9*


(-11)



16.8#


(-38)



26.4



25.7


(-3)



23.2*


(-12)



16.5#


(-38)



. PND 13



36.2



32.4


(-10)



32.6*


(-10)



22.5#


(-38)



35.6



30.3*


(-15)



31.7*


(-11)



22.0#


(-38)



Mean Body weight change (g)



. PND 1-4



+3.3



+3.6



+2.9



+1.6#



+3.3



+3.2



+2.8



+1.8*



 . PND 4 - 7 (preculling)



+7.7



+7.4



+6.8*



+4.0#



+7.5



+6.9



+6.7



+4.1#



. PND 7 - 10



+8.7



+8.9



+8.0



+5.9#



+8.8



+8.4



+7.9



+5.7#



. PND 10 - 13



+9.1



+7.3



+8.8



+6.2*



+9.2



+7.5



+8.7



+6.1*



(): in brackets, percentage (%) difference vs. controls.


a: 5 litters from PND 4


Statistical significance, *: p<0.05; **: p<0.01 and #: p<0.001.


 


Pup Sex Ratio


The mean percentages of male pups (sex ratio) are presented in the following table:


Table 7. Sex Ratio (Mean Percentage of Male Pups)






























Dose level (mg/kg/day)



0



5



10



30



HCD [min.- max.]



PND 1 (%)



52



43



50



58



PND 0: [41.9-50.5]



PND 13 (%)



49



43



49



60



 



 


Pup Development


Anogenital Distance


The mean anogenital distances (AGD) in pups are presented in the following tables:


Table 8. AGD in Male Pups on PND 1






























Dose level
(mg/kg/day)



0



5



10



30



HCD
[min.- max.]



AGD (mm)



3.8



3.9



3.7



3.8



[3.54-5.95]



AGD / (BW)1/3



2.0



2.0



2.0



2.2



[1.78-3.00]



AGD: anogenital distance on PND 1.


(BW)1/3: cube root of body weight recorded on PND 1.


Historical Control Data (HCD): anogenital distance on PND 1 in F1 generation on PND 1 performed in OECD 422 or 421 studies (n= 46 males). 


 


Table 9. AGD in Female Pups on PND 1






























Dose level


(mg/kg/day)



0



5



10



30



HCD
[min.- max.]



AGD (mm)



1.9



2.0



2.0



2.1



[1.64-3.90]



AGD / (BW)1/3



1.0



1.0



1.1



1.2*



[0.82-2.06]



AGD: anogenital distance on PND 1.


(BW)1/3: cube root of body weight recorded on PND 1.


Historical Control Data (HCD): anogenital distance on PND 1 in F1 generation on PND 1 performed in OECD 422 or 421 studies (n= 47 females) 


Statistical significance, *: p<0.05 (individual values were rounded at 0.1 mm nearest to perform statistical analysis).


 


Nipples and Areolae Numbers


The mean numbers of areolae and nipples in male pups on PND 12 are presented in the following table:


Table 10. Mean Numbers of Areolae and Nipples in Male Pups on PND 12.



























Dose level (mg/kg/day)



0



5



10



30



Mean number of areolae



0.0



0.0



0.0



0.0



Mean number of nipples



0.0



0.0



0.0



0.0



 


Parental animals


Table 11. Clinical Signs in F0 Males (Number of Animals Affected per Group)









































Dose level (mg/kg/day)



0



5



10



30



Nb. of males



10



10



10



10



Chromorhynorrhea



 



 



 



1/10


(Day 3)



Chromodacryorrhea



 



 



1/10


(Days 29 to 55)



 



Hypersalivation



 



 



1/10


(Day 37)



6/10


(Days 1 to 50)



 


























































































Dose level (mg/kg/day)



0



5



10



30



Nb. of males



10



10



10



10



Alopecia (forelimbs)



1/10


(Days 29 to 55)



1/10


(Days 36 to 55)



1/10


(Days 8 to 55)



2/10


(Days 15 to 55)



Scabs (interscapular region, neck region, forelimb)



1/10


(Days 22 to 55)



2/10


(Days 29 to 55)



1/10


(Days 29 to 55)



1/10


 (Day 15)



Thin fur



 



1/10


(Days 36 to 55)



 



1/10


(Days 29 to 55)



Fur erected



 



 



 



10/10


(Days 3 to 43)



Wound (interscapular region, neck region)



1/10


(Days 29 to 55)



1/10


(Days 29 to 50)



 



 



Missing teeth



 



1/10


(Days 27 to 29)



 



 



Decreased activity



 



 



 



4/10


(Days 1 to 14)



Hunched posture



 



 



 



2/10


(Days 1 to 35)



Abdominal breathing



 



 



 



7/10


(Days 3 to 4)



Number of affected animals



2/10



3/10



3/10



1010



Nb.: Number.


(): period of occurrence (Study Days).


Test item treatment-related clinical signs recorded in surviving females are presented in the following table.


Table 12. Clinical Signs in Surviving F0 Females (Number of Animals Affected per Group)


























































































Dose level (mg/kg/day)



0



5



10



30



Nb. of surviving females



10



10



10



5



Alopecia (forelimbs)



1/10


(Days 1 to 53)



3/10


(Days 1 to 55)



1/10


(Days 1 to 52)



2/5


(Days 15 to 53)



Chromorhynorrhea



 



 



1/10


(Day 50)



 



Hypersalivation



 



 



2/10


(Days 37 to 50)



5/5


(Days 15 to 50)



Scabs (interscapular region, hindlimb)



 



1/10


(Day 8)



2/10


(Days 1 to 36)



 



Thin fur



 



1/10


(Days 15 to 52)



 



1/5


(Days 13 to 43)



Fur erected



1/10


(Day 19)



1/10


(Day 19)



 



5/5


(Days 2 to 45)



Thin appearance



 



 



 



3/5


(Days 3 to 45)



Hunched posture



 



 



 



5/5


(Days 2 to 45)



Abdominal breathing



 



 



 



3/5


(Days 3 to 4)



Number of affected animals



2/10



5/10



6/10



5/5



Nb.: Number.


(): period of occurrence (Study Days).


 


Table 13. Mean Estrous Cycles





































































Dose level
(mg/kg/day)



0



5



10



30



. Number of cycles



2.9



2.9



2.9



2.7



. Cycle length (days)



4.2



4.0



4.0



4.6



. Rats cycling normally.



10


(100%)



8


(80%)



9


(90%)



8


(80%)



. Number of females with all stages



9


(90%)



10


(100%)



10


(100%)



8


(80%)



. Number of days of diestrus



4.7



4.6



4.3



5.7



. Number of days of proestrus



3.5



3.8



3.4



3.4



. Number of days of estrus



3.6



3.8



3.8



3.0



. Number of days of metestrus



3.2



2.8



3.5



2.9



 


Table 14. Relevant Hematology Findings
































































































































































Sex



Males



 



 



 



Females



 



 



 



Dose level
(mg/kg/day)



0



5



10



30



0



5



10



30



Red blood cells (T/L)



9.52


 



8.78


(-7.8)



8.46**


(-11.2)



7.36***


(-22.8)



7.55


 



7.36


(-2.5)



7.09


(-6.1)



5.67***


(-24.9)



HCD



9.14 [8.74-9.81]



 



 



 



8.37 [7.58-9.31]



 



 



 



Hemoglobin (g/dL)



16.0


[14.7-16.6]



15.2


(-5.0)



14.9


(-6.8)



13.8***


(-13.9)



15.3


 



14.9


(-2.6)



14.1**


(-7.6)



12.6***


(-17.5)



HCD



15.6 [14.7-16.6]



 



 



 



15.8 [14.0-17.5]



 



 



 



Hematocrit (L/L)



0.50


 



0.47


(-6.0)



0.47


(-6.4)



0.43**


(-13.9)



0.47


 



0.45


(-4.7)



0.43**


(-7.7)



0.39***


(-16.7)



HCD



0.48 [0.46-0.51]



 



 



 



0.48 [0.43-0.52]



 



 



 



Mean Cell Volume (fL)



53.1


 



53.9


(+1.6)



55.3


(+4.2)



58.8***


(+10.7)



62.1


 



60.6


(-2.3)



61.2


(-1.4)



69.2**


(+11.4)



HCD



53.0 [50.2-54.9]



 



 



 



57.3 [54.7-61.3]



 



 



 



Mean Corpuscular Hemoglobin (pg)



16.8


 



17.3


(+3.3)



17.6


(+5.1)



18.7***


(+11.6)



20.3


 



20.3


(-0.1)



20.0


(-1.5)



22.3*


(+9.8)



HCD



17.0 [16.1-18.3]



 



 



 



18.9 [17.7-19.8]



 



 



 



Reticulocytes (T/L)



0.20


 



0.22


(+13.1)



0.20


(+1.0)



0.25


(+26.2)



0.27


 



0.18


(-31.8)



0.25


(-6.1)



0.32


(+17.0)



HCD



[na]



 



 



 



[na]



 



 



 



(): in brackets, percentage (%) difference vs. controls.


Statistical significance, *: p<0.05; **: p<0.01 and ***: p<0.001.


HCD: Historical Control Data [minimum and maximum values]


 


Table 15. Relevant Blood Biochemistry Findings.




















































































































Sex



Males



 



 



 



Females



 



 



 



Dose level (mg/kg/day)



0



5



10



30



0



5



10



30



Urea (mmol/L)



3.8



4.2


(+11.1)



4.0


(+5.8)



4.6


(+22.1)



7.8



6.7


(-13.6)



8.0


(+2.3)



7.6


(-2.8)



HCD



4.4 [3.0-7.7]



 



 



 



8.8 [5.1-13.8]



 



 



 



Creatinine (µmol/L)



26.99



30.00


(+11.1)



28.84


(+6.9)



30.58


(+13.3)



31.68



29.86


(-5.7)



32.31


(+2.0)



30.31


(-4.3)



HCD



34.36 [30.19-38.50]



 



 



 



38.70 [30.36-46.01]



 



 



 



Total Bilirubin (µmol/L)



1.08



0.87


(-19.8)



1.39


(+28.7)



2.12**


(x2)



1.10



0.90


(-18.5)



1.45


(+31.5)



3.16***


(x2.9)



HCD



0.69 [0.00-1.90]



 



 



 



0.80 [0.27-1.41]



 



 



 



Cholesterol (mmol/L)



1.60



1.62


(+0.9)



1.55


(-3.1)



2.56**


(+59.4)



2.73



2.20


(-19.4)



3.02


(+10.4)



2.93


(+7.2)



HCD



1.95 [1.41-2.43]



 



 



 



2.74 [1.97-3.38]



 



 



 



(): in brackets, percentage (%) or fold difference vs. controls.


Statistical significance, **: p<0.01 and ***: p<0.001.


HCD: Historical Control Data [minimum and maximum values]


 


Table 16. Relevant Urinary Findings






























































































Sex



Males



 



 



 



Females



 



 



 



Dose level
(mg/kg/day)



0



5



10



30



0



5



10



30



Volume (mL)


 



9.4



7.0


(-25.5)



10.8


(+14.9)



21.2*


(x2.3)



14.8



16.6


(+12.2)



17.6


(+18.9)



16.8


(+13.5)



HCD



8 [4-15]



 



 



 



19 [9-33]



 



 



 



pH



6.5



6.4


(-1.5)



6.8


(+4.6)



7.0*


(+7.7)



6.5



6.9


(+6.2)



6.8


(+4.6)



7.0


(+7.7)



HCD



na



 



 



 



na



 



 



 



Gravity



1.032



1.040


(+0.8)



1.032


(0.0)



1.016**


(-1.6)



1.039



1.034


(-0.5)



1.034


(-0.5)



1.032


(-0.7)



HCD



1039 [1030-1050]



 



 



 



1038 [1020-1050]



 



 



 



(): in brackets, percentage (%) or fold difference vs. controls.


Statistical significance, *: p<0.05 and **: p<0.01.


HCD: Historical Control Data [minimum and maximum values] 


na: not applicable


 


Table 17. Mean (± SD) T4 (ng/mL) and TSH (pg/mL) Concentrations in F0 and F1 animals






























































Dose level mg/kg/day)



0



30



100



300



HCD



T4



 



 



 



 



 



F0 males at termination



45.91±5.978



43.25±4.663


(-5.8)



46.00±2.494


(+0.2)



41.54±5.248


(-9.5)



38.44
[25.60-54.17]



PND 13 pups



35.81±3.242



38.89±6.164


(+8.6)



37.09±7.661


(+3.6)



31.53±3.622


(-11.9)



34.6 [20.1-49.9]



TSH



 



 



 



 



 



F0 males at termination



1348±813.3



1062±658.4


(-21.2)



967±418.0


(-28.3)



1082±686.3


(-19.7)



2283 [627-4455]



PND 13 pups



908±475.4



968±737.8


(+6.5)



760±408.4


(-16.3)



886±314.0


(-2.5)



1560 [421-4038]



(): in brackets, percentage (%) difference vs. controls. No statistical significance vs. controls.


HCD: Historical Control Data [minimum and maximum values] 


 


Table 18. Group Incidences of test item-Related Microscopic Observations in the Mesenteric lymph node and Thymus at Main Necropsy
































































































































































Finding



Male


Dosage (mg/kg/day)



 



 



 



Female


Dosage (mg/kg/day)



 



 



 



 



0



5



10



30



0



5



10



30



No. Animals



10



10



10



10



10



10



10



5



Mesenteric lymph node (examined)



5



1



2



9



5



0



0



5



Erythrophagocytosis



0



0



1



7



1



na



na



4



Grade 1 (minimal)



-



-



-



2



-



na



na



-



Grade 2 (slight)



-



-



-



5



1



na



na



4



Grade 3 (moderate)



-



-



1



-



-



na



na



-



Pigment



-



-



-



2



-



na



na



-



Grade 1 (minimal)



-



-



-



1



-



na



na



-



Grade 2 (slight)



-



-



-



1



-



na



na



-



Thymus (examined)



6



0



0



5



5



0



0



5



Decreased cellularity



0



0



0



0



0



0



0



3



Grade 2 (slight)



-



na



na



-



-



na



na



3



-: finding not observed, na: not applicable.


 


Dose Formulation Analyses


The test item concentrations in the administered test item dose formulations analyzed in Weeks 1, 3 and 7 remained within an acceptable range of variations (-6.4% to +4.3%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was observed in the control dose formulations.

Applicant's summary and conclusion

Conclusions:
Based on these results of the study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 10 mg/kg bw/day in males and 5 mg/kg bw/day in females based on adverse effects (i.e. mortality, clinical signs, low body weights and body weight changes and/or reduced food consumption) observed in males at 30 mg/kg bw/day and in females from 10 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 10 mg/kg/day, based on adverse effects (i.e. dystocia in 1/10 females) at 30 mg/kg bw/day in presence of severe maternal toxicity,
- the No Adverse Effect Level (NOAEL) for F1 development was considered to be 5 mg/kg bw/day, based on adverse findings (i.e. reduced live birth and viability indexes, increased clinical signs and/or lower body weights and body weight gains) observed in pups from 10 mg/kg bw/day in presence of maternal toxicity.
Executive summary:

The objectives of this study were to determine the potential toxicity of the test item, when administered by the oral (gavage) route for at least 28 days to rats, and to evaluate the potential of the test item to affect male and female reproductive performance (such as gonadal function, mating behavior, conception, parturition and early postnatal development). The study was performed according to the OECD TG 422 and in compliance with GLP.


Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, daily by the oral route (gavage) at 5, 10 or 30 mg/kg bw/day. Males were treated for 2 weeks before mating, throughout mating and then until the day before euthanasia (i.e. after a 7-week treatment period). Females were treated for an overall period of 7 to 9 weeks: 2 weeks before pairing, then throughout the mating and gestation periods until PND 13 (Post-Natal Day).


A control group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions.


The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 3 and 7 were determined using a validated HPLC/UV analytical method.


The following parameters and end points were evaluated in this study:


- for parent animals: mortality, clinical observations, functional tests, body weight, food consumption, estrous stages, clinical pathology (hematology, coagulation, clinical chemistry and urinalysis), T4 and TSH thyroid hormones, organ weights, and macroscopic and microscopic examinations,


- for progeny: mortality, clinical observations, abnormal behavior, physical development (i.e. anogenital distance and the number of nipples and areolae in male pups), body weight, T4 and TSH thyroid hormones and macroscopic examination.


F0 animals


The test item concentrations in the administered dose formulations analyzed for all test item-treated groups in Weeks 1, 3 and 7 remained within an acceptable range of variations (-6.4% to +4.3%) when compared with the nominal values (± 15% of the nominal concentrations). No test item was observed in the control dose formulations.


There were no unscheduled deaths in males.


At 30 mg/kg bw/day, on GD (Gestation Day) 24 or PND 1 or 2, four high dose females with poor clinical condition were prematurely euthanized or found dead. The cause of death for two out of them was considered to be ulceration in the stomach in a situation of dystocia for one of them. The cause of death in the two other females was undetermined. At the same dose level, on PND 3, one female was prematurely euthanized because of deaths in the litter that most probably resulted from a lack of maternal care. The early death of 5/10 females was considered to be test item-related and adverse due to the high incidence of premature deaths in this group.


Hypersalivation occurred from 10 mg/kg bw/day in both sexes with a dose-related incidence, but was not considered to be adverse as this finding is commonly observed after gavage administration. At 30 mg/kg bw/day, decreased activity was noted in 4/10 males during the premating period and thin appearance was observed in 3/5 surviving females from premating until the beginning of the lactation period. This was accompanied by fur erected in all animals and hunched posture (2/10 males and 5/5 surviving females) and/or abdominal breathing (7/10 males and 3/5 surviving females) from the premating period; these clinical signs were no longer noted at the end of the study, but were considered to be adverse in a context of low body weight gain and low food intake.


There were no test item-related CNS abnormalities at functional tests, detailed clinical examination, reactivity to manipulation/different stimuli and/or motor activity.


When compared with controls, there was a markedly lower body weight gain (p<0.001) in males or body weight loss (p<0.001) in females at 30 mg/kg bw/day over the first week of the pre-mating period. Slightly to markedly lower body weight gains (up to p<0.001) were recorded in females from 10 mg/kg bw/day during the gestation period and an overall higher mean body weight gain (not statistically significant) was recorded in females from 10 mg/kg bw/day during the lactation period. These variations resulted in lower final body weights in females at 10 and 30 mg/kg/day at the end of the gestation period (-9% and -21% vs. controls, with p<0.05 and p<0.001, respectively) and in females at 30 mg/kg bw/day at the end of the lactation period (-12% vs. controls, p<0.05). The overall body weight gain (+120 g vs. +166 g in controls; p<0.01) and the final body weight were also affected in males (-9%, not statistically significant) at 30 mg/kg bw/day.


These findings correlated with slight to severe (p<0.001) lower food consumption when compared with controls, namely in males and females at 30 mg/kg bw/day during the premating period, in females from 10 mg/kg bw/day during the gestation period and in females at 30 mg/kg/day during the lactation period . These changes (i.e. body weight loss, and low body weight gains, body weights and food consumption) were considered to be adverse.


Other differences from controls were observed (i.e. low body weight gain in females at 5 mg/kg bw/day and in males at 10 mg/kg bw/day and low food consumption in males at 5 and 10 mg/kg bw/day). As these variations were of minimal magnitude and/or did not impact the body weight, they were not considered to be adverse.


Changes among hematological parameters consisted of slightly to moderately lower red blood cell count (p<0.01 to p<0.001) in males from 10 mg/kg bw/day and in females at 30 mg/kg bw/day, slightly to moderately lower hemoglobin level and hematocrit (p<0.01 to p<0.001) in females from 10 mg/kg bw/day and in males at 30 mg/kg bw/day, slightly higher mean cell volume (p<0.01 to p<0.001) and corpuscular hemoglobin (p<0.01 to p<0.001) in males and females at 30 mg/kg bw/day. Reticulocyte counts (not statistically significant) were slightly increased in both sexes at 30 mg/kg bw/day, suggesting an ongoing regeneration of the red blood cells.


The test item induced moderately to markedly changes at 30 mg/kg bw/day among blood biochemistry parameters when compared with controls, namely higher total bilirubin concentration in males and females ( p<0.01 and p<0.001, respectively) and higher cholesterol level in males (p<0.01). Slightly higher urea (not statistically significant) and creatinine concentrations (not statistically significant) were also observed in males at 30 mg/kg bw/day.


Test item-related changes in urinary parameters were observed in males at 30 mg/kg/day and consisted of moderately, but statistically significantly higher pH (p<0.05) and higher urinary volumes (p<0.05) along with lower specific gravity (p<0.01).


None of the changes observed in clinical pathology parameters were considered as adverse in view of their low magnitude and/or the absence of adverse histopathological correlates.


There were no test item-related effects on coagulation parameters in parental animals or on mean T4 and TSH plasma concentrations in parental males at any dose level.


There were no effects on the estrous cycle during the pre-mating period at any dose level. There were no effects on mating or fertility indexes, on pre-coital time intervals (number of days taken to mate) or on reproductive and litter data at any dose level.


The test item induced a slightly lower gestation index (90.0% vs. 100.0% in controls, not statistically significant) as well as a slightly prolonged gestation period (22.0 vs. 21.4 in controls with p<0.05) at 30 mg/kg/day. As these findings were of slight magnitude and/or as individual values were within the range of the control values [21 -22 days] and the historical control data, they were not considered to be adverse.


At final sacrifice of females at 30 mg/kg/day lower body and thymus weights (-15% vs. controls with p<0.01) were observed when compared with concurrent controls. The lower thymus weight correlated with decreased thymus in 4/5 females and decreased lymphoid cellularity in 3/5 females. The thymus findings were considered to be secondary to stress and not adverse.


Minimal to moderate erythrophagocytosis in the mesenteric lymph node was observed in 7/10 males and 4/5 females at 30/mg/kg bw/day, 1/10 males at 10 mg/kg bw/day and 1/10 female controls. Minimal to slight pigment was observed in the mesenteric lymph node of 2/10 males at 30 mg/kg bw/day. The erythrophagocytosis and pigment correlated with red or brown discoloration in the mesenteric lymph node. These findings were not considered to be adverse.


Pups


When compared with controls, there were significant reductions in the live birth (-21.7% vs. controls with p<0.01) and viability (-21.0% vs. controls with p<0.001) indexes, marked increases in the number of pups found dead and cannibalized as well as in the incidence of litters affected (9/9 and 3/9 vs. 1/10 in controls, respectively) at 30 mg/kg bw/day. These findings, which could have resulted from abnormal maternal behavior, were considered to be adverse.


At the same dose level and when compared with controls, increased clinical signs due to lack of maternal care (thin appearance, generalized pallor, abnormal color of the body, blackish color of the abdomen, cold to the touch, abdominal breathing, dehydration, absence of milk in the stomach, stiffness and/or hypoactivity) were observed in pups, along with an increased number of litters affected (8/9 vs. 0/10 in controls). These findings were considered to be adverse.


When compared with controls, there were slightly to severely dose-related lower body weight gains in pups during the lactation period from 10 mg/kg/day (statistically significant up to p<0.05 at 10 mg/kg bw/day and p<0.001 at 30 mg/kg bw/day). This resulted in moderately to severely lower final body weights from 10 mg/kg/day (down to -16% with p<0.001 in female pups at 10 mg/kg bw/day and down to -40% in male pups with p<0.001 at 30 mg/kg bw/day). These findings were considered to be adverse.


There were no test item-related effects on mean body weight or mean body weight change in pups at 5 mg/kg bw/day.


There were no test item-related effects on pup viability at 5 or 10 mg/kg bw/day, no test item external abnormalities or macroscopic lesions and no effects on sex ratio, mean anogenital distance or areolae/nipples in male pups at any dose level.


There were no test item-related effects on mean T4 and TSH plasma concentrations on PND 13 pups at any dose level.


In conclusion and based on these results:


- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 10 mg/kg bw/day in males and 5 mg/kg bw/day in females based on adverse effects
(i.e. mortality, clinical signs, low body weights and body weight changes and/or reduced food consumption) observed in males at 30 mg/kg bw/day and in females from 10 mg/kg bw/day,


- the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 10 mg/kg bw/day, based on adverse effects (i.e. dystocia in 1/10 females) at 30 mg/kg bw/day in presence of severe maternal toxicity,


- the No Adverse Effect Level (NOAEL) for F1 development was considered to be 5 mg/kg bw/day, based on adverse findings (i.e.  reduced live birth and viability indexes, increased clinical signs and/or lower body weights and body weight gains) observed in pups from 10 mg/kg bw/day in presence of maternal toxicity.