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Administrative data

Description of key information

An oral (gavage) combined repeated dose study with a repro/developmental toxicity screening was performed in rat according to OECD TG 422 and in accordance with GLP. 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 bw/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined repeated dose toxicity study with the reproduction/developmental toxicity screening
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October, 2008
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421. Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) 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 perfomed 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
Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because this is a route of administration which is requested by the Regulatory Authorities for this type of test item.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared according to stability data and divided into aliquots.
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 during 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
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 (for details please see "Any other information on materials and methods incl. tables" section): 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 bw/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.
Observations and examinations performed and frequency:
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, Activated partial thromboplastin time, Fibrinogen, Prothrombin time

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/SERUM 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: Not specified
The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for F0 males sampled at termination. Plasma samples obtained 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, o/n
- 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

ESTROUS CYCLICITY: YES
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 am): during the 2 weeks of the pre-treatment period, from the beginning of the dosing period during the pre-mating and mating periods, until the females were mated, on the day of necropsy just before euthanasia (unscheduled or scheduled), to allow correlation with reproductive organs histopathology. Those smears were stained with Harris Schorr.


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
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all animals (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 2 ("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: Yes
Representative samples of tissues identified in Table 3 ("Any other information on materials and methods incl. tables") were collected and preserved. Histopathological evaluation was performed on all tissues listed in Table 3 (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.
Statistics:
Data are expressed as group mean values ± standard deviation (body weight, body weight change, food consumption). 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 and motor activity data were performed by Provantis software and organ weight data by PATHDATA software according to the sequences attached as picture.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg/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/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/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 (+93g vs. +159g 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 (385g vs. 423g in controls; -9%). At 5 mg/kg bw/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
Description (incidence and severity):
Statistically significant lower absolute and relative to body thymus weights were observed in the terminal sacrifice females at 30 mg/kg bw/day when compared to concurrent controls. Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Brown discoloration was observed in the mesenteric lymph node of 1/10 males at 5 mg/kg bw/day, 1/10 males at 10 mg/kg bw/day and 5/10 males and 1/5 females at 30 mg/kg bw/day. The brown discoloration of the mesenteric lymph node in the male at 5 mg/kg/day had no microscopic correlations.
Red discoloration was also observed in the mesenteric lymph node of 1/10 males at 10 mg/kg/day and 4/10 males and 1/5 females at 30 mg/kg bw/day. Black discoloration was observed in the mesenteric lymph node of one female at 30 mg/kg bw/day.
Reduced size in the thymus was observed in 4/5 females at 30 mg/kg bw/day.
The remaining macroscopic findings were not test item-related effects as they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.
Neuropathological findings:
not examined
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
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Effects on oestrous cycle: There were no test item-related effects on estrous cycle during the pre-mating period. At 30 mg/kg bw/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.

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Critical effects observed:
no

Table 1. Clinical Signs in 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 2. Clinical Signs in Surviving 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 3. 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



No statistically significant differences vs. controls.


 


Table 4. 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 5. 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 6. 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 7. Mean (± SD) T4 (ng/mL) and TSH (pg/mL) Concentrations














































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]



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]



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


HCD: Historical Control Data [minimum and maximum values] 


 


Table 8. 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.

Conclusions:
An oral (gavage) combined repeated dose study with a repro/developmental toxicity screening was performed in rat according to OECD TG 422 and in accordance with GLP. 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 bw/day.
Executive summary:

An oral (gavage) combined repeated dose study with a repro/developmental toxicity screening was performed in rat according to OECD TG 422 and in accordance with GLP. Based on these results of the study:


Administration of Tetramethylthiuram monosulphide at 30 mg/kg/day for 7 to 9 weeks was associated with early euthanasia of 5 females at this dose level. The cause of premature euthanasia or death in two of these females was considered to be related to the ulcers observed in the stomach, in a situation of dystocia in one of them. The ulcers were considered to be test item related and a test item relationship could not be excluded for dystocia as this finding was noted at the high dose level only in a context of prolonged gestation period.


Adverse clinical signs were observed at 30 mg/kg/day and consisted of decreased activity, erected fur, hunched posture and/or abdominal breathing in both sexes. These signs were noted together with adverse body weight changes, in males at 30 mg/kg/day and in females at 10 and 30 mg/kg/day leading to adverse lower final body weights in males (-9% vs. controls; not statistically significant) at the end of the study period and in females at the end of the gestation period (-9% and -21% vs. controls at 10 and 30 mg/kg/day, with p<0.05 and p<0.001, respectively) and the end of the lactation period (-12% vs. controls at 30 mg/kg/day, with p<0.5), accompanied by sporadic or continuous adverse lower food consumption.

The clinical pathology changes (lower red blood cell count, lower hemoglobin and hematocrit levels, higher mean cell volume and corpuscular hemoglobin, higher total bilirubin and cholesterol concentrations, increases in mean urea and creatinine levels, higher urine pH and urinary volumes observed in males and/or females from 10 mg/kg/day ) were not considered to be adverse due to their low magnitude and as they were not associated with adverse histopathological correlates. The decreased red blood cell mass was consistent with slight variations observed in reticulocyte counts (+26.2% and + 17.0% vs. controls in males and females at 30 mg/kg/day, respectively; not statistically significant) and may correlate with erythrophagocytosis observed in the mesenteric lymph nodes at 30 mg/kg/day.


The lower thymus weights (-15% vs. controls with p<0.01) observed in females at 30 mg/kg/day correlated with smaller thymus size observed at macroscopic evaluation and decreased cellularity observed at microscopic evaluation in females at 30 mg/kg/day. These findings were considered to be secondary to the poor clinical condition and the associated stress as there was no evidence of a direct toxic effect in other lymphoid system organs. These effects are considered to be test item-related but not adverse.
Erythrophagocytosis and pigment were observed in the medullary sinusoids of the mesenteric lymph node. The pigment was golden brown, which is consistent with hemosiderin, and therefore both findings were considered to be different stages of the same process (red blood cells engulfed by macrophages). Erythrophagocytosis is suggestive of extranodal hemorrhage in the digestive system, which is consistent with the decrease in red blood cells reported in animals at 30 mg/kg/day. At terminal euthanasia, there was no evidence of hemorrhage or inflammation in the tissues examined. Due to the lack of other findings indicative of hemorrhage in other tissues and the minimal to slight severity of most of the microscopic findings, the erythrophagocytosis and pigment in the mesenteric lymph node were considered to be test item-related and non-adverse.


In conclusion and based on these results:
• the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 10 mg/kg/day in males and 5 mg/kg/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/day and in females from 10 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP-compliant study conducted in accordance with a relevant OECD Testing Guideline.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An oral (gavage) combined repeated dose study with a repro/developmental toxicity screening was performed in rat according to OECD TG 422 and in accordance with GLP. Based on these results of the study:


 


Administration of Tetramethylthiuram monosulphide at 30 mg/kg/day for 7 to 9 weeks was associated with early euthanasia of 5 females at this dose level. The cause of premature euthanasia or death in two of these females was considered to be related to the ulcers observed in the stomach, in a situation of dystocia in one of them. The ulcers were considered to be test item related and a test item relationship could not be excluded for dystocia as this finding was noted at the high dose level only in a context of prolonged gestation period.


 


Adverse clinical signs were observed at 30 mg/kg/day and consisted of decreased activity, erected fur, hunched posture and/or abdominal breathing in both sexes. These signs were noted together with adverse body weight changes, in males at 30 mg/kg/day and in females at 10 and 30 mg/kg/day leading to adverse lower final body weights in males (-9% vs. controls; not statistically significant) at the end of the study period and in females at the end of the gestation period (-9% and -21% vs. controls at 10 and 30 mg/kg/day, with p<0.05 and p<0.001, respectively) and the end of the lactation period (-12% vs. controls at 30 mg/kg/day, with p<0.5), accompanied by sporadic or continuous adverse lower food consumption.


 


The clinical pathology changes (lower red blood cell count, lower hemoglobin and hematocrit levels, higher mean cell volume and corpuscular hemoglobin, higher total bilirubin and cholesterol concentrations, increases in mean urea and creatinine levels, higher urine pH and urinary volumes observed in males and/or females from 10 mg/kg/day ) were not considered to be adverse due to their low magnitude and as they were not associated with adverse histopathological correlates. The decreased red blood cell mass was consistent with slight variations observed in reticulocyte counts (+26.2% and + 17.0% vs. controls in males and females at 30 mg/kg/day, respectively; not statistically significant) and may correlate with erythrophagocytosis observed in the mesenteric lymph nodes at 30 mg/kg/day.


 


The lower thymus weights (-15% vs. controls with p<0.01) observed in females at 30 mg/kg/day correlated with smaller thymus size observed at macroscopic evaluation and decreased cellularity observed at microscopic evaluation in females at 30 mg/kg/day. These findings were considered to be secondary to the poor clinical condition and the associated stress as there was no evidence of a direct toxic effect in other lymphoid system organs. These effects are considered to be test item-related but not adverse.


 


Erythrophagocytosis and pigment were observed in the medullary sinusoids of the mesenteric lymph node. The pigment was golden brown, which is consistent with hemosiderin, and therefore both findings were considered to be different stages of the same process (red blood cells engulfed by macrophages). Erythrophagocytosis is suggestive of extranodal hemorrhage in the digestive system, which is consistent with the decrease in red blood cells reported in animals at 30 mg/kg/day. At terminal euthanasia, there was no evidence of hemorrhage or inflammation in the tissues examined. Due to the lack of other findings indicative of hemorrhage in other tissues and the minimal to slight severity of most of the microscopic findings, the erythrophagocytosis and pigment in the mesenteric lymph node were considered to be test item-related and non-adverse.


 


In conclusion and based on these results, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 10 mg/kg/day in males and 5 mg/kg/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/day and in females from 10 mg/kg/day.

Justification for classification or non-classification

In accordance with Regulation (EC) No 1272/2008, no classification is warranted for the registered substance in regards to toxicity following a repeated exposure.

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