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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of an OECD 422 compliant study, the test item administered at 100, 300 and 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. There were no signs of systemic toxicity in male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats: 1000 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-25 to 2019-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Hsd.Han
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, approximately weeks old at the beginning of the pre-mating phase and weeks at mating.
- Weight at study initiation: The weight variation in animals involved at the starting point of the study not exceed ± 20 % of the mean group weight of each sex.
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water from municipal supply
- Acclimation period: days

The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared not longer than for three days before the use.

VEHICLE
- Concentration in vehicle: 20 mg/mL, 60 mg/mL and 200 mg/mL
- Treatment volume: 5 mL dose preparation/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurs or 14 days have elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female will be caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and at least three replicates are measured on 2 occasions, approximately during the first and last treatment weeks. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
Duration of treatment / exposure:
Males dosed for at least 28 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period until the necessary number of pregnant female animals is evident)
Females dosed for 14 days pre-mating, through 14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
corresponding to active ingredient: 80 mg/kg bw/d
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
corresponding to active ingredient: 240 mg/kg bw/d
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
corresponding to active ingredient: 800 mg/kg bw/d
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on findings obtained in a 14-day dose range finding study performed with the substance and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. In case of severe signs of toxicity, the high dose will be reduced.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
Observations on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted on five male and five female animals randomly selected from each group during their respective last exposure week but before the blood sampling.

BODY WEIGHT: Yes
Parental males weighed on the first day of dosing (day 0), weekly thereafter and at termination.
Parental females weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals additionally measured on gestation day 10 in order to give accurate treatment volumes, but these data was evaluated statistically. Body weight data was reported individually for adult animals. Body weight was measured on the day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

FOOD CONSUMPTION: Yes
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase as follows: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals. Fasted body weight was determined for animals selected for toxicity examinations before the necropsy.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles will included in the study preferably. Vaginal smears were prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.
Vaginal smear were prepared on the day of the necropsy.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- testis weight, epididymis weight, prostate weight and seminal vesicles with coagulating glands as a whole
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 and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) presence of gross anomalies. Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete, post-natal day 0) and on postnatal day 4. The anogenital distance of each pup was determined on postnatal day 4. Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum T4 and TSH levels. The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
CLINICAL PATHOLOGY
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry. In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4).

HEMATOLOGY
WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes), Differential white blood cell count

CLINICAL CHEMISTRY
ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration),
UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+ (Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4,TSH) as follows:
- from all dams and at least two pups per litter on day 13 if feasible
- from all parent male animals at termination

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
The following organs were preserved: Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with vagina), Gross lesions, Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines(representative regions: duodenum, ileum, jejunum), Spinal cord
(at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder.

At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals was determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus was weighed. Absolute organ weight was reported. Relative organ weight (to body and brain weights) was calculated and reported.
The thyroid weight was determined after fixation.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups.
Postmortem examinations (offspring):
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test). Dead pups found were subjected to necropsy by a macroscopic examination. Any observed abnormalities were recorded.
Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance
(ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
see table 1
Offspring viability indices:
see table 2
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item related clinical signs in any group, i.e. parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 1000 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
Black colored stool was detected in the bedding material in each cage of the male and female animals at 100, 300 and 1000 mg/kg bw/day groups from Day 1 until termination. The color of stools was probably indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract. Alopecia was noted for one control female animal (1/12) on the hindlimbs and abdomen from gestational day 16 up to the termination of the study. Alopecia is a common dermal change in experimental rats of this strain with similar age.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study (male and female). One female animal administered with 300 mg/kg bw/day died due to over-anesthesia at the blood sampling on lactation day 13.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was undisturbed in male and female animals at 100, 300 and 1000 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 100, 300 and 1000 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods and in female animals during the pre-mating, gestation and lactation periods. Mean body weight gain was statistically significantly lower with respect to the control in the male animals at 300 mg/kg bw/day between Days 34 and 41 and at 1000 mg/kg bw/day between Days 34 and 41 and between Days 0 and 41, as well as in female animals at 300 mg/kg bw/day between gestation days 0 and 7, but remaining within the ranges of the historical control values.
These changes in body mean weight gain at 300 mg/kg bw/day were sporadic and of minor degree and had no influence on the overall body weight gain or body weight. The reduced body weight gain seen in the high dose males did not result in a significant change of the mean body weight. Therefore, this finding was considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no test item related adverse effect in the mean daily food consumption of male or female animals at 100, 300 and 1000 mg/kg bw/day. The mean daily food consumption was comparable in the control and test item treated male animals at 100, 300 and 1000 mg/kg bw/day during pre-mating and post mating periods. In the female animals of the high dose group, a slight but statistically significant higher mean daily food consumption with respect to the control was detected during the course of the first two weeks of gestation period and between lactation days 4 and 13. These changes were considered to be toxicologically not relevant due to minor degree (7-9 %), and were within the ranges of the historical control values.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day. All examined hematological and blood coagulation parameters were comparable with the control in male animals at 100 mg/kg bw/day.
Statistically significant difference with respect their control was detected at the lower mean percentage of monocytes (MONO) in male animals at 300 and 1000 mg/kg bw/day.
The examined hematological and blood coagulation parameters were comparable in female animals in the control and 100 mg/kg bw/day groups. In female animals of the 300 mg/kg bw/day group, statistically significantly lower mean percentage of neutrophil granulocytes (NEU), higher mean percentage of lymphocytes (LYM) and lower mean corpuscular (erythrocyte) hemoglobin content (MCH) were detected when compared to their control.
At 1000 mg/kg bw/day, the mean corpuscular (erythrocyte) hemoglobin content was slightly but statistically significantly lower compared to their control in female animals.
These differences were considered to be toxicologically not relevant due to the minor degree and in the lack of dose dependency (MONO, NEU, LYM, MCH). All values were within the ranges of the historical control data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300, 1000 mg/kg bw/day (male or female).
Statistical significance was detected at lower mean activity of aspartate aminotransferase (AST) in male animals at 100 mg/kg bw/day, at the lower mean cholesterol concentration (CHOL) in male animals at 300 mg/kg bw/day as well as at the lower mean concentrations of creatinine (CREA) and higher mean concentration of albumin (ALB) in female animals at 1000 mg/kg bw/day. The statistically significant differences in AST, CHOL, CREA and ALB with respect to their controls in male or female animals were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.

Serum Thyroid Hormones
The thyroid hormone (free FT4 and TSH) levels were not affected by the test item in parental male animals (100, 300 and 1000 mg/kg bw/day) and in offspring sampled on postnatal day 13. Statistical significance was only detected at the lower mean concentration of free-thyroid hormone level of parental male animals at 100 mg/kg bw/day compared to their control. This concentration was within the ranges of the historical control data.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 100, 300 or 1000 mg/kg bw/day groups at the end of the treatment period (on Day 42 for male animals and on Day 51 for female animal). There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated reproductive and other organs of experimental male and female animals at 1000 mg/kg bw/day.

In the male animals of control and 1000 mg/kg bw/day and groups, the investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases (12/12, each).

The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.The histology picture of epididymides, prostate, seminal vesicles and coagulating glands was normal in all animals in the control and high dose groups (12/12, both). In one animal at 80 mg/kg bw/day (1/1) granulomatous inflammation (one side) in the epididymis was observed. Spermatogenic granulomas in the testis and epididymides are uncommon in the rat. The etiology of many sperm granulomas is unknown, but experimentally it has been shown that they can occur as results of reduced blood flow of the organ. Therefore, this phenomenon was considered as individual disorder, without toxicological significance.
In the female animals belonging to the high dose (1000 mg/kg bw/day) treated and control groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle (12/12, in control and high dose groups). The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male (5/5 in both groups) and female (5/5, in both groups) animals.
In one dam (1/2) at 300 mg/kg bw/day, showing hydrometra at the necropsy, dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In one female animal, which died because of over-anesthesia at 300 mg/kg bw/day, black content in the intestines (1/1) mild alveolar emphysema (1/2) were detected. The ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in this animal.
Black discoloration of the intestinal content in male and female animals at 1000 mg/kg bw/day (5/5 male and 5/5 female) and black pigment in the cytoplasm of some macrophages in the lungs in single female animals at 100 mg/kg bw/day (1/1), at 300 mg/kg bw/day (1/2) without degenerative or inflammatory lesions in the affected tissues (intestinal mucous membrane and lung tissues) were considered to have no toxicological significance. There were no vital inflammatory or other reaction in connection with the discoloration of intestinal content and the alveolar macrophages, therefore these findings were considered as innocuous lesions.
In animals selected for full histological examinations, no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.
Minimal alveolar emphysema in the lungs (1/5 male and 1/5 female control, 1/5 male at 1000 mg/kg bw/day) were detected sporadically. The pulmonary emphysema was considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs in some animal (1/5 control male; 1/5 control female; 1/5 male at 1000 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Both sides pyelectasia was observed in a single male animal at 300 mg/kg bw/day without other histopathological lesions (degeneration, inflammation, fibrosis): Therefore, this renal alteration was considered to be an individual lesion, without toxicological significance. Pyelectasia is a common finding in experimental rats of this strain with similar age.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day). There were no statistically or biologically significant differences between the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) in the examined parameters of estrous cycle: all animal shoved regular cycles, the mean number of cycles, the mean length of cycles, mean number of days in pro-estrous, estrous or diestrus were similar in all groups and there were no animals with prolonged estrous or diestrous during the pre-mating period.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the test item at 100, 300 or 1000 mg/kg bw/day in male or female animals. The copulatory and fertility indices were 100 % in male and female animals in each group. There was no difference between the control and test item treated groups in the gestation index (female animals), in the mean pre-coital interval or mean conceiving days.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed at the highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13. The percentage of offspring with signs (cold and not suckled) was slightly higher than in the control group at 1000 mg/kg bw/day on postnatal day 0. However, these signs were considered to be toxicologically not relevant as the signs were transient – detected only shortly after the delivery – and were not associated with depression on the development of the offspring.
Occasionally, other clinical signs were also observed at 1000 mg/kg bw/day: pale and yellow skin (1 %, both), damaged tail (5 %), which however were considered to have no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality. There were no extrauterine mortality in the control, 300 and 1000 mg/kg bw/day treated groups. Two female pups in two litters were missing at 100 mg/kg bw/day.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices.
One dam of the 300 mg/kg bw/day-dose group was over-anesthetized on lactation day 13, therefore its pups were euthanized on the same day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development of the offspring was undisturbed during the observation period. The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13. Statistical significance was observed at the slightly lower mean body weight of female pups at 300 mg/kg bw/day. The value was below the historical control data values, but as no dose-dependency was observed, this finding was not considered toxicologically relevant. The slightly higher mean body weight of male pups of the 1000 mg/kg bw/day group on postnatal day 4 comparing to their control was also statistically significant. These differences with respect to the control were minor and were considered to be toxicologically not relevant.
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:
no effects observed
Description (incidence and severity):
Sex distribution
There were no test item related differences between the control and all test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not adversely affected by the test item treatment at 100, 300 and 1000 mg/kg bw/day.
Statistical significance was observed at the shorter mean absolute and normalized anogenital distances with respect to their control at 100 and 300 mg/kg bw/day (male and female) and at 1000 mg/kg bw/day (female). All values were within the ranges of the historical control values.
In the lack of dose relevance and the minor degree of differences, these findings were considered to be toxicologically not relevant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on postnatal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects observed up to the highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present study, the test item administered at 100, 300 and 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. There were no signs of systemic toxicity in male or female animals at 100, 300 or 1000 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male rats: 1000 mg/kg bw/day
NOAEL for systemic toxicity of female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day
Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 1000 mg/kg bw/day compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

Solubilised Sulphur Black 1 was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 20, 60 and 200 mg/mL , corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.

The test item was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL in a refrigerator (at 5 ± 3 °C) for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. The test item concentrations in the dosing formulations varied within the range of 101 % and 111 % in comparison to the nominal values) and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-18, i.e. up to the day before necropsy (altogether for 50-63 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam. The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-8 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 3-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology examination.

All parental animals were subjected to gross pathology one day after the last treatment – except for female animal subjected to early necropsy on lactation day 13 due to over anesthesia. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination. Histopathology examination was performed on ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in over-anesthetized dam at 300 mg/kg bw/day.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

There was no test item related mortality at any dose level (100, 300 and 1000 mg/kg bw/day). One dam at 300 mg/kg bw/day died due to over-anesthesia at the blood sampling on lactation day 13. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period. The body weight development was not adversely affected by the test item in male or in female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals). The mean daily food consumption was similar in male or female animals in control and at 100, 300 and 1000 mg/kg bw/day during the entire study. A test item influence on the estrous cycle was not found at any dose level (100, 300 and 1000 mg/kg bw/day). There were no significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (100, 300 and 1000 mg/kg bw/day). Hematological and blood coagulation investigation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day (male or female). There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female). There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose (parental male or 13-day offspring). Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 300 or 1000 mg/kg bw/day. Gray-black, black or gray discoloration of several organs (lungs, mesenteric lymph nodes, testes, uterus or kidneys) and discoloration of intestinal content (in whole gastrointestinal tract, in cecum and colon or in stomach) were observed in the test item treated animals at 100, 300 or 1000 mg/kg bw/day (male or female) with a variable incidence. There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides or seminal vesicles with coagulating gland as a whole in male animals at any dose level. Minor changes in the weights of the kidneys in male animals at 300 and 1000 mg/kg bw/day and in female animals at 1000 mg/kg bw/day compared to their control were considered to be of little or no biological relevance in the lack of any related findings. There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) of male or female animals administered with 1000 mg/kg bw/day. Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 1000 mg/kg bw/day. Black discoloration of organs and black content in the gastrointestinal tract were considered as innocuous lesions in the lack of degenerative or inflammatory lesions in the affected tissues (intestinal mucous membrane and lung tissues) and can be most likely attributed to the black color of the test item.

The offspring’s development was undisturbed at 100, 300 and 1000 mg/kg bw/day from birth to post-natal day 13. No effects on the mortality, clinical signs, body weight, anogenital distance (male and female) or nipple retention (male) were detected.

Under the conditions of the present study, the test item administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. There were no signs of systemic toxicity in male or female animals at any dose level tested.

The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats: 1000 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 422

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 1000 mg/kg bw/day compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

Solubilised Sulphur Black 1 was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 20, 60 and 200 mg/mL , corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.

The test item was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL in a refrigerator (at 5 ± 3 °C) for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. The test item concentrations in the dosing formulations varied within the range of 101 % and 111 % in comparison to the nominal values) and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-18, i.e. up to the day before necropsy (altogether for 50-63 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam. The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-8 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 3-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology examination.

All parental animals were subjected to gross pathology one day after the last treatment – except for female animal subjected to early necropsy on lactation day 13 due to over anesthesia. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed. Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in over-anesthetized dam at 300 mg/kg bw/day

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

There was no test item related mortality at any dose level (100, 300 and 1000 mg/kg bw/day). One dam at 300 mg/kg bw/day died due to over-anesthesia at the blood sampling on lactation day 13. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period. The body weight development was not adversely affected by the test item in male or in female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals). The mean daily food consumption was similar in male or female animals in control and at 100, 300 and 1000 mg/kg bw/day during the entire study. A test item influence on the estrous cycle was not found at any dose level (100, 300 and 1000 mg/kg bw/day). There were no significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (100, 300 and 1000 mg/kg bw/day). Hematological and blood coagulation investigation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day (male or female). There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female). There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose (parental male or 13-day offspring). Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 300 or 1000 mg/kg bw/day. Gray-black, black or gray discoloration of several organs (lungs, mesenteric lymph nodes, testes, uterus or kidneys) and discoloration of intestinal content (in whole gastrointestinal tract, in cecum and colon or in stomach) were observed in the test item treated animals at 100, 300 or 1000 mg/kg bw/day (male or female) with a variable incidence. There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides or seminal vesicles with coagulating gland as a whole in male animals at any dose level. Minor changes in the weights of the kidneys in male animals at 300 and 1000 mg/kg bw/day and in female animals at 1000 mg/kg bw/day compared to their control were considered to be of little or no biological relevance in the lack of any related findings. There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) of male or female animals administered with 1000 mg/kg bw/day. Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 1000 mg/kg bw/day. Black discoloration of organs and black content in the gastrointestinal tract were considered as innocuous lesions in the lack of degenerative or inflammatory lesions in the affected tissues (intestinal mucous membrane and lung tissues) and can be most likely attributed to the black color of the test item.

The offspring’s development was undisturbed at 100, 300 and 1000 mg/kg bw/day from birth to post-natal day 13. No effects on the mortality, clinical signs, body weight, anogenital distance (male and female) or nipple retention (male) were detected.

Under the conditions of the present study, the test item administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. There were no signs of systemic toxicity in male or female animals at any dose level tested. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats: 1000 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

Under the conditions of the present study, the test item administered at 100, 300 and 1000 mg/kg bw/day by oral gavage did not adversely influence the development of the F1 offspring from birth to post-natal day 13.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats: 1000 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification as toxic to reproduction according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.

Additional information

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