Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-21 to 2019-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-21 to 2019-05-24

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

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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 large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: Young adult rats, at least 10 weeks old at starting and 14 weeks at mating.
- Weight at study initiation:
The weight variation does not exceed ± 20 per cent of the mean weight at starting of administration
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals/ cage
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats and mice (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: tap water ad libitum
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 1 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle (1 % methylcellulose) in concentrations of 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 22.09, 66.28 and 220.92 mg/mL). Formulations were prepared in the formulation laboratory of Test Facility beforehand not longer than for three days and were stored at 5 ± 3 °C until use.

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 was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 mL of each dose group (20, 60 and 200 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 97 % at ca. 1 mg/mL and 101 % at ca. 200 mg/mL).

Duration of treatment / exposure:

Males were dosed altogether for 49 days (14 days pre-mating and 14 days mating plus an extended post-mating period until the necessary number of pregnant female animals is evident).
Females were dosed for altogether for 51-56 days (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 (actual dose received)

Dose / conc.:

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

Remarks:

corresponding to 100 mg dye/kg bw/d

Dose / conc.:

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

Remarks:

corresponding to 300 mg dye/kg bw/d

Dose / conc.:

1 105.4 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 1000 mg dye/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 was adjusted.

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 was 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 are 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 were weighed on the first day of dosing (day 0), weekly thereafter and at termination.
Parental females were 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 is 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 were 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 was observed, and any abnormality was recorded including details of the location, colour, 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 was 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) will be 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 are 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. the 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.
Piloerection, decreased activity and bloody vaginal orifice was observed for one female animal in the 100 mg/kg bw/day group (no. 225) on gestation days 22 and 23 due to prolonged and difficult delivery. These observations were considered to be individual signs.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality in the 100, 300 or 1000 mg/kg bw/day treatment groups during the course of study (male and female).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was not affected in male and female animals at 100, 300 or 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 and female animals.
A slight but statistically significant difference with respect to their control was detected at the higher mean body weight gain in male animals at 100 mg/kg bw/day between Days 41 and 48 and at the lower mean body weight gain in female animals at 100 and 1000 mg/kg bw/day on the first week of treatment. These slight changes were considered to be indicative of biological variation and not related to the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related changes in the mean daily food consumption of male or female animals at 100, 300 or 1000 mg/kg bw/day.
Statistical significance with respect to the control was detected in female animals at 1000 mg/kg bw/day at the slightly lower mean daily food consumption during the first week of pre-mating period.
The mean daily food consumption was comparable in the control and test item treated animals at 100, 300 or 1000 mg/kg bw/day during pre-mating and post mating periods in male animals and during the course of gestation and lactation periods in female animals.

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.
The examined hematological parameters were comparable in male animals in the control and 100 and 300 mg/kg bw/day groups.
In male animals at 1000 mg/kg bw/day, statistical significance was noted for the higher mean white blood cell count (WBC), platelet count (PLT) and percentage of reticulocytes (RET).
In female animals, statistical significances were observed at the higher mean red blood cell (erythrocyte) count (RBC), higher mean hematocrit value (HCT) and higher mean concentration of hemoglobin (HGB) at 100 mg/kg bw/day.
The mean red blood cell count and hematocrit value were higher and the mean corpuscular hemoglobin concentration (MCHC) was lower than in the control in female animals at 300 mg/kg bw/day.
In female animals at 1000 mg/kg bw/day, higher mean red blood cell count, higher mean hematocrit value and lower mean corpuscular hemoglobin concentration were observed in comparison with the control.
The above-mentioned statistical significances indicated only slight differences between the control and test item treated groups (WBC, RET, PLT in male animals; RBC, HCT, HGB and MCHC in female animals) the values were within or marginal to the historical control ranges. There were no related changes in organ pathology (male and female) or dose relevance (female) therefore these findings were considered to have no or little toxicological relevance.

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 or 1000 mg/kg bw/day (male or female).
The mean concentration of cholesterol (CHOL) slightly exceeded the control value in the male animals at 100 mg/kg bw/day groups. Statistically significance with respect to the control was observed at the lower mean concentration of total bilirubin (TBIL) in male animals at 300 mg/kg bw/day. In male animals at 1000 mg/kg bw/day, the mean activity of alanine aminotransferase (ALT) and mean cholesterol concentration were higher than in the control group. The examined clinical chemistry parameters were similar in female animals of the control and 100 and 1000 mg/kg bw/day groups. In the female animals at 300 mg/kg bw/day, statistically significantly higher mean concentration of glucose (GLUC) was observed when compared to the control.
These statistically significant differences with respect to their controls were considered to have little or no toxicological importance because all these changes were with low degree (ALT, TBIL, CHOL and GLUC) or values – mean and individual – remained mainly within the historical control ranges or there was no dose relevance. Moreover, there were no histological changes in related organs.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional observation battery did not demonstrate any alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, control, 100, 300 or 1000 mg/kg bw/day groups, on Day 42).
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):

There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 1000 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (1000 mg/kg bw/day).
In the male animals belonging to the treated and control groups (12/12 control; 12/12 at 1000 mg/kg bw/day and 1/1 male (mating pair of a not delivered female) at 300 mg/kg bw/day), 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. 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 histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 12/12 at 1000 mg/kg bw/day and 1/1 not delivered female at 300 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated 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 was normal in all cases as well.
Dilatation of uterine horns was observed in some female animals as follows: 1/12 control dam; 1/1 dam at 100 mg/kg bw/day; 1/1 not delivered female at 300 mg/kg bw/day, 3/12 dams at 1000 mg/kg bw/day (histological processing was only performed in one animal showing macroscopic finding and in one not delivered female animal in the low and mid dose group).
This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In dam No 428, the macroscopic observation of a hard, pea-sized formation in the left uterine horn was not confirmed: no abnormal finding correlating with the necropsy observation was detected.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in the control, 1/5 male and 1/5 female at 1000 mg/kg bw/day), acute hemorrhage in the lungs (2/5 control males) and in the thymus (1/1 male at 100 mg/kg bw/day and 1/1 male at 300 mg/kg bw/day) were detected sporadically. The pulmonary emphysema and hemorrhages in the lungs and thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (2/5 control females; 2/5 males and 1/5 female at 1000 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in the animals. The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum thyroid hormones
There were no significant differences with respect to the control in the TSH thyroid hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.
Statistically significant difference with respect to the control was noted for the higher mean thyroid hormone (free T4) level in PN13 offspring at 1000 mg/kg bw/day. This difference was considered to be toxicologically not relevant as the individual values were within the historical control range.

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).
At 300 mg/kg bw/day, the mean number of cycles of animals was slightly lower and the mean number of days in diestrus was higher than in the control group in the pre-mating period. These changes were not considered to be related to the treatment, but indicative of biological variation. There were no differences between the control and treated groups (100, 300 and 1000 mg/kg bw/day) in number or percentage of animals with regular or irregular cycles, in the mean length of cycles, mean number of days in pro-estrous or estrous during the pre-mating period. 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 during the pre-experimental period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the treatment with the test item at 100, 300 or 1000 mg/kg bw/day in male or female animals. There was a statistical difference between the control and the 300 mg/kg bw/day group of female animals in the gestation index as one pregnant female animal in the mid dose group did not deliver. This minor change was considered to be an isolated finding and not related to the test item. There was no difference between the control and test item treated groups in copulatory and fertility indices (male and female animals), in the pre-coital interval and conceiving days.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: no adverse effects observed up to and including 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 comparable in the control and test item treated groups. These signs were considered to be toxicologically not relevant as those 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: pale (1% in the control), smaller than normal pup (1% in the control; 2% at 1000 mg/kg bw/day), cyanotic skin (1% at 300 mg/kg bw/day), hemorrhage on the head (1% at 300 mg/kg bw/day; 1% at 1000 mg/kg bw/day), 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 significant differences between the control and test item treated groups (100, 300 or 1000 mg/kg bw/day) in the mean number of dead offspring (including missing pups) per litter.
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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development of the offspring was unaffected by the test item.
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. Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly higher mean weight of male pups at 300 mg/kg bw/day on postnatal day 4. The difference with respect to the control was minor and it was 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 test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
Statistically significant difference with respect to the control was detected at the slightly lower number of male pups and at the slightly higher number of female pups at 1000 mg/kg bw/day. The difference was with minor degree and the mean number of male and female pups per litter did not show statistical significance. Therefore, it was considered to be toxicologically not relevant.

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.
In female pups, statistical significance was observed at the longer mean absolute and normalized anogenital distance at 100, 300 and 1000 mg/kg bw/day.
As there was no dose relevance, and the mean value of the control is relatively low, these differences with respect to the control were not considered to be toxicologically 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:

no effects observed

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
There were no macroscopic changes in the organs or tissues of four stillborn offspring (3/4 at 1000 mg/kg bw/day subjected to necropsy on postnatal day 0). One stillborn pup (1/4 at 1000 mg/kg bw/day, subjected to necropsy on postnatal day 0 was smaller than normal and was partially cannibalized.
In seven pups at 100 mg/kg bw/day, autolyzed visceral organs were detected on post-natal day 0. Three pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day was partially cannibalized on post-natal day 0. Therefore, determination of stillborn or liveborn was not feasible for these eleven pups due to autolysis or cannibalism.
In two pups found dead at 100 mg/kg bw/day (2/2) on the day of birth, autolyzed visceral organs were detected.
One pup found dead in the control group (1/1) was partially cannibalized on post-natal day 17.

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 and including the highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of an OECD 422 compliant study, the test item administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; corresponding to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day, respectively) by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/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 the 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 (corrected doses; corresponding to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day, respectively) 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.

The test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) 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 calculated by the active ingredient content and corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (1 % methylcellulose) 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 in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day and 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 94 % and 105 % 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 49 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 51-56 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 (two weeks pre-mating period) and during the mating period until evidence of copulation.

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 (T4 – Thyroxine, free tetra-iodothyronine – and TSH – thyroid-stimulating hormone) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/ post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides of adult male animals were determined. 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, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). In addition, these organs were processed histologically in not delivered female and male cohabited with in the mid dose group. 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 full histopathology examination.

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 mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study (male and female). Clinical signs of systemic toxicity 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. Test item related changes in the body weight or body weight gain were not detected. The body weight development was not affected and it was comparable in the control and test item treated groups. 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 (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals). 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 evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day. 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 (T4 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. There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level. The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 and 1000 mg/kg bw/day groups at the end of the treatment period. Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 1000 mg/kg bw/day groups.

No adverse effect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Under the conditions of the present study, the test item administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; respectively to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day) by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

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

NOAEL for systemic toxicity of male/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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity w ith the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

EC Number:

290-904-3

EC Name:

Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Cas Number:

90268-98-7

Molecular formula:

Molecular formula is not available

IUPAC Name:

Reaction product of aniline, 4-nitrobenzenamine, p-phenylenediamine and p-toluidine with sodium polysulfide

Test material form:

solid: particulate/powder

Details on test material:

Test item: Yellow 22
Appearance: ocher clay, solid
CAS No. 90268-98-7
EC No. 290-904-3
Storage: room temperature

Test animals

Species:

rat

Strain:

Wistar

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 large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Male animals: Young adult rats, at least 10 weeks old at starting and 14 weeks at mating.
- Weight at study initiation: The weight variation does not exceed ± 20 per cent of the mean weight at starting of administration
- Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals/ cage
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: tap water ad libitum
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally, by gavage. The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.

Vehicle:

methylcellulose

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of diet: Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at 5 +/- 3 °C until use.

- VEHICLE
- Justification for use and choice of vehicle: 1 % methylcellulose is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 22.09, 66.28 and 220.92 mg/mL)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 mL of each formulation (20, 60 and 200 mg/mL) to be administered to the animals and five aliquots of 5 mL control substance (vehicle) were taken. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 97 % at ca. 1 mg/mL and 101 % at ca. 200 mg/mL).

Duration of treatment / exposure:

Males were dosed altogether for 49 days (14 days pre-mating and 14 days mating plus an extended post-mating period until the necessary number of pregnant female animals is evident).
Females were dosed for altogether for 51-56 days (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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Remarks:

corresponding to 100 mg dye/kg bw/d

Doses / concentrations 3

Dose / conc.:

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

Remarks:

corresponding to 300 mg dye/kg bw/d

Doses / concentrations 4

Dose / conc.:

1 105.4 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 1000 mg dye/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 levels were chosen on the basis of the results of a preliminary dose range finding study (Study no. 805-400-2616). 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. The mid dose was interpolated geometrically.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day
- Parameters checked in table No.1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), weekly thereafter and at termination.
Parental females were 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 was additionally determined on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight data were reported individually for adult animals. Individual body weight change was calculated.
Body weight was measured on 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 (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the terminal necropsy
- Anaesthetic used for blood collection: Yes, Isofluran
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table No.2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the terminal necropsy
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy consists of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size.

HISTOPATHOLOGY: Yes
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 are weighed. Absolute organ weight is 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.

Other examinations:

Organ Weight
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals was determined. Absolute organ weight was reported. Relative organ weight (to body and brain weight) was calculated and reported. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed. The thyroid weight was determined – if relevant – after fixation. Paired organs were weighed together; absolute organ weight was reported. Relative organ weight (to body and brain weight) was calculated and reported.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- Body weight
- Food consumption
- Hematology
- Blood coagulation
- Clinical chemistry
- Organ weight

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related clinical signs in any group, i.e. the 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.
Piloerection, decreased activity and bloody vaginal orifice was observed for one female animal in the 100 mg/kg bw/day group (no. 225) on gestation days 22 and 23 due to prolonged and difficult delivery. These observations were considered to be individual signs.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality in the 100, 300 or 1000 mg/kg bw/day treatment groups during the course of study (male and female).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was not affected in male and female animals at 100, 300 or 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 and female animals.
A slight but statistically significant difference with respect to their control was detected at the higher mean body weight gain in male animals at 100 mg/kg bw/day between Days 41 and 48 and at the lower mean body weight gain in female animals at 100 and 1000 mg/kg bw/day on the first week of treatment. These slight changes were considered to be indicative of biological variation and not related to the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related changes in the mean daily food consumption of male or female animals at 100, 300 or 1000 mg/kg bw/day.
Statistical significance with respect to the control was detected in female animals at 1000 mg/kg bw/day at the slightly lower mean daily food consumption during the first week of pre-mating period.
The mean daily food consumption was comparable in the control and test item treated animals at 100, 300 or 1000 mg/kg bw/day during pre-mating and post mating periods in male animals and during the course of gestation and lactation periods in female animals.

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.
The examined hematological parameters were comparable in male animals in the control and 100 and 300 mg/kg bw/day groups.
In male animals at 1000 mg/kg bw/day, statistical significance was noted for the higher mean white blood cell count (WBC), platelet count (PLT) and percentage of reticulocytes (RET).
In female animals, statistical significances were observed at the higher mean red blood cell (erythrocyte) count (RBC), higher mean hematocrit value (HCT) and higher mean concentration of hemoglobin (HGB) at 100 mg/kg bw/day.
The mean red blood cell count and hematocrit value were higher and the mean corpuscular hemoglobin concentration (MCHC) was lower than in the control in female animals at 300 mg/kg bw/day.
In female animals at 1000 mg/kg bw/day, higher mean red blood cell count, higher mean hematocrit value and lower mean corpuscular hemoglobin concentration were observed in comparison with the control.
The above-mentioned statistical significances indicated only slight differences between the control and test item treated groups (WBC, RET, PLT in male animals; RBC, HCT, HGB and MCHC in female animals). The values were within or marginall to the historical control ranges. There were no related changes in organ pathology (male and female) or dose relevance (female) therefore these findings were considered to have no or little toxicological relevance.

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 or 1000 mg/kg bw/day (male or female).
The mean concentration of cholesterol (CHOL) slightly exceeded the control value in the male animals at 100 mg/kg bw/day groups. Statistically significance with respect to the control was observed at the lower mean concentration of total bilirubin (TBIL) in male animals at 300 mg/kg bw/day. In male animals at 1000 mg/kg bw/day, the mean activity of alanine aminotransferase (ALT) and mean cholesterol concentration were higher than in the control group. The examined clinical chemistry parameters were similar in female animals of the control and 100 and 1000 mg/kg bw/day groups. In the female animals at 300 mg/kg bw/day, statistically significantly higher mean concentration of glucose (GLUC) was observed when compared to the control.
These statistically significant differences with respect to their controls were considered to have no toxicological importance because all these changes were with low degree (ALT, TBIL, CHOL and GLUC) or values – mean and individual – remained mainly within the historical control ranges or there was no dose response. Moreover, there were no histological changes in related organs.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional observation battery did not demonstrate any alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, control, 100, 300 or 1000 mg/kg bw/day groups, on Day 42).
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

Description (incidence and severity):

There were no toxicologically relevant differences between the control and test item treated male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day) in the examined organ weights.
The weights of the examined organs were comparable in male animals in the control and 100 and 1000 mg/kg bw/day groups. In the male animals at 300 mg/kg bw/day, the mean kidney weights (absolute) and the mean weights of adrenal glands (absolute and relative body weight) were lower than in the control group.
In the female animals, there were no statistically or biologically significant differences between the control and the 100, 300 or 1000 mg/kg bw/day groups in the weights of the examined organs at the end of the treatment period.
The differences with respect to the control in mid dose treated male animals were judged to be toxicologically not relevant as there was no dose response.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Specific macroscopic alterations indicative of test item effect was not observed in the organs or tissues at any dose levels 100, 300 or 1000 mg/kg bw/day at the necropsy.
There were no macroscopic findings in the organs or tissues in male animals in the control and 1000 mg/kg bw/day groups (12/12, each).
Hemorrhage in the thymus was observed in one male at 100 mg/kg bw/day (1/12) and in two males at 300 mg/kg bw/day (2/12).
Moderate or marked hydrometra was noted for two dams in the control group (2/12) and the right eye of one control dam (1/12) damaged at blood sample collection
Marked hydrometra was observed in one dam at 100 mg/kg bw/day (1/12).
There were no macroscopic findings in the organs or tissues in dams at 300 mg/kg bw/day groups (11/11). Marked hydrometra was noted for not delivered pregnant female animal (1/1).
In one female animal at 1000 mg/kg bw/day, a hard, pea-sized formation in the left uterine horn (1/12) was observed. Marked or moderate hydrometra (3/12) was additionally observed in the highest tested dose. The right eye of one dam at 1000 mg/kg bw/day (1/12) was damaged during blood sample collection. There was no abnormal histopathological finding which could correlate to the formation in the uterus.
Hemorrhage in the thymus was due to circulatory disturbance developed during exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) these findings were judged to be toxicologically not relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 1000 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (1000 mg/kg bw/day).
In the male animals belonging to the treated and control groups (12/12 control; 12/12 at 1000 mg/kg bw/day and 1/1 male (mating pair of a not delivered female) at 300 mg/kg bw/day), 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. 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 histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 12/12 at 1000 mg/kg bw/day and 1/1 not delivered female at 300 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated 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 was normal in all cases as well.
Dilatation of uterine horns was observed in some female animals as follows: 1/12 control dam; 1/1 dam at 100 mg/kg bw/day; 1/1 not delivered female at 300 mg/kg bw/day, 3/12 dams at 1000 mg/kg bw/day (histological processing was only performed in one animal showing macroscopic finding and in one not delivered female animal in the low and mid dose group).
This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In dam No 428, the macroscopic observation of a hard, pea-sized formation in the left uterine horn was not confirmed: no abnormal finding correlating with the necropsy observation was detected.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in the control, 1/5 male and 1/5 female at 1000 mg/kg bw/day), acute hemorrhage in the lungs (2/5 control males) and in the thymus (1/1 male at 100 mg/kg bw/day and 1/1 male at 300 mg/kg bw/day) were detected sporadically. The pulmonary emphysema and hemorrhages in the lungs and thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (2/5 control females; 2/5 males and 1/5 female at 1000 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in the animals. The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum thyroid hormones
There were no significant differences with respect to the control in the TSH thyroid hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.
Statistically significant difference with respect to the control was noted for the higher mean thyroid hormone (free T4) level in PN13 offspring at 1000 mg/kg bw/day. This difference was considered to be toxicologically not relevant as the individual values were within the historical control range.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: no adverse effects observed up to and including the highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of an OECD 422 compliant study, the test item administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; corresponding to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day, respectively) by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/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 the 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 (corrected doses; corresponding to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day, respectively) 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.

The test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) 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 calculated by the active ingredient content and corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (1 % methylcellulose) 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 in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day and 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 94 % and 105 % 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 49 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 51-56 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 (two weeks pre-mating period) and during the mating period until evidence of copulation.

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 (T4 – Thyroxine, free tetra-iodothyronine – and TSH – thyroid-stimulating hormone) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/ post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides of adult male animals were determined. 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, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). In addition, these organs were processed histologically in not delivered female and male cohabited with in the mid dose group. 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 full histopathology examination.

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 mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study (male and female). Clinical signs of systemic toxicity 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. Test item related changes in the body weight or body weight gain were not detected. The body weight development was not affected and it was comparable in the control and test item treated groups. 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 (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals). 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 evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day. 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 (T4 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. There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level. The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 and 1000 mg/kg bw/day groups at the end of the treatment period. Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 1000 mg/kg bw/day groups.

No adverse effect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Under the conditions of the present study, the test item administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; respectively to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day) by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

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

NOAEL for systemic toxicity of male/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