Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a study according OECD TG 422 performed with the read across substance Blue Sema (CAS No.1040873 -93 -5) 100, 300 or 1000 mg dye/kg bw/day (corresponding to 0, 115, 345 and 1150 mg test item/kg bw) were administered to rats by oral gavage. Neither signs of systemic toxicity nor adverse influence on the reproductive performance (gonad function, mating behavior, conception, parturition) were detected.

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. The NOAEL for reproductive performance of male/ female rats was 1000 mg/kg bw/day and the NOAEL for F1 Offspring was 1000 mg/kg bw/day.

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 February 2018 - 7 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
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:
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. certificate)
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 and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 80 – 83 days (males and females)
- Weight at study initiation: 302 – 366 g for male animals; 205 – 250 g for female animals
- 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: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water ad libitum
- Acclimation period: 20 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:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- 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
- Concentration in vehicle:
20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 23, 69 and 230 mg/mL)
- Amount of vehicle: 5 mL/kg bw
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 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: 95 % at ca. 1 mg/mL and 103 % at ca. 200 mg/mL).
Homogeneity: the maximal difference between the measured concentrations was ≤ 9 % (9-10 replicates), therefore the Blue Sema in distilled water formulations were considered to be homogeneous.
Duration of treatment / exposure:
Male animals were dosed for 50 days (14 days pre-mating and 1-6 days mating in animals plus 30-35 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-6 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)
Remarks:
corresponding to 0 mg dye/kg bw/d
Dose / conc.:
115 mg/kg bw/day (actual dose received)
Remarks:
corresponding to 100 mg dye/kg bw/d
Dose / conc.:
345 mg/kg bw/day (actual dose received)
Remarks:
corresponding to 300 mg dye/kg bw/d
Dose / conc.:
1 150 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. 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:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- 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 weights of the female animals were additionally determined on gestational day 10 in order to give accurate treatment volumes. 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 be determined by weekly interval during post-mating period.

WATER CONSUMPTION: No
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 weight 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, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

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):
SACRIFICE
- Male animals: All surviving animals were sacrificed after the last litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the 14 day lactation period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal viscera.

HISTOPATHOLOGY
- The tissues indicated in Table No. 2 were prepared for microscopic examination.

ORGAN WEIGHTS
- 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 were determined. Absolute organ weight were reported. Relative organ weights (to body and brain weight) were 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 weights were reported. Relative organ weights (to body and brain weight) were calculated and reported.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
Statistics:
The statistical evaluation 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) 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 was performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Reproductive indices:
See table No. 3
Offspring viability indices:
See table No. 4
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Alopecia on the head was noted for one female control animal (1/12) on Days 7-13 in the pre-mating period and on gestation days 0-12. This observation was detected at the weekly detailed clinical observations on Days 7 and 13 and on gestation days 0 and 7. Clinical signs were noted for one male animal (1/12) at 100 mg/kg bw/day as follows: moderately decreased activity between Days 13 and 19, abnormal limb position on Days 14-19, swelling on the chest (~1cm in diameter) on Days 14-21 and a scar at the same place on the chest between Days 17-33. This animal was symptom-free from Day 34 until the end of the study. At 300 mg/kg bw/day, piloerection was observed in one female animal on gestation days 22-23 until lactation day 1. In this group alopecia on the abdomen was observed for one female on gestation days 19-21 and on lactation days 0-14, until termination. Alopecia and dermal/ subcutaneous alterations (swelling, wound) in single animals were individual findings observed in non-treated rats and were independent from the treatment. Piloerection was due to the elaborated delivery of one female animal in the mid dose group and was considered not to be test item related. There were no clinical signs in animals administered 1000 mg/kg bw/day at the daily or at the detailed weekly clinical observations during the entire study.
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:
effects observed, non-treatment-related
Description (incidence and severity):
A slight but statistically significant difference with respect to the control was only detected at the higher mean body weight in female animals at 1000 mg/kg bw/day on lactation day 13. This slight change was considered to be indicative of biological variation and not related to the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance with respect to the control was detected in female animals at 100 mg/kg bw/day at the slightly higher mean daily food consumption between gestation days 0-7 and 14-21. The mean daily food consumption was comparable in the control and test item treated animals at 300 or 1000 mg/kg bw/day during pre-mating and post mating periods in male animals and during the course of pre-mating, gestation and lactation periods in female animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All examined hematology parameters were comparable in male animals in the control and in all test item treated groups. Slight but statistically significant differences with respect to the controls were noted for two parameters in female animals at 100 and 1000 mg/kg bw/day: lower mean corpuscular (erythrocyte) volume (MCV) and higher mean red blood cell (erythrocyte) count. These differences were considered to be toxicologically not relevant due to the minor degree and the lack of dose dependency.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
All examined clinical chemistry parameters were comparable in male animals in the control and in all test item treated groups. In the female animals, statistically significant difference with respect to the control was observed at the slightly higher mean concentration of sodium (Na+) and albumin (ALB) at 300 mg/kg bw/day and at the lower mean Urea concentration (UREA) at 100 mg/kg bw/day. These slight, but statistically significant differences were considered to have no toxicological relevance because all these changes were with low degree and there was no dose dependency.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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). One male animal at 300 mg/kg bw/day had smaller than normal seminal vesicle (one side). Histological examination revealed decreased amount of secrete in the ductuli, without any other pathological lesion (inflammation, fibrosis etc. (1/1). This finding was considered as individual disorder without toxicological relevance. In one female animal (1/1) at 300 mg/kg bw/day, dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was associated with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance. In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 control male, 2/5 males and 2/5 females at 1000 mg/kg bw/day), acute hemorrhage in the lungs (1/1 male at 300 mg/kg bw/day; 1/5 male at 1000 mg/kg bw/day; lungs were evaluated histologically in one male animal in mid dose group on the basis of macroscopic finding) and in the thymus (1/1 male at 100 mg/kg bw/day; the thymus was evaluated histologically in one male animal in low dose group on the basis of macroscopic finding) 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 (1/5 control male; 1/5 male at 1000 mg/kg bw/day; 1/5 control female) is a physiological immune-morphological phenomenon, occurring also in nontreated rats and has no toxicological relevance.
The macroscopic findings in the intestines (blue discoloration of content in cecum and colon in male animals – 1/12 at 300 mg/kg bw/day; 5/12 at 1000 mg/kg bw/day – and thickened wall of the smaller than normal caecum in two female animals – 1/12 at 300 mg/kg bw/day; 1/12 at 1000 mg/kg bw/day) were not correlated with abnormal histological finding in the mucous membrane of the affected large intestines. Hypoplasia of hair follicles was observed in one female animal at 1000 mg/kg bw/day (1/12) in accordance with focal alopecia on the abdominal region. In the lack of inflammatory lesions, Trichophyton or Microsporum bodies or Demodex mites, this finding was considered as an individual, non-infectious lesion, having no toxicological relevance.
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):
There were no significant differences with respect to the control in the thyroid hormone (free T4 and TSH) levels in parental male animals at any dose level.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle was similar in the control and test item administered animals at 100, 300 and 1000 mg/kg bw/day.
There were no statistically significant or biologically relevant 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 number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus, in number or percentage of animals in prolonged estrous or diestrous during the pre-experimental and pre-mating periods.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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.
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 no difference between the control and test item treated groups in copulatory and fertility indices (male and female animals), in the pre-coital interval, conceiving days and gestation indices.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
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) at 100, 300 and 1000 mg/kg bw/day was higher than in the control group on postnatal day 0. However, these signs were considered to be toxicologically not relevant due to the lack of dose dependency and 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: cyanotic skin (1% at 100 mg/kg bw/day; 1% at 1000 mg/kg bw/day), hemorrhage on the neck (1 % at 1000 mg/kg bw/day), smaller than normal pup (1% in the control; 1% at 300 mg/kg bw/day), uncleaned pup (1 % at 300 mg/kg bw/day) and intact umbilical cord (1 % at 300 mg/kg bw/day). These findings were considered to have no toxicological relevance.
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 and cannibalized pups) per litter.
Body weight and weight changes:
no effects observed
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:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no significant differences with respect to the control in the thyroid hormone (free T4 and TSH) levels in offspring sampled on postnatal day 13 at any dose level.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The test item administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; respectively to uncorrected doses of 115, 345 and 1149 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.
Executive summary:

A study according OECD TG 422 was performed 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 to rats. Blue Sema was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 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 (distilled water) treated animals (n= 12/sex) served as a control. Blue Sema was stable in distilled water in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day and in a refrigerator for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Blue Sema concentrations in the dosing formulations varied within the range between 89% and 109% of 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 50 days). Females were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-57 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 and TSH) 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).

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.

No mortality was observed 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 the detailed weekly clinical observations nor 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. 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 and 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. Dark blue content was observed in the cecum and colon of some male animals at 1000 mg/kg bw/day and of one male at 300 mg/kg bw/day.

The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 or 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.

Based on these observations the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats was 1000 mg/kg bw/day and the NOAEL for F1 Offspring was 1000 mg/kg bw/day.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For justification please refer to Read Across Statement in IUCLID section 13.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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:
OECD TG 422 (Read Across)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A study according to OECD TG 422 with the read across substance Blue Sema (CAS No. 1040873-93 -5) is available. Blue Sema was administered orally (by gavage) once daily at doses of 0, 100,300 and 1000 mg dye /kg bw/day(corresponding t0 0, 115, 345 and 1150 mg test item/kg bw/d)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 (distilled water) treated animals (n=12/sex) served as a control. Blue Sema was stable in distilled water in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day and in a refrigerator for 3 days.The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Blue Sema concentrations in the dosing formulations varied within the range between 89% and 109% of 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 50 days). Females were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-57 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 and TSH) 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).

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.

No mortality was observed 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 the detailed weekly clinical observations nor 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. 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 and 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. Dark blue content was observed in the cecum and colon of some male animals at 1000 mg/kg bw/day and of one male at 300 mg/kg bw/day.

The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 or 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.

Based on these observations the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats was 1000 mg/kg bw/day and the NOAEL for F1 Offspring was 1000 mg/kg bw/day.

Effects on developmental toxicity

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:
OECD TG 422 (Read Across)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available information is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.