Registration Dossier

Administrative data

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.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25 January 2018 - 23 March 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
14 days exposure instead of 28 days
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
yes
Remarks:
only 14 days exposure
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
yes
Remarks:
only 14 days exposure
Qualifier:
according to
Guideline:
other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents, EPA Health Effects Test Guidelines
Version / remarks:
2000
Deviations:
yes
Remarks:
only 14 days exposure
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests and is a well-known laboratory model with sufficient historical data.
Sex:
male/female
Details on test animals 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: 48 – 53 days, Female animals: 63 – 68 days
- Weight at study initiation: 202 – 223 g for male animals, 145 – 176 g for female animals
- Housing: 2-3 animals of the same sex/ cage (Type II polypropylene/ polycarbonate)
- 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: 13 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 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
- 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)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical dose verification of the formulations was performed once during the study. Five aliquots of 5 mL of each formulation to be administered to the animals (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: 95 % at ca. 1 mg/mL and 103 % at ca. 200 mg/mL). The test substance proved to be stable in distilled water at the intended concentrations at room temperature for 24 hours and at 5+/-3 °C for three days.
Duration of treatment / exposure:
14 days
Frequency of treatment:
7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 0, 100, 300 and 1000 mg/kg bw/day was based on the literary data of chemically similar compounds. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.

Positive control:
no
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: Day 0 (prior to study start) and twice weekly (on Days 0, 4, 7, 10 and 13)

FOOD CONSUMPTION: Yes
- Time schedule: determined once weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

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: all
- 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: all
- Parameters checked in table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
The organs listed in table 4 were removed and preserved.

HISTOPATHOLOGY: No
The organs listed in table 4 were removed and preserved. Histopathological examinations were not performed because no adverse effects were observed up to the top dose applied.
Other examinations:
Organ Weight
The following organ weights were determined and recorded: paired organs were weighed together: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands, as a whole, adrenal glands.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data: body weight, food consumption, hematology, blood coagulation, clinical chemistry and organ weight.
The heterogeneity 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 was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Clinical signs:
no effects observed
Description (incidence and severity):
The test item did not cause clinical signs at 100, 300 or 1000 mg/kg bw/day in surviving male or female animals during the course of 14-day administration. The behavior and physical condition of all animals (male and female) were normal during the 14-day observation period. Clinical signs of dead female animal (see below) were not considered to be related to the test item due its singular occurrence. Transiently diarrhea was noted for one male animal at 300 mg/kg bw/day (1/5) on Day 2 but there were no clinical signs during the following days.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal (1/5, no. 42) at 1000 mg/kg bw/day was found dead on Day 10. Preceding clinical signs – decreased activity, dyspnea and noisy breathing – and decrease in the body weight were noted for this animal from Day 4 up to and including Day 9. The organs and tissues were significantly autolyzed therefore detection of necropsy findings was not feasible. There was no mortality in the control, 100 or 300 mg/kg bw/day groups during the 14-day treatment period (male and female).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (100, 300 or 1000 mg/kg bw/day) throughout the entire observation period. The mean body weight and mean body weight gain was similar in male animals in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) during the entire observation period. The body weight of the dead female animal decreased between Days 4 and 7. A slight, but statistically significant difference with respect to the control was detected at the slightly higher mean bodyweight gain of female animals at 100 mg/kg bw/day between Days 4 and 7. 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):
no effects observed
Description (incidence and severity):
There were no test item related changes in the mean daily food consumption at 100, 300 or 1000 mg/kg bw/day. The mean daily food consumption was similar in the control and test item treated groups (male and female) during the 14-day observation period.
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):
There were no test item related alterations in the examined hematological parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day compared to their controls.
Slight but statistically significant differences compared to the control group were noted in some of the white blood cell parameters in male animals as follows: elevated mean white blood cell count (WBC) and lower mean percentage of neutrophil granulocytes (NEU) at 100 and 1000 mg/kg bw/day; lower mean percentage of basophil granulocytes (BASO) at 300 mg/kg bw/day; slightly higher mean percentage of lymphocytes (LYM) and lower mean percentages of monocytes (MONO) at 1000 mg/kg bw/day. In the female animals, statistical significance was detected at the slightly lower mean percentage of reticulocytes (RET) at 100 and 300 mg/kg bw/day as well as at the slightly longer mean prothrombin time (PT) at 100 mg/kg bw/day when compared to the concurrent control. These slight but statistically significant differences with respect to control (WBC, NEU, BASO, LYM and MONO in male animals and RET, PT in female animals) were considered to be toxicologically not relevant as these were at a low magnitude, were not related to doses or were only present in the low or mid dose groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry evaluation did not reveal test item related changes in the investigated parameters at 100, 300 or 1000 mg/kg bw/day (male and female). The examined clinical chemistry parameters were similar in male animals in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day). Slight but statistically significant differences with respect to the control were detected at the lower mean concentration of albumin (ALB) and albumin: globulin ratio (A/G) at 100 mg/kg bw/day and at the higher mean glucose concentration at 300 and 1000 mg/kg bw/day. These findings in the clinical chemistry parameters were judged to be indicative of biological variation and of no biological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related organ weight changes were not observed in male and female animals at 100, 300 or 1000 mg/kg bw/day. In male animals, statistical significances were noted for the slightly lower heart weights (absolute and relative to body and brain weights) at 100 and 300 mg/kg bw/day and at the higher mean weights of seminal vesicle-coagulating gland-prostate complex (absolute and relative to body and brain weights) at 100 mg/kg bw/day when compared to the relevant control. In the female animals, there were no statistically or biologically significant differences between the control and treated groups (100, 300 or 1000 mg/kg bw/day) in the weights of examined organs. Changes in the weights of the above mentioned organs were of low degree and not related to doses. Therefore these findings were judged to be toxicologically not relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related macroscopic findings were observed in male or female animals (100, 300 or 1000 mg/kg bw/day). In the female animal found dead, autolysis of visceral organs was seen therefore macroscopic observation of tissues was not possible. There were no macroscopic changes in the organs or tissues of male animals in the control 100, 300 or 1000 mg/kg bw/day groups at the necropsy. Slight or moderate hydrometra was observed in single female animals in control (1/5) group and at 300 mg/kg bw/day (1/5). Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs it was judged to be toxicologically not relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of the present study, the test item did not cause adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day.
Executive summary:

A study was conducted to obtain first information on the toxic potential of the test item in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study according to TG OECD 422). Doses of 0 (vehicle only), 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/day; corrected doses) were orally administered (gavage) to four groups of Hsd.Han:Wistar rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg. Blue Sema was administered in concentrations of 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 23, 69 and 230 mg/mL). A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. Detailed clinical observations were performed daily after the treatment and before the necropsy. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed. One male animal at 1000 mg/kg bw/day was found dead on study Day 10. Dyspnea, noisy breathing and decreased activity were observed on the preceding day (from Days 4 to 9).The test item did not induce clinical signs at 100, 300 or 1000 mg/kg bw/day in surviving male or female animals during the two weeks treatment period. No test item-related body weight or body weight gain changes were observed at 100, 300 or 1000 mg/kg bw/day in male or female animals. The mean daily food consumption was comparable in the control and test item treated groups – 100, 300 or 1000 mg/kg bw/day, male and female. Hematological evaluation did not reveal test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day (male and female). There were no test item related alterations in the examined clinical chemistry parameters (100, 300 or 1000 mg/kg bw/day, male or female). Specific macroscopic findings were not detected in the organs or tissues of male or female animals at 100, 300 or 1000 mg/kg bw/day. The weights of examined organs were not affected by the treatment with 100, 300 or 1000 mg/kg bw/day dose of the test item. Therefore, under the conditions of the present study, the test item did not cause adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day. A NOAEL of 1000 mg/kg bw/d was determined.

Endpoint:
short-term repeated dose toxicity: oral
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 w ith the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
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
Details on route of administration:
The test item was administered orally, by gavage. The route of application is selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.
Vehicle:
water
Details on oral 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

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:
no
Observations and examinations performed and frequency:
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 weight of the female animals were additionally weighed 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 also be 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:
- Time schedule for collection: Blood samples were collected for determination of serum levels of thyroid hormones (T4, TSH) from all male animals at termination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.

HISTOPATHOLOGY: Yes
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, adrenal glands and pituitary and all organs showing macroscopic lesions of all adult animals were preserved. All organs showing macroscopic lesions and the the organs listed in Table No. 4. were preserved for five male and five female animals randomly selected from each group.
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 were determined. Absolute organ weight were reported. Relative organ weight (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 weight will be reported. Relative organ weight (to body and brain weight) were calculated and reported.
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.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
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
Description (incidence and severity):
In the male animals, there were no statistically significant or biologically relevant 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.
In the female animals at 300 mg/kg bw/day, the mean liver weights (absolute and relative to body weight) were slightly higher than in the control group.
In the female animals at 300 and 1000 mg/kg bw/day, statistical significance was noted for the slightly higher mean kidney weights (absolute only) when compared to the control group. A dose-dependent increase was observed; however, the increased kidney weight was not correlated with any histopathological findings.
These slight differences with respect to the control were considered to have no toxicological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic findings in the organs or tissues in male animals in the control group (12/12).
In one male animal dosed with 100 mg/kg bw/day (1/12), hemorrhage in the thymus was observed.
At 300 mg/kg bw/day, in one male animal (1/12), the left side seminal vesicle was smaller than normal. Pea-size hemorrhage on the right lung and dark blue content in the cecum and colon were observed in one male (1/12) of this dose.
In five male animals at 1000 mg/kg bw/day (5/12), dark blue content in the cecum and colon was observed. One of these males (1/12) had pea-size hemorrhage on one right lobe of lungs. There were no macroscopic findings in the organs or tissues in female animals in the control and 100 mg/kg bw/day groups (12/12 in each).
In one female animal at 300 mg/kg bw/day, marked hydrometra was observed (1/12). Alopecia on the abdomen was noted for single female animals at 300 mg/kg bw/day (1/12) and at 1000 mg/kg bw/day (1/12). The cecum was smaller than normal and its wall was thickened at 300 and 1000 mg/kg bw/day (1/12, both).
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats.
The smaller than normal seminal vesicle had decreased amount of secrete, without any other pathological lesion.
Due to the lack of related histopathological alterations (inflammatory or other pathological lesion) these findings were considered to be toxicologically not relevant.
The macroscopically blue discoloration of intestinal content and the thickened cecum wall were not correlated with abnormal histological finding in the mucous membrane, therefore it was considered to have no toxicological relevance.
Neuropathological findings:
not examined
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 non-treated 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 or in offspring sampled on postnatal day 13 at any dose level.

Offspring:
There was no test item related effect on offspring’s extra uterine mortality. Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13. The anogenital distances (in male or female offspring) or nipple retention (male) were similar in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day). The body weight development of the offspring was unaffected by the test item. Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
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
Critical effects observed:
no
Conclusions:
Blue Sema 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 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.
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:
short-term repeated dose toxicity: oral
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: other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting 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: other: absence of test item related adverse effects
Key result
Critical effects observed:
no
Endpoint conclusion
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)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No information on the repeated dose toxicity of the test item is available. However, suitable and reliable data on Blue Sema (CAS No. 1040873 -93 -5) is available which can be used in a read-across approach.

A study was conducted to obtain first information on the toxic potential of the test item in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study according to TG OECD 422). Doses of 0 (vehicle only), 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/day; corrected doses) were orally administered (gavage) to four groups of Hsd.Han:Wistar rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg. Blue Sema was administered in concentrations of 20, 60 and 200 mg/mL by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 23, 69 and 230 mg/mL). A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. Detailed clinical observations were performed daily after the treatment and before the necropsy. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed.One male animal at 1000 mg/kg bw/day was found dead on study Day 10. Dyspnea, noisy breathing and decreased activity were observed on the preceding day (from Days 4 to 9).The test item did not induce clinical signs at 100, 300 or 1000 mg/kg bw/day in surviving male or female animals during the two weeks treatment period. No test item-related body weight or body weight gain changes were observed at 100, 300 or 1000 mg/kg bw/day in male or female animals. The mean daily food consumption was comparable in the control and test item treated groups – 100, 300 or 1000 mg/kg bw/day, male and female. Hematological evaluation did not reveal test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day (male and female). There were no test item related alterations in the examined clinical chemistry parameters (100, 300 or 1000 mg/kg bw/day, male or female). Specific macroscopic findings were not detected in the organs or tissues of male or female animals at 100, 300 or 1000 mg/kg bw/day. The weights of examined organs were not affected by the treatment with 100, 300 or 1000 mg/kg bw/day dose of the test item. Therefore, under the conditions of the present study, the test item did not cause adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day. A NOAEL of 1000 mg/kg bw/d was determined.

In a study according to OECD TG 422 performed with the read across substance Blue Sema (CAS No. 1040873 -93 -5) the substancewas administered orally (by gavage) once daily at doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day (corresponding to 0, 115, 345 and 1150 mg test item/kg bw)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

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) 1272/2008 (CLP). As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008 as amended for the twelfth time in Regulation (EU) No 2019/521.