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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 11 to June 05, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
validated analytical method

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Direct Blue 267
IUPAC Name:
Direct Blue 267

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop ZRT. 1103 Budapest, Hungary.
- Hygienic level: SPF (specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females: nulliparous, non-pregnant females.
- Age at study initiation: male animals 94 - 99 days; female animals 94 - 99 days.
- Weight at study initiation: male animals 362 - 450 g; female animals 215 - 264 g. The weight variation did not exceed ± 20 % of the mean weight.
- Housing: before mating 2 animals of the same sex/cage; during mating period 1 male and 1 female / cage; pregnant females individually; males after mating 2 animals / cage.
- Cage type: type III polypropylene/polycarbonate (22 × 32 × 19 cm).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
- Water: animals received tap water, ad libitum, as for human consumption from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service.
- Acclimation period: 34 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
FORMULATION
The test item was formulated in the vehicle (distilled water) in concentrations of 18, 53 and 159 mg/ml by the active ingredient content (corresponding to to uncorrected concentrations of 20, 60 and 180 mg/ml, respectively).
Details on mating procedure:
Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 5 ml of each formulation and five aliquots of 5 ml control substance (vehicle) were taken and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 91 and 103 % of the nominal values at both analytical occasions.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. Recovery of the test item from water at ca. 1 and ca. 200 mg/ml concentrations in water was 101 and 99 % of nominal concentrations, respectively.
The test item was stable in water at 1 and 200 mg/ml concentrations at room temperature for one day (100 % and 101 % of nominal concentrations, respectively) and in a refrigerator (5 ± 3 °C) for three days (98 % and 97 % of nominal concentrations, respectively).
Duration of treatment / exposure:
48-55 days
Frequency of treatment:
7 days per week
Details on study schedule:
- Mating phase: started after 14-days treatment (pre-mating) period.
- Male treatment: males were dosed for 48 days (14 days pre-mating and 1-4 days mating plus 30-33 days of post-mating period until the necessary number of pregnant female animals was evident); then they were sacrificed.
- Female treatment: females were dosed for 14 days pre-mating, through 1-4 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice. The day of birth (viz. when parturition is complete) was defined as day 0 post-partum.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
corresponding to 88.3 mg a.i./kg bw/day
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
corresponding to 265 mg a.i./kg bw/day
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Remarks:
corresponding to 795 mg a.i./kg bw/day
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of the experimental period: the experimental period involved 34 days of acclimatization (including 14 days for examination of estrous cycle) and 48-55 days treatment/observation period (depending on the effectiveness of mating) and necropsy days.

Examinations

Parental animals: Observations and examinations:
MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time, considering the peak period of anticipated effects after dosing.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT
All parental animals were weighed with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically. Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION
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, and post-mating days 20, 27, 34, 41 and 48 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

EXAMINATION OF PLACENT SIGNS
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If negative on day 13, the examination was repeated on day 14 of gestation.

THYROID HORMONES
Blood samples for assessment of serum thyroid hormones (T4 and TSH) were collected from all parental male and female animals. All animals were subjected to a full detailed gross necropsy. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all males were weighed.
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH) as follows:
- from all dams and at least two pups per litter on post-natal day 13;
- from all parent male animals at termination on day 48;
- from all non-pregnant females at termination on day 48.
Blood samples collected for thyroid hormones (T4 and TSH) measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A (no anticoagulant). Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. All samples for T4 and TSH determination were stored between minus 15 and minus 20 °C until measurement.
Blood samples from the day 13 pups and the adult males were assessed for serum levels of thyroid hormones (T4 and TSH). Further assessment of T4 and TSH in blood samples from the dams and day 4 pups were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals). The quantitative determination of thyroid hormones (T4 and TSH) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.

OBSERVATION OF THE DELIVERY PROCESS AND CARE OF OFFSPRING
Delivery process was observed as carefully as possible. All observations and any evidence of abnormal deliveries were recorded. The duration of gestation was recorded and is calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment started from each animal for two weeks. Animals exhibited typical 4-5 days cycles were included in the study preferably. Vaginal smears were also 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 mating.
Vaginal smear also was prepared on the day of the necropsy.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope.
Litter observations:
LITTER EXAMINATION
Each litter was examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities. On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
Live pups were counted, sexed and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on days 4 and 13 post-partum with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
All litters were checked and recorded daily for the number of viable and dead pups.

THYROID HORMONES
Blood samples for serum T4 and TSH assessment were pooled from at least two pups per litter on postnatal day 4 (if it was feasible) and on postnatal day 13.
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH) as follows:
- from at least two pups per litter on post-natal day 4 (if it was feasible) - pup blood was pooled by litter;
- from all dams and at least two pups per litter on post-natal day 13;
- from all parent male animals at termination on day 48;
- from all non-pregnant females at termination on day 48.
Blood samples collected for thyroid hormones (T4 and TSH) measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A (no anticoagulant). Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. All samples for T4 and TSH determination were stored between minus 15 and minus 20 °C until measurement.
Blood samples from the day 13 pups and the adult males were assessed for serum levels of thyroid hormones (T4 and TSH). Further assessment of T4 and TSH in blood samples from the dams and day 4 pups were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals). The quantitative determination of thyroid hormones (T4 and TSH) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.
Postmortem examinations (parental animals):
SACRIFICE
All other animals (one day after the last treatment) were euthanized by exsanguination after verification of deep narcosis by Isofluran CP®.

GROSS NECROPSY
Gross necropsy was performed on each animal.
One female animal (no. 429) at 795 mg/kg bw/day was found dead and was subjected to necropsy on Day 46.
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.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with pharynx.
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
All organs showing macroscopic lesions were preserved in 4 % buffered formaldehyde solution.
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved.

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 adult male animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported. The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examinations were performed on the ovaries, testes, epididymides – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure – in the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
These organs were also processed and examined histologically in non-pregnant females and males these females cohabited with in the low and mid dose groups.
In addition, the following organs were processed and examined due to the macroscopic findings:
- trachea of one male (no. 308) in the mid dose group,
- liver of one male (no. 401, erythematous, small lobe) in the high dose group,
- uterus of dams and non-pregnant females (no. 129, 130, 221, 223, 226, 227, 229, 231, 322, 323, 327, 328, 329, 428, 431) in the control, low, mid and high doses,
- mammary gland of one female (no. 422, swollen teats) in the high dose group,
Full histopathology was performed on one found dead female (no. 429) in the high dose.
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Postmortem examinations (offspring):
GROSS NECROPSY
Dead pups were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4 and the litter weight was calculated for evaluation on postnatal day 4.
The number of nipples/areolae in male pups was counted on postnatal day 13.

MILK CONSUMPTION
The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
- Copulatory index: measure of animals’ ability to mate.
- Fertility index: measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
- Gestation index: measure of pregnancy that provides at least one live pup.
Offspring viability indices:
- Post-implantation / pre-natal mortality (intrauterine mortality)
- Post-natal mortality / extra-uterine mortality
- Survival Index
- Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of systemic toxicity related to the test item were not detected at any dose level either at the daily or at the detailed weekly clinical observations.
Alopecia on the chest (1 cm in diameter) was noted for one female animal in the control group from gestation day 12 until termination.
Alopecia (at first on the hindlimbs only, later on the forelimbs and abdomen) was observed in one female animal at 265 mg/kg bw/day from day 15 until termination.
In male animals at 265 mg/kg bw/day, nuzzling up the bedding material (6/12) was observed from days 3-6 to days 13-18.
At 795 mg/kg bw/day, nuzzling up the bedding material was transiently noted for 12/12 male animals and 12/12 female animals (males: from days 3-6 to days 18-21, females: from days 3-5 to days 12-13). Blue colored eyes, scrotum, forelimbs and hindlimbs were observed in this group for 12/12 male animals between days 41 and 47. Swelling was observed around the two posterior teats on the left side in the inguinal region in one female animal (1/12) in the high dose group on lactation days 14 and 15.
One female (1/12) at 795 mg/kg bw/day was found dead – as described above – on lactation day 8 (day 46).
Dark stool was detected in the bedding material in each cage of the 88.3, 265 or 795 mg/kg bw/day groups (male and female) from day 1 until termination. The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.

The behavior and physical condition of animals were considered to be normal in most of male and female animals in the control, at 88.3, 265 and 795 mg/kg bw/day during the detailed weekly clinical observations.
Alopecia – as described above – was detected in two female animals (1/12 in control group, 1/12 at 265 mg/kg bw/day) at the detailed weekly observation, too.
At 795 mg/kg bw/day, blue colored eyes, scrotum, forelimbs and hindlimbs were observed in all male animals (12/12) on days 41 and 48.
Piloerection was observed in one female animal (1/12) at 795 mg/kg bw/day on lactation day 4. This animal was found dead on lactation day 8 – as described above.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality at 88.3, 265 or 795 mg/kg bw/day groups during the course of study (male and female).
One female animal (no. 429) at 795 mg/kg bw/day was found dead on lactation day 8 (day 46). Piloerection, decreased body tone, red discharge on the hair around the eyes, nose and on the legs was observed in this dam from lactation day 4 or 5 until lactation day 7. Histological examination revealed serious diffuse catarrhal-purulent pneumonia as the probable cause of death. This lesion could be considered as an individual disease and not related to the test item. Dark stool was detected in the bedding material in the cage from day 1 until the death. The color of stools was indicative of presence of the test item or its metabolite(s) in the intestinal tract. Nuzzling up the bedding material was also detected for this animal between day 5 and 12 in the premating period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or in female animals at 88.3, 265 or 795 mg/kg bw/day during the entire treatment period. Slightly depressed body weight gain was observed at 795 mg/kg bw/day in male animals resulting in slight body weight difference with respect to their control (<10 %). These slight changes in bodyweight gain were considered to be toxicologically not relevant.
The mean body weight and body weight gain were similar in male animals in the control and at 88.3 and 265 mg/kg bw/day groups.
Statistical significance with respect to the control was detected in male animals at 795 mg/kg bw/day at the lower mean body weight gain between days 27 and 34 and between days 0 and 48 (summarized body weight gain).
The mean body weight and body weight gain were similar in female animals in the control and in the 88.3 and 265 mg/kg bw/day groups during the course of premating, gestation and lactation periods.
Statistical significance was observed at the higher mean body weight gain in dames at 795 mg/kg bw/day between gestation days 7 and 14.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item treatment related changes in the food consumption of male or female animals at any dose level (88.3, 265 and 795 mg/kg bw/day).
The mean food consumption was comparable in male and female animals in the control and 88.3, 265 and 795 mg/kg bw/day groups during the entire observation period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examinations revealed test item related changes in the testes and epididymides at 795 mg/kg bw/day.
Tubular degeneration and necrosis (i.e. degeneration and necrosis of all cellular components of all tubuli) in both testes of all males was observed at 795 mg/kg bw/day (12/12). The interstitium of testes was not affected. Slight fibrosis and focal mineralization were observed in the altered tubuli. The diameter of tubuli and the weight of the organ were also decreased.
Lack of mature spermatozoa and decrease of diameter of tubuli was noted in the tubuli of epididymides of all males at 795 mg/kg bw/day (12/12).
The histological picture of accessory sex glands (prostate, seminal vesicles with coagulating glands) was normal.
Focal inflammation in the trachea was observed in one male animal (1/1) at 265 mg/kg bw/day.
Congestion in the liver was noted for one male animal (1/1) at 795 mg/kg bw/day.
No histological lesions (degeneration, inflammation etc.) in the different visceral organs or stomach, small or large intestines (see the histological results of dead animal no.: 429) were detected in connection with the macroscopically observed blue discoloration of different tissues.
No testicular or other test item related lesions were observed in the investigated organs of male animals, in the control (12/12), at 88.3 mg/kg bw/day (1/1) or 265 mg/kg bw/day (3/3).

In the female animals, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was observed in several female animals (1 dam and 1 non-pregnant female in the control group; 5 dams at 88.3 mg/kg bw/day; 3 dams and 2 non-pregnant females at 265 mg/kg bw/day; 2 dams at 795 mg/kg bw/day). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and could be in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
One female animal at 795 mg/kg bw/day (animal no. 429) was found dead during the study on lactation day 8 (day 46). Histological examination revealed serious diffuse catarrhal-purulent pneumonia as the probable cause of death. This lesion could be considered as an individual disease without toxicological significance. No degenerative or other possible treatment related lesions were detectable in the different organs of this animal. Zonal congestion was observed in the kidney of this animal.
Mastitis was detected of one female (1/1) at 795 mg/kg bw/day. This was considered an individual disease.
No histological lesions (degeneration, inflammation etc.) in the different visceral organs or stomach, small or large intestines were detected in connection with the macroscopically observed blue discoloration of the different tissues of several animals based on the examination of the dead animal (animal no. 429, full histopathology performed).
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4 and TSH) levels in parental male animals or in PN13 offspring at any dose levels.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Test item influence on the estrous cycle was not detected at any dose level (88.3, 265 and 795 mg/kg bw/day).
The number and percentage of animals with irregular cycles and in prolonged diestrous were higher than in the control group at 265 and 795 mg/kg bw/day during the pre-experimental period; the percentage incidence of these findings resulted to be reduced during the pre-mating period.
There were no statistically or biologically significant differences between the control and test item treated groups (88.3, 265 and 795 mg/kg bw/day) in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrous during the pre-experimental and pre-mating periods.
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 in male or female animals at 88.3, 265 or 795 mg/kg bw/day.
There were no significant differences between the control and test item treated groups (88.3, 265 or 795 mg/kg bw/day) in the copulatory and fertility indices (males and females) and in the gestation indices (females).
The percentage of pregnant females, dams delivered, pregnants with liveborn(s) and the pre-coital interval and the mean number of conceiving days were comparable in female animals of control and test item treated animals.

Details on results (P0)

DELIVERY DATA OF DAMS - effects observed, non-treatment-related
There were no relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (88.3, 265 or 795 mg/kg bw/day).
The mean number of implantation sites per dams were comparable in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the percentage of viable pups on post-partum day 0, in the percentage of dams with viable offspring on post-partal day 0 or in the live birth indices.
The mean number of total births and liveborns was slightly lower in the control group than in dams at 795 mg/kg bw/day without statistical significance. These values met well the historical control data. The mean number of stillborn and the mean post-implantation loss was slightly higher at 795 mg/kg bw/day when compared to the control group without statistical significance. Therefore, these minor differences between the control and test item treated groups were considered to have no or little toxicological relevance.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 265 - < 795 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: During necropsy it was found that the reproductive organs of all males of the high dose group were blue coloured - indicating accumulation of the test item and/or its metabolite(s).
Key result
Dose descriptor:
NOAEL
Effect level:
795 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: systemic toxicity
Remarks on result:
other: highest tested dose
Key result
Dose descriptor:
NOAEL
Effect level:
795 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: highest tested dose

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
795 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
seminiferous tubules
testes
other: During necropsy it was found that the reproductive organs of all males of the high dose group were blue coloured - indicating accumulation of the test item and/or its metabolite(s).
Treatment related:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of offspring with signs at 795 mg/kg bw/day was higher than in the control group on postnatal day 0. However, the observed signs were considered to be toxicologically not relevant as they were transient – detected only shortly after the delivery – and were not associated with depression on the development of the offspring.
Occasionally, other clinical signs were also observed: pale and cyanotic skin, which however were considered to have no toxicological relevance.
For one dam at 795 mg/kg bw/day (no. 424) 6 pups were uncleaned; inadequate nursing was observed for this dam.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
There were no statistically significant differences between the control and test item treated groups (88.3, 265 or 795 mg/kg bw/day) in the mean number of dead offspring (including missing pups) per litter. The mean number of dead pup was higher than in the control in the high dose group between post-natal day 0 and 13. The pup mortality (total of 14 dead pups) in this group belonged to only two dams: one of them (no. 429c) was found dead – due to an individual disease without toxicological relevance – on lactation day 8 and its 8 pups also died by this day; the other dam (no. 424) lost weight in the lactation period, the nursing was inadequate and its 6 pups died on lactation days 1-2. Therefore, this difference was considered to have no or little toxicological relevance.
There were no significant differences between the control and test item treated (88.3, 265 or 795 mg/kg bw/day) groups in the survival indices. However, the survival index on post-natal day 4 was slightly lower in the high dose group than in the control based on the above-mentioned case (no. 424).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and pup weights as well as the mean litter weight gain and pup weight gain were similar in the control and in all test item treated groups (88.3, 265 and 795 mg/kg bw/day) on postnatal days 0, 4 and 13.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not affected by the test item treatment at 88.3, 265 and 795 mg/kg bw/day.
Statistical significances were observed at the shorter anogenital distances (AGD) in male pups of the 88.3 (absolute and normalized AGD), 265 (normalized AGD) and 795 mg/kg bw/day (absolute and normalized AGD), and in female pups at 88.3 and 795 mg/kg bw/day (absolute and normalized AGD). These minor changes, in the lack of dose relationship, were considered to be indicative of biological variations and not related to the test item.
Nipples/areoles were not visible in any of the examined male offspring in the control or 88.3, 265 or 795 mg/kg bw/day groups on postnatal day 13.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
Slightly autolyzed visceral organs and empty stomach were detected in one dead pup (1/1) in the control on post-natal day 1.
One male pup (1/2) at 265 mg/kg bw/day was pale and partially cannibalized on postnatal day 0.
There were no macroscopic changes in the organs or tissues of one dead pup (1/2) at 265 mg/kg bw/day on post-natal day 0 and three dead pups (3/9) at 795 mg/kg bw/day on post-natal day 2.
Six pups of one dam at 795 mg/kg bw/day (6/9) was cachectic with empty stomach at the time of necropsy on postnatal day 7 or 8.
Four pups (one male at 265 mg/kg bw/day and three females at 795 mg/kg bw/day) had no macroscopic changes in the organs or tissues and the lung flotation test was negative referring to intrauterine death of these offspring subjected to necropsy on postnatal day 0.
Description (incidence and severity):
SEX DISTRIBUTION
There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0, 4 or 13.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 795 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: highest tested dose

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
795 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

Applicant's summary and conclusion

Conclusions:
NOAEL (male rats) = 265 mg a.i./kg bw/day (toxicity observed in male reproductive organs at 795 mg/kg/day)
NOAEL (female rats) = 795 mg a.i./kg bw/day
NOAEL for F1 offspring: 795 mg a.i./kg bw/day
NOAEL for reproductive performance of male/female rats: 795 mg a.i./kg bw/day (reproductive performance of male rats is evaluated upon 14-18 days of dosing).
Executive summary:

The toxic potential of test item on reproduction and development was investigated, when repeatedly administered orally (by gavage) to rats at doses of 88.3, 265 and 795 mg/kg bw/day as a.i. corresponding to doses of 100, 300 and 900 mg/kg bw/day as test material.

Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered orally once daily at the concentrations of 18, 53 and 159 mg/ml as active ingredient content, corresponding to test substance concentrations of 20, 60 and 180 mg/ml. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the vehicle for the test item was analytically verified and the substance resulted to be stable; the concentration of test item in the dosing formulations was checked two times during the study and the test item concentrations varied in the acceptable range between 91 % and 103 % of the nominal values, 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 were dosed after mating up to the day before necropsy (altogether for 48 days). Females were additionally dosed through the gestation period and up to lactation days 12 -15, i.e. up to the day before necropsy (altogether for 48 -55 days; one female animal at 795 mg/kg bw/day was administered for 46 days due to its early death). 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 mating. Blood samples were collected for 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 on post-partum/post-natal day 13 and from all parent male animals and from non-pregnant females at termination (between days 48 and 55).

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.

All parental animals were subjected to gross pathology one day after the last treatment. Body weight, brain weight and weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole 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 (at day 13) for the possible subsequent histopathological examination.

Histopathology examination was performed on ovaries, testes, epididymides in the control and high dose groups. These organs were also processed and examined histologically in non-pregnant females and males these females cohabited with in the low and mid dose groups.

Full histopathology was performed in dead dam (no. 429) at 795 mg/kg bw/day.

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

There was no test item related mortality at any dose level (88.3, 265 and 795 mg/kg bw/day). One female animal at 795 mg/kg bw/day died on lactation day 8 (day 46) due to serious diffuse catarrhal-purulent pneumonia; the lesion could be considered as an individual disease and not related to the test item.

Clinical signs of systemic toxicity related to test item were not detected at any dose level. Body weight development and daily food consumption were not adversely affected.

A test item influence on the estrous cycle was not detected at any dose level (88.3, 265 and 795 mg/kg bw/day).

There were no toxicologically relevant differences between control and treated groups in the evaluated delivery parameters of dams.

There were no test item related differences between control and treated groups in the examined parameters of reproductive performance of male and female animals.

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).

Smaller than normal testes and epididymides (male) and erythema between the cortex and medulla of the kidneys (male and female) were observed at 795 mg/kg bw/day as test item related changes. It has to be noted that testes and epididymides of all high dose males (12/12) was described during necropsy as blue colored - indicating accumulation of the test item and/or its metabolite(s). Also kidney as well as other visceral organs were blue colored in all males and females of the high dose group. Presence of the test item or its metabolite(s) in the stomach and/or intestines were detected in several male and female animals (265 and 795 mg/kg bw/day) at the gross macroscopic observation.

The weight of testes, epididymides and seminal vesicle was slightly lower than in the control group in male animals at 795 mg/kg bw/day in accordance with the necropsy and histopathology findings.

Histopathological examinations revealed degeneration and necrosis of different stages of spermatogenesis in the testes of all male animals, accompanied with lack of mature spermatozoa in the tubuli of epididymides at 795 mg/kg bw/day. Degeneration and necrosis of all cellular components of all tubuli, slight fibrosis and focal mineralization in the tubuli in both testes, decreased diameter of tubuli in the testes and epididymides were observed in these animals.

All such findings in males only were observed at the high dose level where blue coloration of reproductive organs indicated accumulation of the test item and/or its metabolite(s).

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

Conclusion

Under the conditions, the substance caused changes in the testes (smaller than normal, reduced weight, tubular degeneration and necrosis), in epididymides (smaller than normal, reduced weight, lack of mature spermatozoa, decreased diameter of tubuli) and in seminal vesicles (reduced weight) at 795 mg/kg bw/day (corrected dose respectively to uncorrected dose of 900 mg/kg bw/day) administered by oral gavage to Han:WIST rats. However, blue coloration of the reproductive organs indicated accumulation

of the test item and/or its metabolite(s).

There were no test item related changes in male or female animals at 88.3 or 265 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

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

NOAEL (male rats) = 265 mg a.i./kg bw/day

NOAEL (female rats) = 795 mg a.i./kg bw/day

NOAEL for reproductive performance (male/female rats) = 795 mg a.i./kg bw/day

NOAEL for F1 offspring = 795 mg a.i./kg bw/day