Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted on 28th July 2015

Deviations:

yes

Remarks:

not impacting the study results

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Direct Red 253

IUPAC Name:

Direct Red 253

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o.
- Age: males, females: sexually adult, 7-9 weeks on arrival.
- Weight at study initiation: males 383 - 3978 g; females 250 - 261 g.
- Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period; one male and one female in one cage, pregnant females; individually, offspring; with mother, satellite animals; 2 rats of the same sex in one cage.
- Bedding: sterilized soft wood fibers Lignocel.
- Diet: complete pelleted diet for rats and mice in SPF breeding - Altromin for Rats/Mice.
- Water: drinking water ad libitum.
- Acclimation period: at least 6 days. During the acclimatisation period the health condition of all animals was controlled daily. In all females cyclicity of oestrous cycle was monitored.
- Rationale for animal selection: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex did not exceed ± 20% of

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70 %
- Photoperiod:12 hour light / 12 hour dark the mean weight.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro injectione

Details on exposure:

PREPARATION OF APPLICATION FORM
The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test solution was dissolved in ultrasonic bath for a 30 minutes and then the solution was stirred by magnetic stirrer (800 rpm) for 40 minutes. The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.

Details on mating procedure:

Animals were mated from the 29th day of study. Mating 1: 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY AND HOMOGENEITY
The stability and the homogeneity of application form were determined in testing laboratory analytical laboratories (Analytical Group I).
Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a high-performance liquid chromatography based on a method developed at the test facility. Both application forms 10 mg and 1000 mg /10 ml of the test substance at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

Duration of treatment / exposure:

Parental males: totally 49 days of administration
Parental females: 13 days, pre-mating period
Non-pregnant females (with and without evidence of copulation): 13 days, pre-mating period
Satellite males and females: totally 49 days of administration + 14 days of observation

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control and satellite groups

Doses / concentrations 2

Dose / conc.:

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

Remarks:

tested group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

tested group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

tested and satellite groups

No. of animals per sex per dose:

12 females and 12 males per group
6 males and 6 females per satellite group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a Dose-Range Finding Experiment was performed. According to results of Dose-Range Finding Experiment the following dose levels 250, 500 and 1000 mg/kg/day were chosen for the main test.

Examinations

Parental animals: Observations and examinations:

Functional observations, haematology, biochemistry, full biometry and full pathology for evaluation of repeated dose toxicity will be performed only in 6 males + 6 females of each dose level and in satellite animals.

MORTALITY
All rats during the treatment periods were examined for vitality or mortality twice daily.

HEALTH CONDITION CONTROL
Experimental data collection: daily, during the acclimatization and the experimental part.

CLINICAL OBSERVATIONS
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 – 13.00 p.m.) – at the time of expectation of probable maximal effect of the test substance. Animals were observed in natural conditions in their cages.

Experimental data collection: males and females daily during the administration period.

DETAILED CLINICAL OBSERVATIONS
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina

Experimental data collection: before the first application and then weekly (except the mating period).

FUNCTIONAL OBSERVATION
This observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period.
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.

Experimental data collection: at the end of administration/observation period.

BODY WEIGHT
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too. Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

Experimental data collection:
males and satellite animals - the first day of administration and then weekly
females - the first day of administration and then weekly
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 1st, 4th day, 12th day and 13th day

FOOD CONSUMPTION
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food conversion in % (weight increment/food consumption x 100) was calculated for animals of Repeated Dose Toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females.

Experimental data collection: weekly and on the same days as body weight (except the mating period); satellite males and females weekly.

WATER CONSUMPTION
The drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

Experimental data collection: satellite males and females twice a week.

HAEMATOLOGY
Parameters: Total erythrocyte count , Mean corpuscular volume, Haematocrit, Haemoglobin concentration, Total leucocyte count, Total platelets count, Partial thromboplastin time, Prothrombin time, Fibrinogen, Granulocytes, Lymphocytes, Monocytes.
Reticulocytes were examined by light microscope. Blood and reticulocyte staining solution e.g. New Methylene Blue were mixed and incubated briefly at room temperature. Smears were made on microscope slides, air dried and evaluated under oil immersion on a light microscope.

Blood collection for haematology and biochemistry:
parental males – 64th day of study
satellite males – 78th day of study
parental females - 13th day of lactation period
satellite females – 78th day of study

BIOCHEMICAL ANALYSIS
Parameters: Protein total, Alkaline phosphatase, Cholesterol total, Triglycerides, Alanine aminotranferase, Aspartate aminotransferase, Creatinine, Urea, Albumin, Bilirubin total, Glucose, Calcium, Phosphorus, Cholinesterase, Bile acids, Sodium, Potassium and Chloride.
Blood samples from the day 13 the parental males were assessed for serum levels of thyroid hormone thyroxine (T4) by ELISA kit.

URINALYSIS
This examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for long time). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 ml of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined: Colour, Cloud, Odour, Glucose, Protein, Bilirubin, Urobilinogen, pH, Specific gravity, Blood, Ketones, Nitrite and Leucocytes.

Experimental data collection: only males – 63rd and 77th day of study. Experimental data collection: the last day of administration/observation period.

PATHOLOGICAL EXAMINATION
Experimental data collection: males and nonpregnant females after the end of application period; parental females on the 13th day of lactation; satellite animals after the end of observation period.

Oestrous cyclicity (parental animals):

Vaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of the oestrous cycle of females. Only females with regular cyclicity were put into the study.
Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa.
Vaginal smears were made also on necropsy day to determine the stage of oestrous cycle. Vaginal smears were prepared and examined according to internal SOP No. M/74.

Sperm parameters (parental animals):

In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/82.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared
sperm solution. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm solution was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck – were recorded

Litter observations:

CLINICAL OBSERVATIONS
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Experimental data collection: as soon as possible after delivery and then daily.

BODY WEIGHT
Experimental data collection:
pups (litters) - 1st , 4th day, 12th day and 13th day
pups – individually – 4th day of lactation

HAEMATOLOGY
Blood collection for haematology and biochemistry: 2 pups per litter 4th day of lactation; 2 pups per litter 13th day of lactation.

BIOCHEMICAL ANALYSIS
Parameters: Protein total, Alkaline phosphatase, Cholesterol total, Triglycerides, Alanine aminotranferase, Aspartate aminotransferase, Creatinine, Urea, Albumin, Bilirubin total, Glucose, Calcium, Phosphorus, Cholinesterase, Bile acids, Sodium, Potassium and Chloride.
Blood samples from the day 13 pups were assessed for serum levels of thyroid hormone thyroxine (T4) by ELISA kit.

PATHOLOGICAL EXAMINATION
Experimental data collection: 2 pups per litter on the 4th day of lactation; parental females and pups on the 13th day of lactation.

ANOGENITAL DISTANCE (AGD)
The AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalized to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.

NIPPLES EXAMINATION
The presence and number of nipples in male pups were counted on day 13 of lactation.

Postmortem examinations (parental animals):

NECROPSY
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4 % formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Experimental data collection:
parental males – 64th day of study
parental females - 13th day of lactation period
non-pregnant females – 26th day after the end of mating period or confirmed mating
satellite males and females – 78th day of study

BIOMETRY OF ORGANS
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected.
The absolute weights of testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

HISTOPATHOLOGY
The tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
Detailed histological examination was performed on testes of all high dose and control animals (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).
Samples of the following tissues and organs were collected at necropsy and fixed: Pituitary gland, Ovaries, Uterus, Cervix of uterus, Vagina, Epididymis, Prostate gland + Seminal vesicles, Testes, Thyroid gland and All gross lesions.

Postmortem examinations (offspring):

NECROPSY
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4 % formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Experimental data collection: 2 pups per litter – 4th day of lactation, other pups - 13th day of lactation

BIOMETRY OF ORGANS
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected.
The absolute weights of testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used.
Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.
The results statistically significant on probability level 0.05 are indicated.

Reproductive indices:

Pre-implantation loss: number of corpora lutea – number of implantations
Post-implantation loss: number of implantations – number of live births
Post-natal loss: number of live births – number of live pups at postnatal day 13

Male mating index: number of males with confirmed mating x 100; number of males cohabited.
Female mating index: number of sperm-positive females x 100; number of females cohabited
Male fertility index: number of males impregnating a females x 100; number of males cohabited
Female fertility index: number of pregnant females x 100; number of sperm-positive females
Gestation index: number of females with live born pups x 100; number of pregnant females

Offspring viability indices:

Survival index: number of live pups on day 13 post partum x 100; number of pups born alive

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Parental Males: in control males and treated males of all dose levels no signs of diseases were recorded during the whole study. Only coloured faeces were recorded. No clinical changes after application of the test substance were recorded in all treated animals. Only coloured faeces were recorded.

Parental Females: in control females and treated females of all dose levels no signs of diseases were recorded during the whole study. Only coloured faeces were recorded. No clinical changes after application of the test substance were recorded. Only coloured faeces were recorded.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Parental Males: there were no unscheduled deaths during the whole study.

Parental Females: female No.142 (the dose level 500 mg/kg/day) died on the sixteenth day of application due to intubation error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Findings considered of no toxicological significance.

Parental Males: body weight was slightly decreased in males at the highest dose level. Body weight increment of all treated males was not adversely affected by the test substance.

Parental Females Pre-mating period: Mean body weight of treated females in the groups 500 and 1000 mg/kg was slightly lover compared to the control group.
The mean body weight increments of the treated females of the dose level 1000 mg/kg was negative within the 1st week of application and vice versa the highest within the second week of application compared to control animals.

Parental Females Pregnancy
Females without parturition (non-pregnant females) were not included in the evaluation of mean body weight increments during pregnancy. Mean body weight of all treated groups was comparable to the control group except the pregnant female of the dose level 1000 mg/kg. The mean body weight increments of all treated pregnant females were similar to the control females.

Parental Females Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. Mean body weight of all treated groups was decreased in comparison with the control group. The mean body weight increments of treated mothers at the highest dose level were comparable to control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Parental Males: the mean food consumption of treated males was similar to control group.

Parental Females Pre-mating period: the mean food consumption of treated females at the highest dose level in pre-mating period was slightly decreased in comparison with the control animals.

Parental Females Pregnancy: females without parturition (non-pregnant females) were not included in the evaluation of food consumption during pregnancy.
The mean food consumption of pregnant females treated by the test substance was slightly decreased within the 1st and 2nd week of pregnancy but at the end of pregnancy period mean food consumption of all treated groups of females was higher in comparison with the control group.

Parental Females Lactation: only mothers (females with live pups born) were included in evaluation of food consumption during lactation period. The mean food consumptions of treated mothers at the end of lactation period were quite well balanced compared to control mothers.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Findings considered of no toxicological significance.

Parental Males - hormone T4
Blood samples from the all adult parental males were assessed for serum levels of thyroid hormone thyroxine (T4).
Mean concentration of males at the dose level 250 mg/kg/day only was significantly decreased. Concentration of other dosed groups were similar to the control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Findings considered of no toxicological significance.

Parental Males
Microscopic examination of testes, epididymis, seminal vesicles, prostate gland, coagulation glands, thyroid gland and pituitary gland did not reveal presence of treatment related changes, that is why only organs with macroscopical changes were examined at the lowest and middle dose level groups. Only one male at the dose level 500 mg/kg/day was examined – macroscopic finding: reduced right seminal vesicle. Histopathological examination did not record any finding.
Mild hydronephrosis was revealed in the kidneys of 1-1 males. Tubular atrophy of testes was found in 1-0 males. Focal chronic inflammation of prostate gland was observed in 1-1 males as a spontaneous finding. Other histological findings were observed either in control animals only, or in both control and treated animals, or they were of an incidental nature, consequence of euthanasia.
The test substance orally administered to rats did not cause any pathological changes in the male genital organs and in pituitary gland.

Parental Females
The incidence of affected females is expressed in numeric form and ranged in sequence of
the dose levels 0-1000 mg/kg/day further in the text.

In uterus the changes related to previous pregnancy were found in both controls and treated animals: accumulation of lipophages and siderophages in mesometrium in 11-9 females, hemosiderin in mucosa in 11-11 females. Also lobular hyperplasia of mammary gland recorded in 7-7 females and mucosal hypertrophy with mucification of vagina in 1-3 females can be related to the previous pregnancy and lactation.
Mild hydronephrosis was revealed in the kidneys of 1-4 males.
Other microscopical changes observed in organs occurred only sporadically or they did not relate to test substance treatment.
The test substance orally administered to rats did not cause any pathological changes in the female genital organs and in pituitary gland.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Before beginning of treatment, oestrous cycle of all females was monitored. Vaginal smears of all females were monitored daily for two weeks. Only females with regular cyclicity were select for the reproduction part of study.
Irregular cycle was demonstrated in one female only – this female was not included in groups for reproduction part of study.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Slightly increased presence of sperms with changed motility was recorded in treated males in comparison with the control males. Presence of “no progressive motility” was detected in all treated groups. Increased percentage of affected sperms was recorded at sperm morphology examination of treated males. Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.

Reproductive performance:

no effects observed

Description (incidence and severity):

Statistically significant differences were not recorded.

The total number of live pups at first check of litter after parturition, on the 4th day and 13th day of lactation at the dose level 250 and 1000 mg/kg was increased compared to control group. The difference in the number of pups of the 4th and 13th day of lactation was not caused by the application of the test substance but by the sacrificing of pups for eventual T4 analysis on 4th day of lactation.
Mean number of pups per litter in all treated mothers was similar in comparison with the control mothers except the mothers at the dose level 500 mg/kg. There the mean number of pups per litter was increased.

Evidence of copulation was found out in all females. Decreased number of females achieving pregnancy was recorded in the control and the middle dose level. All females delivered pups. No abortion was recorded. The lowest number of females bearing live pups was recorded at the dose level 500 mg/kg/day what corresponds to the number of pregnant females. Mean number of corpora lutea was slightly decreased in all treated groups. The mean number of implantations was similar in all groups. Mean number of live pups at birth and on day 4 and 13 after parturition in mothers was slightly increased in females at the dose level 500 mg/kg/day.

Details on results (P0)

REPRODUCTIVE PARAMETERS
Evidence of copulation was found out in all females.
Decreased number of females achieving pregnancy was recorded in the control and the middle dose level. All females delivered pups. No abortion was recorded.
The lowest number of females bearing live pups was recorded at the dose level 500 mg/kg/day what corresponds to the number of pregnant females.
Mean number of corpora lutea was slightly decreased in all treated groups. The mean number of implantations was similar in all groups.
Mean number of live pups at birth and on day 4 and 13 after parturition in mothers was slightly increased in females at the dose level 500 mg/kg/day.
Measurement of an anogenital distance in pups showed unaffected difference between males and females and was similar in all groups. No difference in pup weight at the time of anogenital distance measurement was recorded among the groups.
No treatment-related findings were observed in pups at macroscopical examination.

FERTILITY PARAMETERS
Mating indexes show that mating was not negatively affected by the test substance treatment.
Fertility indexes were lower in animals at the dose level 500 mg/kg/day; in other treated groups was higher compared to control group.
In all treated groups the pre-implantation losses were lower in comparison with the control group.
Gestation index did not show negative effect of the test substance treatment.
Survivor index in treated groups was similar with the control group.
Post-implantation losses were higher at the dose level 250 mg/kg. Similar or lower post-implantation losses in comparison with the control group were recorded at the dose levels 500 and 1000 mg/kg/day.
Post-natal losses were similar in all groups of animals.

Effect levels (P0)

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 adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The presence and number of nipples in male pups were counted on day 13 of lactation - the presence of nipples in male pups was not recorded.
The anogenital distance (AGD) of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalized to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of pup body weight.
No differences in postnatal development of pups were observed at the treated groups compared to the control group.

Description (incidence and severity):

Statistically significant differences were not recorded.

Slightly decreased mean weight of litter within all weighing intervals was recorded at the dose levels 250 and 1000 mg/kg.
The mean body weight of pup from treated groups was slightly decreased in comparison with the control pups. The mean body weight of pups among the treated groups was well balanced within all weighing intervals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Blood samples from the day13 pups were assessed for serum levels for thyroid hormone (T4). Pup blood was pooled by litter.
No significant differences were recorded in pups from treated groups in comparison with the control pups.

Description (incidence and severity):

The macroscopic examination was performed in all pups. Macroscopical findings were observed sporadically and did not relate to the test substance administration.

Control: 149 pups were examined – in 3 pups from 3 litters macroscopic changes were found out (empty stomach, female pup without tail, dead male – autolysis of organs). No macroscopical findings were recorded in others pups.
250 mg/kg/day: 169 pups were examined – in 5 pups from 4 litters macroscopic changes were found out (2 stillborn pups in one litter, cannibalism of one pup in 3 other litters). No macroscopical findings were recorded in others pups.
500 mg/kg/day: 148 pups were examined – in 2 pups from 2 litters macroscopic changes were found out (empty stomach, cannibalism). No macroscopical findings were recorded in others pups.
1000 mg/kg: 161 pups were examined - in 2 pups from 1 litter macroscopic changes were found out (cannibalism). No macroscopical findings were recorded in others pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

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 adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for reproduction and development was established as 1000 mg/kg body weight/day.

Executive summary:

The substance was tested for reproduction and subacute toxicity using the OECD No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on 28th July 2015.

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment (see the Annex 2) and approved by Sponsor.

The treated groups were administered daily for the following periods: males and females 2 weeks prior to the mating period and during the mating period; pregnant females during pregnancy and till the 12th day of lactation; males after mating period (totally for 49 days); nonpregnant females (mated females without parturition) for 25 days after the confirmed mating; non-mated females for 25 days after the end of mating period. After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.   

Repeated oral administration of test item to rats by gavage at the dose levels of 250, 500 and 1000 mg/kg/day did not cause any mortality.

Parental males and females

The number of females achieving pregnancy and microscopical structure of reproductive organs in both parental males and females seem to be not affected by the substance administration.

The mean body weight of parental animals was not significantly affected by the test substance treatment except in females within lactation period. The body weight of treated mothers at the end of lactation period was decreased in comparison with the control females.

Slightly increased presence of sperms with changed motility was recorded in all treated males in comparison with the control males. Increased percentage of affected sperms was recorded at sperm morphology examination of treated males. Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.

All parental males were assessed for serum levels of thyroid hormone thyroxine (T4). Significantly decreased concentration of hormone was recorded in males at the dose level 250 mg/kg/day. This change can be considered to be incidental, concentration of other dosed groups was similar to the control group.

Statistically significant changes of absolute and relative weights of genital organs in males and females were recorded sporadically. Decreased absolute weight of prostate gland+seminal vesicels was recorded in males at the dose level 500 and 1000 mg/kg. Decreased absolute and relative weights of pituitary gland were recorded in males at the dose level 500 mg/kg/day. In females decreased absolute weight of ovaries was recorded in females at the dose level 1000 mg/kg/day and relative weight of ovaries in females at the dose level 250 mg/kg/day.

No macroscopical findings related to the test substance treatment were recorded during the pathological examination of treated males and females.

Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg (the highest dose level) did not cause any pathological changes in the male and female genital organs pituitary and thyroid gland. No findings related to the test substance treatment were recorded, that is why only organs with macroscopical changes were examined at the other dose level groups.

 

Pups

Number and sex ration of pups were not significantly affected by the test substance administration. No differences in postnatal developmental were observed in pups at the treated groups – presence of nipples in male pups was not recorded and anogenital distance in treated male and female pups in comparison with the control pups was similar. Concentration of thyroxine hormone T4 in day 13 pups from the treated groups was similar with the control group.

Microscopical evaluation did not show any findings related to the test substance treatment.

Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy were not affected by the test substance administration.

 

Conclusion

The NOAEL (No Observed Adverse Effect Level) for reproduction and development was established as 1000 mg/kg body weight/day.