Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL reproduction and development: 1000 mg/kg body weight/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 30th, 2015 to July 29th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 28th July 2015
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Laboratory rat has been chosen because our testing laboratory has long experience with this species.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500.
- Age at study initiation: males, females: sexually adult, 7-9 weeks on arrival.
- 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.
- Bedding: sterilized soft wood fibers Lignocel.
- Diet: complete pelleted diet for rats and mice in SPF breeding - Altromin for rats/Mice (Altromin Spezialfutter GmbH & Co.).
- Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law.
- Acclimation period: at least 6 days. During the acclimatisation period the health condition of all animals was controlled daily. Normal course of the oestrus cycle of all females was controlled during 14 days before start of application. Females with abnormal oestrus cycle were removed from mating.ù

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30-70 %
- Photoperiod: 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
APPLICATION FORM
- Stability and homogeneity: determined by means of measuring of a peak area of the test substance by a liquid chromatography based on a method developed at the test facility. Test item in vehicle at defined laboratory conditions resulted to be homogenous and stable at least for 120 minutes from the finalization of application form preparation.
- Preparation: the application form for analysis was prepared in the same manner as for application to animals – i.e.solution in water for injection. Concentration Level 10 mg/10ml: ca 0.1 g of the test substance was weighed into a 250 ml glass beaker calibrated to 100 ml and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for a 30 min. The solution was stirred by magnetic stirrer (750 rpm) for 20 minutes. Concentration Level 1000 mg/10 ml: ca10 g of the test substance was weighed into a 250 ml glass beaker calibrated to 100 ml and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for a 30 min. The solution was stirred by magnetic stirrer (750 rpm) for 40 minutes.
- Concentration: 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.
Details on mating procedure:
- M/F ratio per cage: mating 1: 1 (one male to one female) was used in this study.
- Proof of pregnancy: vaginal smear referred to as day of pregnancy. Each morning the females were examined for presence of spermatozoa in vaginal smears.
Duration of treatment / exposure:
Totally 49 days of administration.
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Details on study schedule:
Parental males: 14 days of pre-exposure period; from 15th to 28th day pre-mating period, administration; from 29th to 42nd day mating; from 43rd to 63rd day administration period; 64th day necropsy.
Parental females: 14 days pre-exposure period; from 15th to 28th day pre -mating period, administration; from 29th to 42nd day mating; gestation followed by lactation day 12 post partum.
Non-pregnant females (without evidence of copulation): 14 days pre-exposure period; from 15th to 28th day pre -mating period, administration; from 29th to 42nd day mating; 25 days after the end of mating period.
Non-pregnant females (with evidence of copulation): 14 days pre-exposure period; from 15th to 28th day pre -mating period, administration; from 29th to 42nd day mating; 25th day after confirmed mating.
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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:
12 females and 12 males per group
Control animals:
yes
Details on study design:
DOSE-RANGE FINDING EXPERIMENT
- Age at study initiation: 9 weeks on arrival.
- Number of animals: 5 males and 5 females per group.
- Acclimatization: at least 5 days.
- Dose selection: according to results of Dose-Range Finding Experiment the following dose levels – 125, 500 and 1000 mg/kg/day were chosenfor the main test. The dose levels were approved by Sponsor.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS
- Data Collection: health condition control daily during the acclimatization and the experimental part.
- Data Collection: males and females daily during the administration period.
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before application, during application and immediately after application. 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.). Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATIONS
- Data Collection: before the first application and then weekly (except the mating period).
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, 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.

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

BODY WEIGHT
- Data Collection: males the first day of administration and then weekly; females the first day of administration and then weekly; during pregnancy at 0, 7th, 14th, 20th day; during lactation at 1st, 4th day, 12th day and 13th day.
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.

FOOD CONSUMPTION
- Data Collection: weekly and on the same days as body weight (except the mating period).
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.
In pre-mating period the food consumption and conversion of females was calculated from values of all females.

FUNCTIONAL OBSERVATION
- Data Collection: at the end of administration/observation period.
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.

HAEMATOLOGY
- Data Collection: males and nonpregnant females after the end of application period; parental females on the 13th dy of lactation.
- 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.
This examination was performed only in 6 males and 6 females of each group. The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation system. Reticulocytes count was examined according to the internal SOP No. M/2. Haematology analysers Celltac alfa and Coagulometer ACL were used for examination and the following parameters were determined

CLINICAL CHEMISTRY
- Data Collection: males and nonpregnant females after the end of application period; 2 parental females on the 13th day of lactation.
- Parameters: protein total, alkaline phosphatise, cholesterol total, triglycerides, alanine aminotranferase, aspartate aminotransferase, creatinine, urea, albumin, bilirubin total, glucose, calcium, phosphorus, cholinesterase, bile acids, sodium, potassium, chloride.
This examination was performed only in 6 males and 6 females of each group. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.
Blood samples from the day 13 the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) by ELISA kit (Biovendor, Brno, Czech republic).
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
Total protein, total bilirubin, urea, creatinine, glucose, transaminases (AST, ALT), cholesterol, albumin, alkaline phosphatase (ALP), phosphorus and calcium, triglycerides, cholinesterase, bile acidswill be measured by automatic clinical chemistry analyser ACCENT-200 (Cormay, Poland); sodium, potassium and chloride ions will be measured by automatic electrolyte analyser with ion-selective electrodes SPOTCHEMTM EL SE-1520 (Arkray, Inc.).

URINALYSIS
- Data Collection: the last day of administration/observation period, only males.
- Parameter: volume, colour, cloud, odour, glucose, protein, bilirubin, urobilinogen, pH, specific gravity, blood, ketones, nitrite, leucocytes.
This examination was performed only in 6 males of each group; 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 by 2 ml of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined by analyser PocketChem PU-4210 and 4010 (Arkray, Inc., Japan).
Oestrous cyclicity (parental animals):
VAGINAL SMEARS
Data collection: daily from 1st to 14th day of study; daily in mating period (max 14 days); on necropsy day.
Vaginal smears were made from the 1sttill the 14th day of study (pre-exposure period) for monitoring of 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 have been 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):
- Data collection: parental males during and after necropsy.
- Parameters examined: in all males the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/45 and 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 suspension. 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 suspension 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
- Data Collection: health condition control daily during the acclimatization and the experimental part.
- Data Collection: pups as soon as possible after delivery and then daily.
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.

BODY WEIGHT
- Data Collection: pups, litters, at 1st, 4th day, 12th day and 13th day; pups, individually, at 4th day of lactation.
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).

ANOGENITAL DISTANCE
Pups's anogenital distance measurement was performed on 4th day of lactation. The AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used.

CLINICAL CHEMISTRY
- Data Collection: 2 pups per litter on the 4th day of lactation; pups on the 13th day of lactation.
- Parameters: protein total, alkaline phosphatise, cholesterol total, triglycerides, alanine aminotranferase, aspartate aminotransferase, creatinine, urea, albumin, bilirubin total, glucose, calcium, phosphorus, cholinesterase, bile acids, sodium, potassium, chloride.
The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.
Blood samples from the day 13 pups were assessed for serum levels of thyroid hormone thyroxine (T4 total) by ELISA kit (Biovendor, Brno, Czech republic).
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
Total protein, total bilirubin, urea, creatinine, glucose, transaminases (AST, ALT), cholesterol, albumin, alkaline phosphatase (ALP), phosphorus and calcium, triglycerides, cholinesterase, bile acidswill be measured by automatic clinical chemistry analyser ACCENT-200 (Cormay, Poland); sodium, potassium and chloride ions will be measured by automatic electrolyte analyser with ion-selective electrodes SPOTCHEMTM EL SE-1520 (Arkray, Inc.).
Postmortem examinations (parental animals):
SACRIFICE
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla.

PATHOLOGY
- Data collection: males and non-pregnant females after the end of application period. parental females on the 13th day of lactation.
Full pathology was performed only in 6 males + 6 females of each dose level.
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.

ORGAN WEIGHTS
- Data collection: during necropsy.
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 thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

HISTOPATHOLOGICAL EXAMINATION
- Data collection: after necropsy
- Organs for histopathological examination: pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland + seminal vesicles, testes, all gross lesions, thyroid gland.
The mentioned 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).
Postmortem examinations (offspring):
PATHOLOGY
- Data collection: 2 pups per litter on the 4th day of lactation; pups on the 13th day of lactation.
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.

ORGAN WEIGHTS
- Data collection: during necropsy.
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 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

HISTOPATHOLOGICAL EXAMINATION
- Data collection: after necropsy
- Organs for histopathological examination: pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland + seminal vesicles, testes, all gross lesions, thyroid gland.
The mentioned 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).
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.
Reproductive indices:
FERTILITY PARAMETERS
Male mating index, female mating index, male fertility index, female fertility index, gestation index, survival index.

LOSS OF OFFSPRING
Pre-implantation loss, post-implantation loss, post-natal loss.
Description (incidence and severity):
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 in treated males during the whole study.

No clinical changes after application of the test substance were recorded in all treated animals. Only coloured faeces were recorded during whole study.

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 in treated females.

No clinical changes after application of the test substance were recorded in treated females. Only coloured faeces were recorded.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the main study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Different body weight before application was caused by sequential putting animals to the study. Body weight of treated males was relatively well-balanced in comparison with control males and without statistical significance. Body weight increment of all treated males was not adversely affected by the test substance.

Females
Pre-mating period
Different body weight before application was caused by sequential putting animals on the study. Mean body weight of treated females was similar compared to the control group.
The mean body weight increment of treated females was variable before mating in comparison with control group.

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 during whole pregnancy.
The mean body weight increments of all treated pregnant females were similar to the control 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 females at the dose level 500 mg/kg/day was decreased on the 1st day of lactation. The body weight of females was similar with control females during the rest of lactation period.
The mean body weight increments of treated mothers were comparable to control animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males
The mean food consumption of treated males was slightly higher in males at the dose level 500 and 1000 mg/kg/day.

Females
Pre-mating period
The mean food consumption of treated females at the middle and the highest dose level was slightly increased in pre-mating period in comparison with the control animals.

Pregnancy
Females without parturition (non-pregnant or aborted 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 similar in comparison with the control group.

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 were slightly higher during the first four days of lactation. At the end of lactation period treated mothers consumptions were quite well balanced compared to control mothers.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Blood samples from the all adult parental males were assessed for serum levels for thyroid hormone thyroxine (T4 total).
Mean concentration of T4 hormone at the dose level 125 mg/kg/day was insignificantly increased in comparison with other test groups. Concentration of others dosed groups were similar to the control group.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The statistical analysis of the data showed no statistically significantly changed absolute weight of organs in comparison with control group. Also relative weight of these organs was not significantly changed.

Females
Non-pregnant females were not included to calculation of means and evaluation of biometry results.
The statistical analysis of the data showed no statistically significantly changed absolute weight of organs in comparison with control group. Also relative weight of these organs was not significantly changed.
Only slightly increased absolute weight of pituitary gland was observed but relative weight was without any changes. Weight of other organs was not changed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Control: no macroscopical findings were recorded in 12 males.
125 mg/kg/day: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract, coloured content and mucous membrane of stomach and coloured kidneys in all males were recorded.
500 mg/kg/day: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract, coloured content and mucous membrane of stomach and coloured kidneys in all males were recorded. Colouration of submandibular lymph nodes was recorded in four males.
1000 mg/kg: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract, coloured content and mucous membrane of stomach, coloured kidneys and mesenteric lymph nodes in all males were recorded. Colouration of paraaortal and submandibular lymph nodes was observed in two respectively three males. Urine in urinary bladder coloured by the test substance was recorded in six males.

Females
Control: no macroscopical findings were recorded in 12 females.
125 mg/kg/day: findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract, coloured content and mucous membrane of stomach and coloured kidneys in all females were recorded. Colouration of submandibular lymph nodes was recorded in four females and colouration of mesenteric lymph node was detected in one female. Haemorhage in right uterine horn was observed in one female.
500 mg/kg/day: mostly findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract, coloured content and mucous membrane of stomach and coloured kidneys in all females were recorded. Colouration of submandibular lymph nodes was recorded in seven females and colouration of liver was also observed in one female.
Focal congestion of mucous membrane of stomach was observed in one female.
1000 mg/kg: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract, coloured content and mucous membrane of stomach and coloured kidneys in all females were recorded. Colouration of liver was observed in two females and colouration of uterus three females. Submandibular or mesenteric lymph nodes were coloured in two respectively in three females.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males
The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-125-500-1000 mg/kg/day further in the text.
Microscopic examination of reproductive organs and pituitary gland did not reveal presence of treatment related changes, only the spontaneous changes were found in both controls and treated animals. This was the reason why only organs with macroscopical changes were examined at the dose level 125 and 500 mg/kg/day. Tubular degeneration or atrophy (mostly in sporadic tubules) was detected in two control males and in two treated males at the highest dose level. In prostate gland there was found out: acinar atrophy in two control and one treated males, focal interstitial oedema in two treated males and chronic inflammation in two control as well as treated males. Desquamation of epithelium of seminal vesicles was detected only in one treated male.
In 6-2-0-0 males no histological changes were detected.
Other microscopical changes observed in organs occurred only sporadically (hydronephrosis and hyaline droplets in epithelium of cortical tubules and basophilic tubules of kidneys, hyperplasia of submandibular lymph nodes) and did not relate to the test substance treatment.
The test substance orally administered to rats caused pathological changes connected with colouration of test substance practically in all treated males mostly at the middle and the highest dose level. Blue pigment in epithelium of cortical tubules in kidneys (very sporadic intensity 0-0-10-0, sporadic 0-0-0-8, mild 0-0-0-3, medium 0-0-0-1), macrophages containing blue pigment in mesenteric lymph nodes (0-4-6-12), in submandibular lymph nodes (0-0-0-2) and in paraaortal lymph nodes (0-0-0-1), blue pigment in enterocytes of large intestines (0-10-12-5) and lamina propria of small intestinal villi (0-0-10-9) and blue pigment in enterocytes of caecum (0-0-0-2) was detected.

Females
The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-125-500-1000 mg/kg/day further in the text.
Microscopic examination of reproductive organs and pituitary gland did not reveal presence of treatment related changes, only the changes related to previous pregnancy were found in both controls and treated animals. This was the reason why only organs with macroscopical changes were examined at the dose level 125 and 500 mg/kg/day. Accumulation of lipophages and/or siderophages in mesometrium and/or endometrium was detected in 10 control females and in 7 treated females at the highest dose level. Hemosiderin in mucosa was observed in eight high dosed females and hydrometra in one control female.
In 1-10-0-0 females no histological changes were detected.
Other microscopical changes observed in organs occurred only sporadically (cyst in adenohypophysis and cystic dilatation of the endometrial glands containing blood of uterus, follicular hyperplasia of submandibular lymph nodes) and did not relate to the test substance treatment.
The test substance orally administered to rats caused pathological changes connected with colouration of test substance practically in all treated females mostly at the middle and the highest dose level. Blue pigment in epithelium of cortical tubules in kidneys (very sporadic intensity 0-0-3-0, sporadic 0-0-8-5, mild 0-0-0-7), macrophages containing blue pigment in mesenteric lymph nodes (0-1-4-10) and blue pigment in enterocytes (0-0-3-4) and lamina propria of intestinal villi (0-0-5-8) of intestines was detected.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cycles were monitored before treatment starts to select for the study females with regular cyclicity. Vaginal smears of all females were monitored daily for two weeks.
It was found that all the females had regular oestrus cycle.
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 to a small degree in all treated groups.
Increased percentage of affected sperms was recorded at sperm morphology examination at the dose level 125 and 1000 mg/kg/day. Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Evidence of copulation was found out in all females.
Decreased number of females achieving pregnancy was recorded in the control and the highest dose level. All pregnant females delivered pups - no abortion was recorded.
The lowest number of females bearing live pups was recorded at the dose level 1000 mg/kg/day and in control females which correspond to the number of pregnant females.
Mean number of corpora lutea was statistically significantly increased in all treated females and implantations was significantly increased just in females at the middle dose level.
Mean number of live pups at birth and on day 4 after parturition in mothers was slightly increased in treated females. One stillborn pup was detected at the dose level 500 mg/kg/day.
More male pups than female pups were observed in control females and in females at the dose level 1000 mg/kg/day. At the dose levels 125 and 500 mg/kg/day were more female pups.
Weight of litters was higher in all treated groups but mean weight of pup was similar.
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 control and in animals at the dose level 1000 mg/kg/day.
The value of pre-implantation losses was lower in animals at the dose level 500 and 1000 mg/kg/day in comparison with the control group. At the dose level 125 mg/kg/day was the value similar compared with control group.
Post-implantation losses were higher in treated animals mostly at the dose level 500 mg/kg/day. Post-natal losses were slightly higher in all treated groups of animals.
Dose descriptor:
NOAEC
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproduction
Critical effects observed:
no
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
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 125 and 500 mg/kg/day was increased compared to control group. Total number of pups at the dose level 1000 mg/kg/day was the same as in control group.
Mean number of pups per litter in all treated mothers was slightly higher in comparison with the control mothers.
More male pups than female pups were observed in control females and in females at the dose level 1000 mg/kg/day. At the dose levels 125 and 500 mg/kg/day were more female pups.
Statistically significant differences were not recorded.
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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Increased mean weight of litter within all weighing intervals was recorded mostly at the dose levels 500 and 1000 mg/kg/day, slightly increased also at the dose level 125 mg/kg/day.
The mean body weight of pups from all treated groups was well balanced within the all lactation period.
Statistically significant differences were not recorded.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Blood samples from the 13 day old 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.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination was performed in all pups. Macroscopical findings were observed sporadically and did not related to the test substance administration.
Control: 146 pups were examined – partial cannibalism of 1 pup was observed. No macroscopical findings were recorded in others pups.
125 mg/kg/day: 168 pups were examined - Just one pup without tail was found out in one litter. No macroscopical findings were recorded in others pups.
500 mg/kg/day: 179 pups were examined- One pup was stillborn. No macroscopical findings were recorded in others pups.
1000 mg/kg: 149 pups were examined - No macroscopical findings were recorded in pups.
Other effects:
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 4th 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.

No differences in postnatal development of pups were observed at the control and treated groups.
Dose descriptor:
NOAEC
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: development
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
NOAEL (49d) (males and females): 1000 mg/kg bw/day
Executive summary:

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

The test substance was administered to rats in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups; each main group consisted of 12 males and 12 females. Main groups contained 3 treated groups (doses 125, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only).

During the study clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. The functional observation was performed at the end of application and observation period. Vaginal smears were prepared daily, 2 weeks before start of administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy day. Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups, measurement of anogenital distance, nipple retention) were also recorded.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from adult animals were removed for weighing and histopathological examination. The thyroid gland was taken out, weighed and histologically examined in selected pups.

The test substance treatment did not affect physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period) and absolute and relative weight of reproductive organs was not affected by the test substance treatment at all dose levels 125, 500 and 1000 mg/kg/day. Numbers of corpora lutea, implantations and pups were not influenced by the test substance treatment at any dose level too. No adverse effect of the test substance treatment was observed during biochemical examination of parental males (concentration of thyroxine hormone) not even during pathological and histopathological examination of reproductive organs in treated parental animals. Also evaluation of body weight of pups, development of pups, concentration of thyroid hormone (T4) in pups and macroscopical examination of pups did not reveal any influence of the test substance treatment at any dose level.

Slightly increased percentage of affected sperms and increased presence of sperms with changed motility was recorded at the dose level 125 mg/kg/day but male ability to produce sperm that can fertilise eggs was not affected.

Results of sperm morphology in males of the dose level 500 mg/kg/day was similar in comparison with control group. Slightly increased presence of sperms with changed motility was recorded at this dose level but male ability to produce sperm that can fertilise eggs was not affected.

Slightly increased percentage of affected sperms and increased presence of sperms with changed motility was recorded at the dose level 1000 mg/kg/day but male ability to produce sperm that can fertilise eggs was not affected.

 

Conclusions

The NOAEL (No Observed Adverse Effect Level) for reproduction and development was established as 1000 mg/kg body weight/day. All changes observed in parental males and females and in their offspring at all dose levels were considered to be of no toxicological significance.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was perfromed on Direct Blue 199.

The test substance was administered to rats in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups; each main group consisted of 12 males and 12 females. Main groups contained 3 treated groups (doses 125, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only).

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from adult animals were removed for weighing and histopathological examination. The thyroid gland was taken out, weighed and histologically examined in selected pups.

The test substance treatment did not affect physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period) and absolute and relative weight of reproductive organs was not affected by the test substance treatment at all dose levels 125, 500 and 1000 mg/kg/day. Numbers of corpora lutea, implantations and pups were not influenced by the test substance treatment at any dose level too. No adverse effect of the test substance treatment was observed during biochemical examination of parental males (concentration of thyroxine hormone) not even during pathological and histopathological examination of reproductive organs in treated parental animals.

Slightly increased percentage of affected sperms and increased presence of sperms with changed motility was recorded at the dose level 125 mg/kg/day but male ability to produce sperm that can fertilise eggs was not affected. Results of sperm morphology in males of the dose level 500 mg/kg/day was similar in comparison with control group. Slightly increased presence of sperms with changed motility was recorded at this dose level but male ability to produce sperm that can fertilise eggs was not affected. Slightly increased percentage of affected sperms and increased presence of sperms with changed motility was recorded at the dose level 1000 mg/kg/day but male ability to produce sperm that can fertilise eggs was not affected.

Effects on developmental toxicity

Description of key information

NOAEL reproduction and development: 1000 mg/kg body weight/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance was administered to rats in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups; each main group consisted of 12 males and 12 females. Main groups contained 3 treated groups (doses 125, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only).

The test substance treatment did not affect physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period) and absolute and relative weight of reproductive organs was not affected by the test substance treatment at all dose levels 125, 500 and 1000 mg/kg/day. Numbers of corpora lutea, implantations and pups were not influenced by the test substance treatment at any dose level too. No adverse effect of the test substance treatment was observed during biochemical examination of parental males (concentration of thyroxine hormone) not even during pathological and histopathological examination of reproductive organs in treated parental animals. Also evaluation of body weight of pups, development of pups, concentration of thyroid hormone (T4) in pups and macroscopical examination of pups did not reveal any influence of the test substance treatment at any dose level.

Justification for classification or non-classification

According to CLP Regulation (EC 1272/2008), 3.7 Reproductive toxicity section, reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

Based on the available information, the substance does not meet the criteria to be classified for reproductive toxicity, according to CLP Regulation (EC 1272/2008).