Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted by the Council on 29th July 2016
Deviations:
yes
Remarks:
see Any other information on materials and methods
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
A laboratory rat has been chosen because the 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,
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 7 - 9 wks
- Weight at study initiation: males: cca 383 g; females: cca 255 g
- Housing: SPF conditions; 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
- Diet (e.g. ad libitum): complete pelleted diet for rats and mice in SPF breeding - Altromin for Rats/Mice
- Water (e.g. ad libitum): drinking water ad libitum,
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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 10 minutes (it was necessary to stir the solution with a glass stick) and then the solution was stirred by magnetic stirrer (700 rpm) for 20 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.
The test substance was administered to the stomach by gavage. Oral way of administration was chosen according to the guideline and it was approved by Sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am). The vehicle control group was administered by water for injection in the same volume.
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:
ANALYTICAL METHOD
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.

THE PREPARATION OF APPLICATION FORM
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/10 mL
Ca 0.1 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The test substance was dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer for 5 minutes at 200 rpm.
Concentration level 1000 mg/10 mL
Ca 10 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was slowly replenished by the vehicle. The test substance was dissolved in ultrasonic bath together with careful mixing with glass rod for 10 minutes. After this the application form was stirred by magnetic stirrer for 20 min at 700 rpm.

THE HOMOGENEITY AND STABILITY OF THE APPLICATION FORM
The first sampling (time interval 0 min) for the measuring of homogeneity and stability was carried out after 5 minutes of ultrasonication and 5 minutes of mixing by the magnetic stirrer (200 rpm) for conc. level 10 mg /10 mL and after 10 minutes of ultrasonication together with careful mixing with glass rod and 20 minutes of mixing by the magnetic stirrer (700 rpm) for conc. level 1000 mg /10 mL.
Homogeneity
Conc. level 10 mg/10 mL: The samples were taken after 5 minutes in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (200 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.
Conc. level 1000 mg/10 mL: The samples were taken after 10 minutes in ultrasonic bath together with careful mixing with glass rod and 20 minutes of mixing by magnetic stirrer (700 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.
Stability
The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.
Conc. level 10 mg/10 mL: Time interval 0 min represents for this concentration the time after 5 minutes of ultrasonication and 5 minutes of mixing by magnetic stirrer at 200 rpm.
Conc. level 1000 mg/10 mL: Time interval 0 min represents for this concentration the time after 10 minutes of ultrasonication together with careful mixing with glass rod and 20 minutes of mixing by magnetic stirrer at 700 rpm.

RESULTS OF ANALYSIS
It follows from the results of analyses (homogeneity and stability) that the both application forms (10 mg and 1000 mg /10 mL) of the test substance, Acid Brown 235, 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:
Males: 49 days; 63 days in satellite group
Females: according to mating, gestation and lactation period; 63 days in satellite group
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am). The vehicle control group was administered by water for injection in the same volume.
Details on study schedule:
Health condition control: daily - during the acclimatization and the experimental part
Body weight:
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
pups (litters) - 1st , 4th day, 12th day and 13th day
pups – individually – 4th day of lactation
Food consumption: weekly and on the same days as body weight (except the mating period); satellite males and females – weekly
Water consumption: satellite males and females – twice a week
Clinical observations:
males and females - daily during the administration period
pups - as soon as possible after delivery and then daily
Mortality control: twice daily
Detailed clinical observation: before the first application and then weekly (except the mating period)
Functional observation: at the end of administration/observation period
Laboratory examinations:
vaginal smears:
daily – 1st – 14th day of study
daily in mating period (max. 14 days)
on necropsy day
urinalysis: the last day of administration/observation period – only males
haematology:
males – after the end of application period
parental females – on the 13th day of lactation
satellite animals – after the end of observation period
biochemistry:
males and nonpregnant females – after the end of application period
2 pups per litter - on the 4th day of lactation
parental females and pups – on the 13th day of lactation
satellite animals – after the end of observation period
anogenital distance measurement: pups– 4th day of lactation
pathological examination:
males and nonpregnant females – after the end of application period
2 pups per litter - on the 4th day of lactation
parental females and pups – on the 13th day of lactation
satellite animals – after the end of observation period
weight of organs: during necropsy
sperm observation: parental males – during and after necropsy (not performed in satellite males)
histopathological examination: after necropsy
Dose / conc.:
250 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, 6 males and 6 females per satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
PREPARATION OF EXPERIMENTAL ANIMALS
During the acclimatisation period the health condition of all animals was controlled daily. Normal course of the oestrus cycle of all females was control during 14 days before start of application. Females with abnormal oestrus cycle were removed from mating. Then the animals were randomly divided into the control and test groups and they were marked individually.

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.

METHODS OF INVESTIGATION
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.

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.

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.

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

Health Condition Control
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, during application and immediately after application.

Clinical Observations of Males and Females
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.). Animals were observed in natural conditions in their cages.

Clinical Observation of Pups
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.

Detailed Clinical Observation
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.

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.

Examination of vaginal smears
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.

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.

Haematological examination
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. 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.

Biochemical examination
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum. 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 acids, sodium, potassium and chloride ions.
Blood samples from the day 13 pups and the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) by ELISA kit (Biovendor, Brno, Czech Republic). Treatment related changes of T4 total blood serum levels, of thyroid gland weight and microscopical structure were not observed in the day 13 pups therefore assessment of blood serum level of T4 total was not performed in day 4 pups.

Anogenital distance (AGD) measurement
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.

Pathological examination
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.

Observation of sperm
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.

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.

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 liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded (Repeated Dose Toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (Reproduction part of study – all animals). 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.

Histopathological examination
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.
In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. No macroscopical findings were reported at low and middle dose level, treatment-related changes were not recorded in high dosed animals, therefore histological examination was not performed at low and middle dose levels.
In Reproductive Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals.
Treatment-related changes were not observed at the high dose group therefore detailed histological examination of testes was performed only for all high dose and control males from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

DATA PROCESSING
All the primary data (body weight, food consumption, water consumption, health condition control, general clinical observation, detailed clinical observation, functional observation, haematological examination, biochemistry, urinalysis, examination of vaginal smears, sperm examination, biometry of organs, necropsy findings, histopathological examination) were recorded in special protocols. These primary data were used for calculations and processed to tables.
For the evaluation of Repeated Dose Toxicity part of study the first-six males and the first-six birth giving mothers of each basic group and satellite males and females were used. For the evaluation of Reproduction Toxicity part of study all males and females of basic groups were used.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
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
pups (litters) - 1st , 4th day, 12th day and 13th day
pups – individually – 4th day of lactation

FOOD CONSUMPTION:
- Food consumption: Yes
- Body weight gain: Yes
- Time schedule for examinations: weekly and on the same days as body weight (except the mating period); satellite males and females – weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: satellite males and females – twice a week
Oestrous cyclicity (parental animals):
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 four females – they were not included in groups for reproduction part of study.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations: testes weight, sperm motility, sperm morphology
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: 64th day
- Maternal animals: Parental females: day 12 post partum; Non-pregnant females (without evidence of copulation): 25 days after the end of mating period; Non-pregnant females (with evidence of copulation): 25th day after confirmed mating

GROSS 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
Pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland + seminal vesicles, testes, thyroid gland and all gross lesions 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.
The full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Treatment-related changes were not observed at the high dose group therefore detailed histological examination of testes was performed only for all high dose and control males (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed at day 13 of lactation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: number and sex ratio of pups, body weight, presence of nipples in male pups, anogenital distance, concentration of thyroxine hormone T4 in day 13 pups, microscopical evaluation of thyroid gland, macroscopic examination was performed in all pups.
Statistics:
STATISTICAL ANALYSIS
For statistical evaluation the software Statgraphic Centurion (version XVII) 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.
As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved then non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.
For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).
Reproductive indices:
see Fertility parameters table in attached tables
Offspring viability indices:
see Reproduction data table in attached tables
Clinical signs:
no effects observed
Description (incidence and severity):
At all control and treated and satellite males and females, no clinical changes were recorded during the whole study.
The activity (poise, gait, reaction to handling) of all males and females of all treated groups and satellite groups was similar during the study and not different from the activity of males of the control group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths of male during the main study.
One female (dose level 500 mg/kg/day/day) died due to intubation error on day 34 of application period during the main study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males
Body weight was decreased in all treated groups of males during the whole study. The total body weight gain among the treated groups was similar.
Body weight increment of all treated males was comparable to the control males and not adversely affected by the test substance treatment.
Females
Pre-mating period
Mean body weight of treated females in all dose levels was similar to control groups of animals.
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 similar or slightly higher in comparison with the control group.
The mean body weight increments of all treated pregnant females were similar or higher 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 all treated groups was well-balanced with the control group.
The mean body weight increments of treated mothers were not negatively influenced by the test substance treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males
No differences in mean food consumption were observed between treated and control males.
Females
Pre-mating period
The mean food consumption of treated females at all dose levels in pre-mating period was comparable or slightly increased in comparison with the control animals.
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 comparable 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 in lactation period were comparable or slightly increased compared to control mothers.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Blood samples from the all adult parental males were assessed for serum levels of thyroid hormone thyroxine (T4). Statistically significant differences were not detected in treated males. Mean concentrations of T4 hormone at all dose levels were similar to the control group. Concentration of thyroxine hormone was in historical control range.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Blood samples from the all adult parental males were assessed for serum levels of thyroid hormone thyroxine (T4). Statistically significant differences were not detected in treated males. Mean concentrations of T4 hormone at all dose levels were similar to the control group. Concentration of thyroxine hormone was in historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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. Organs with macroscopical changes were not recorded in the middle and low dose level that is why no histopathological examination were performed there.
The incidence of affected animals is expressed in numeric form and ranged in sequence of the dose levels 0-1000 in main groups.
In 6-7 males no histological findings were diagnosed.
Mild hydronephrosis was revealed in the kidneys of 1-2 males. Focal chronic inflammation of prostate gland was recorded in 2-1 males and tubular atrophy of testes in 1-2 males. Oligospermia was recorded in 0-2 males.
Other histological findings were observed either in control animals only, or in both control and treated animals, or they were of an incidental nature.
The test substance orally administered to rats did not cause any pathological changes in the male genital organs and in pituitary gland.
Females
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-1000 mg/kg/day/day.
In 1-0 females no histological findings were diagnosed.
In uterus the changes related to previous pregnancy were found in both control and treated animals: accumulation of lipophages and siderophages in mesometrium in 7-9 females, hemosiderin in mucosa in 6-9 females.
Lobular hyperplasia of mammary gland was noted in 6-6 females. Hydrometra of uterus was recorded in 1-2 females.
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.
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
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 four females – they were not included in groups for reproduction part of study.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Motility of sperms was not significantly changed in treated males compared to control.
The portion of affected sperms in treated males recorded at sperm morphology examination was comparable to the control 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):
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.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect related to the test substance observed
Key result
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean weight of litter at the dose level 250 mg/kg/day was decreased compared to control group. On the contrary statistically significantly increased mean body weight of pup was recorded only at the dose level 250 mg/kg/day on 12th day of lactation. In other dose levels the mean values were comparable with the control group. The mean body weight of pup at all treated groups were comparable with the value of control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
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.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute weight of thyroid gland of one male and one female pup per litter (on the 13th day of lactation) was recorded. No statistically significant changes in thyroid gland weight were recorded. The thyroid gland weight of pups from treated groups was slightly increased in comparison with control group of pups.
Histopathological examination of thyroid gland of pups did not reveal any pathologic findings.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination was performed in all pups.
Control: 92 pups were examined – no macroscopical findings were recorded.
250 mg/kg/day/day: 99 pups were examined – no macroscopical findings were recorded
500 mg/kg/day/day: 107 pups were examined – no macroscopical findings were recorded
1000 mg/kg/day: 116 pups were examined - 1 female from one litter was without tail; no macroscopical findings were recorded in other pups.
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathological examination of thyroid gland of pups did not reveal any pathologic findings.
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Number and sex ratio 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 not influenced by the test substance treatment. Microscopical evaluation of thyroid gland did not show any findings related to the test substance treatment.
Key result
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: no adverse effect related to the test substance observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg/day body weight/day. No biologically significant changes of reproduction parameters were observed.
Executive summary:

The test substance, Acid Brown 235, 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 29th July 2016. 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/day 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/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment 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.   

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 parental animals and pups were removed for weighing and histopathological examination.

Repeated oral administration of Acid Brown 235 to rats by gavage at the dose levels of 250, 500 and 1000 mg/kg/day/day did not cause any mortality.

Parental males and females:

The number of females achieving pregnancy was the worse in control group. Microscopic 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. The necropsy weight was decreased in males and on contrary increased in females in comparison with the control animals.

Observation of sperm in parental males did not show negative effect. 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). The concentration of hormone of treated males was comparable to control males

Statistically significant changes of absolute and relative weights of genital organs in males and females were not recorded. Only statistically significantly increased absolute and relative weight of pituitary gland in parental males at all dose levels was recorded. 

Macroscopic findings related to the test substance colour only were recorded during the pathological examination of treated males and females. Other findings were sporadic and not related to the test substance treatment.

Microscopic evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day (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.

Pups:

Number and sex ratio of pups were not significantly affected by the test substance administration. No differences in postnatal development 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 not influenced by the test substance treatment. Microscopic evaluation of thyroid gland 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.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg/day body weight/day. No biologically significant changes of reproduction parameters were observed.

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

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
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

Justification for classification or non-classification

The combined repeated dose toxicity study with reproduction/developmental toxicity study did not reveal any adverse effects associated with administration of the test substance. Classification criteria according to Regulation (EC) No. 1272/2008 did not fulfilled.