Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Testslim
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:
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
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: cca 383 g (males), cca 255 g (females)
- 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
- 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
Details on route of administration:
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.
Vehicle:
physiological saline
Details on oral 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.
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.
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.
Observations and examinations performed and frequency:
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
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
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).
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical changes were recorded in control and treated females.
The activity (poise, gait, reaction to handling) of all males and females of all treated and satellite groups was similar during the study and not different from the activity of males of the control group.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Body weight of males in all treated and satellite groups was slightly decreased in comparison with satellite control males for the whole time of application and during recovery period.
Statistically significant differences were not found.
Weight increments in treated males and satellite treated males were variable and not adversely influenced by the test substance administration.
Weight increments of satellite treated males were not adversely influenced by the test substance treatment. Mean body weight increment of treated males was slightly decreased in comparison with the control group most of time. At the end of recovery period the weight increment of treated males was higher compared to control males.
Females
The body weight of dosed females was not adversely influenced by the test substance administration.
Slightly decreased body weight compared to control animals was recorded in animals at the dose level 250 mg/kg/day in period before mating and during the pregnancy period.
Well-balanced body weight of treated females and control females was noted during the lactation period.
Statistically significant differences in necropsy body weight were not found in treated females.
Body weight increments of treated females were higher compared to control females within the 1st and 2nd week of application. Low or negative body weight increment in treated females after the 1st week of application can be considered to be adaptive process to the test substance administration.
Satellite females
Body weight of satellite treated females was higher in comparison with the control animals for the whole time of application and recovery period. Statistically significant differences in necropsy body weight were not found in satellite treated females.
Weight increments of satellite treated females were higher in comparison with the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males
The food consumption of treated males and satellite treated males was comparable with control males.
Females
In pre-mating period the food consumption of treated females was slightly higher in comparison with control females. During pregnancy and lactation period the food consumptions of treated females were comparable with the control females.
Satellite females
The food consumption of satellite treated females was most of time slightly higher in comparison with the control group for the time of application period. Decreased food consumption of satellite treated females was recorded at the end of recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Satellite males
The water consumption of satellite treated males was comparable to satellite control group during the whole application period of study. During recovery period (8th and 9th week) the water consumption of satellite treated males was variable in comparison with the satellite control males.
Satellite females
The water consumption of satellite treated females was comparable to satellite control group during the whole application period of study. During recovery period (8th and 9th week) the water consumption of satellite treated females was little higher in comparison with the satellite control females.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Red blood components (total erythrocyte count, mean corpuscular volume, haematocrit, haemoglobin concentration, reticulocytes) of treated males were not adversely influenced by administration of the test substance.
The platelet count of treated males at the dose level 1000 mg/kg/day was slightly (insignificantly) decreased in comparison with the control group of males.
The value of total leucocyte count of treated males in comparison with the value of the control group were not significantly influenced by the test substance treatment. Increased value of portion of granulocytes (insignificant) and on contrary decreased portion value of monocytes (statistically significant) were recorded in treated males at the dose level 250 and 1000 mg/kg/day. The portion value of lymphocytes of treated males was comparable to the value of control group.
Haemocoagulation parameters (activated partial tromboplastin time, prothrombin time) were slightly increased in treated males in comparison with the control males. The value of activated partial tromboplastin time of males at the dose level 1000 mg/kg/day was statistically significantly increased in comparison with the value of control males. The concentration of fibrinogen of dosed males was comparable with the value recorded in control males.
All parameters were in historical control range.
Satellite males
Statistically significant differences were not found out in treated males. All haematological parameters of treated males were similar with the control males.
Females
Parameters of red blood component (total erythrocyte count, mean corpuscular volume, haematocrit, haemoglobin concentration, reticulocytes) were slightly increased in females at the dose levels 500 and 1000 mg/kg/day. The values of haemoglobin concentration and haematocrite were statistically significantly increased at the dose level 1000 mg/kg/day in comparison with the value of control females. Platelet count of treated females at all dose levels was decreased (dose-dependently) compared to control group of females.
The value of total leucocyte count was well-balanced among the control and treated groups of females. The portion of lymphocytes was statistically significantly increased in females at the dose level 250 mg/kg/day. On contrary the portion of granulocytes at the dose level 250 mg/kg/day was statistically significantly decreased in comparison with the control group.
Coagulation parameters (activated partial tromboplastin time and prothrombin time) of treated animals were quite well-balanced with the control animals, only the value of fibrinogen in females at all dose levels was statistically significantly decreased compared to control group (without dose dependency).
All parameters were in historical control range.
Satellite females
Statistically significantly changed value of mean corpuscular volume was recorded in satellite treated females.
Other parameters were similar in satellite control and treated females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The concentration of creatinine in males at all dose level was statistically significantly decreased in comparison with the control group. Also concentration of sodium ions was statistically significantly decreased in males at the dose level 1000 mg/kg/day. All changes were in historical control range (and reversible).
Values of other biochemical parameters of treated males were similar to the control group.
All parameters were in historical control range.
Satellite males
Statistically significantly decreased values of cholinesterase and concentration of chloride ions, were recorded in satellite treated males. All changed values were still in the range of historical control values.
Values of other biochemical parameters of satellite treated males were similar to the satellite control group.
Females
Statistically significant differences were found out only in females at the dose level 1000 mg/kg/day – the concentration of calcium was statistically significantly decreased (still in historical control range) in comparison with the control group. Other biochemical parameters of treated females were similar with the control females.
All parameters were in historical control range.
Satellite females
Differences in values of biochemical parameters between control females and satellite group did not show any significant differences except the value of total protein, glucose and concentration of potassium ions. These values were statistically significantly increased in satellite treated females.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis was performed only in males (six of each group) during the last week of the study. Statistical evaluation was performed for pH and volume of urine.
Males
Volume of urine was increased in all treated groups of males in comparison with the control group. Statistically significant differences were recorded in volume of urine in males at the dose levels 500 and 1000 mg/kg/day/day.
Value of urine pH at treated groups of males was comparable to control group.
Presence of proteins was recorded sporadically at the dose levels 1000 mg/kg/day and leucocytes were recorded in treated males at the dose levels 250 and 1000 mg/kg/day.
Satellite males
Slightly increased volume of urine was recorded in treated group of males compared to control group. Value of urine pH was in control and treated group similar.
Presence of leucocytes was recorded in both groups – control and treated.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Males
Reactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The activity – number of upstanding was quite well-balanced. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated males.
Satellite males
No significant differences were detected in examined parameters.
Females
Reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control females. The activity – number of upstanding was quite well-balanced. The values of grip strength of pectoral and pelvic legs were without significant differences.
Satellite females
No significant differences were detected in examined parameters.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The absolute weight of thymus was slightly decreased in males at the dose level 500 mg/kg/day. The absolute weight of testes was decreased in males at all treated groups - statistically significantly in males at the dose level 500 mg/kg/day. The weight of pituitary gland was statistically significantly increased in males at all dose levels in comparison with the control group (without dose dependency). Weight of other organs was similar in treated and control males.
Statistically significant differences were recorded in relative weight of pituitary gland in males at all dose levels. Relative weight of pituitary gland was increased (without dose dependency). Significantly increased weight of kidneys in treated animals compared to control animals was recorded in dose level 1000 mg/kg/day. Relative weights of other organs of treated males were similar to control males.
Satellite males
Statistically significantly changed values were not recorded in satellite treated males. The absolute weight of thymus was slightly decreased in treated satellite males. The weight of testes was increased in treated males in comparison with the control group of males.
No statistically significantly changed weights of organs in satellite treated males were recorded. Insignificantly increased relative weight of testes, epididymides and prostate gland was recorded.
Females
Statistically significant differences were not recorded in absolute weight of organs in treated females compared to control females. Absolute weight of thymus was increased in all groups of treated females – especially in females at the dose levels 250 mg/kg/day (compared to control females).
Statistically significant differences were recorded in relative weight of adrenal glands in females at dose levels 250 and 1000 mg/kg/day in comparison with the control group. Relative weight of thymus was increased (not statistically significantly) in comparison with the control group in females of the dose level 250 and 1000 mg/kg/day. Weights of other organs of treated females were quite well-balanced in treated and control animals.
Satellite females
Statistically significantly decreased weight of pituitary gland of satellite treated females was recorded in comparison with the control group. Absolute weights of others organs were similar in satellite treated and control females.
Statistically significantly decreased relative weight of pituitary gland in treated satellite females was recorded. Relative weights of organs were similar in satellite treated and control females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Control: no macroscopic findings were recorded in all six males.
250 mg/kg/day/day: content of stomach coloured by the test substance was recorded in three of the six males.
500 mg/kg/day/day: coloured content in the stomach and intestines in all six males were recorded
1000 mg/kg/day: coloured content in the stomach and intestines in all six males were recorded; coloured urine in two males, coloured mucosa of forestomach in three males and reduced testes and epididymides in one of the six males were recorded.
Satellite males
Control satellite: markedly reduced testes and epididymides in one male and reduced left testis in other one of the six males were recorded.
1000 mg/kg/day/day satellite: no macroscopic findings were recorded.
Females
Control: no macroscopic findings were recorded in all six females.
250 mg/kg/day/day: no macroscopic findings were recorded in all six females.
500 mg/kg/day/day: coloured content of intestines was recorded in one of the six females.
1000 mg/kg/day: coloured content of stomach and intestines in five of the six females was recorded.
Satellite females
Control satellite: no macroscopic findings were recorded.
1000 mg/kg/day/day satellite: no macroscopic findings were recorded except dilatation of uterus in 1 female.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Full histopathology of the preserved organs and tissues was performed for high dose and control animals and satellite animals. No findings related to the test substance treatment were recorded. Macroscopic changes at the low and the middle dose levels were not found out that is why organs from these level groups were not microscopically examined.
Males
In 1-2 males no histological findings were diagnosed.
Incidence of microscopic findings was very sporadic. Mild hydronephrosis was revealed in the kidneys of 1-2 males. Tubular atrophy of testes was recorded in 1-1 males; oligospermia in 0-1 males. Inflammation of prostate gland was observed in 1-1 males. Other histological findings were incidental, recorded in treated males as well as in control males.
Satellite males
In 2-2 satellite males no histological findings were diagnosed.
Focal chronic inflammation of prostate gland in 0-1 male and in testes tubular atrophy in 2-1 males were reported. Mild hydronephrosis was revealed in the kidneys of 1-1 males. Other histological findings were incidental, recorded in control males only.
Females
In 0-0 female no histological findings were diagnosed. In uterus and vagina, the changes related to previous pregnancy were found in both control and treated animals: accumulation of lipophages and siderophages in mesometrium in 6-6 females, hemosiderin in mucosa in 5-6 females. Lobular hyperplasia of mammary gland was recorded in 6-6 females. Other histological findings were incidental, recorded in treated females as well as in control females.
Satellite females
In 1-4 female no histological findings were diagnosed. Follicular cysts on ovaries were recorded in 3-2 females. Siderosis in spleen was noted in 2-1 females. Other findings were observed in control animals only. They were of spontaneous character.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Haematological examination of males and females showed several haematological parameters affected by treatment at all dose levels. All values were in range of historical control. No treatment-related changes were observed so all changes can be considered to be of no toxicological significance (absence of a treatment-related signs).
In males these changes of haematological parameters were recorded: prolonged time of activated partial thromboplastin time at the dose level 1000 mg/kg/day and decrease of portion of monocytes at the dose levels 250 and 1000 mg/kg/day. In females these changes were recorded: increased haematocrit and concentration of haemoglobin, decreased concentration of fibrinogen at the dose level 1000 mg/kg; decreased concentration of fibrinogen and portion of granulocytes, increased portion of lymphocytes in females at the dose level 250 mg/kg/day; and decreased concentration of fibrinogen was also recorded in females at the dose level 500 mg/kg/day. Increased value of mean corpuscular volume vas observed in females at the treated satellite group.
Biochemical examination showed only sporadic changed values (all in range of historical control). In males, decreased values of concentration of creatinine was noted in males at all treated groups (dose dependent, in range of historical control value). Decreased portion of sodium concentration was recorded in males at the dose level 1000 mg/kg/day (reversible). In females only one statistically significant change was detected: decreased concentration of calcium at the dose level 1000 mg/kg/day (reversible). In satellite treated females increased concentration of glucose and concentration of potassium were recorded.
Urinalysis did not manifest the influence of the test substance administration on value of pH urine. The volume of urine was increased in males at all dose levels.
During biometry of organs, statistically significant changes in absolute weights of pituitary gland in males at all dose levels (without dose dependency) and decreased absolute weight of testes in males at dose level 500 mg/kg/day were recorded. Relative weight of pituitary gland was also statistically significantly increased in males at all dose levels, but in satellite treated males this relative weight of pituitary gland in treated males was the same as in satellite control males. Statistically significantly increased relative weight of kidneys was recorded in males at dose level 1000 mg/kg/day. In satellite treated males no significantly changed absolute or relative weight of organs was reported. In treated females no statistically significantly changed absolute weight of organs in comparison with the control group was recorded. Relative weight of adrenal glands was statistically significantly deceased in females at the dose levels 250 and 1000 mg/kg/day. At the end of recovery period no statistically significant changes in relative weight of organs were recorded.
Statistically significantly increased absolute and relative weight of pituitary gland and on contrary insignificantly decreased absolute weight of pituitary gland in females were not connected with any histopathological findings.
Macroscopic changes related to test substance colour were recorded (coloured content of stomach and intestines was observed in males and females). Other findings were recorded only in males at the dose level 1000 mg/kg/day (reduced testes and epididymides in one male and coloured mucosa of forestomach in three males) and had probably spontaneous character.
Microscopic evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs. Mild hydronephrosis of kidneys was reported in dosed animals as well as in a control animals.
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 observrd
Key result
Critical effects observed:
no

The tables are enclosed in the attached document.

Conclusions:
The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES and FEMALES was established as 1000 mg/kg/day body weight/day. No biologically significant signs of toxicity 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 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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.