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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29.8. 2012 - 18.4.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Solvent Red 19E
- Physical state: dark viscous liquid/borderline waxy solid
- Analytical purity: 90% (w/w)
- Impurities (identity and concentrations): Solvent Red24 (CAS 85-83-6) 2% (w/w)
- Lot/batch No.: S2409
- Expiration date of the lot/batch: unlisted
- Storage condition of test material: in the dark place at laboratory temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks (on arrival)
- Weight at study initiation: 270 - 315 g
- Fasting period before study: No
- Housing: 2 rats of the same sex in one plastic cage containing sterilised clean shavings of soft wood
- Diet (e.g. ad libitum): complete pelleted diet for rats (ad libitum)
- Water (e.g. ad libitum): free access to drinking water (ad libitum)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Relative humidity (%): 30-70 %
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

STUDY TIME SCHEDULE
Administration started: 16.10.2012
Examination of blood and necropsies: 25.11. – 06.12.2012, 10. – 12.12.2012 (satellite groups)
End of histopathological examination: 18.04.2013

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered to the stomach by gavage as the solution in olive oil. 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 concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by olive oil in the same volume. The application form (test substance solution in olive oil) was prepared daily just before administration.
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The application form was prepared by mixing with olive oil. Two concentrations of application form were prepared (1g /L and 100 g/L).

1 g/L: Ca. 0.1 g of the test substance was weighted into a 150 mL glass beaker calibrated to 100 ml and the beaker was replenished by the vehicle (olive oil). The solution was stirred by magnetic stirrer (650 rpm) for 60 minutes.

100 g/L: Ca. 10 g of the test substance was weighted into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (650 rpm) for 60 minutes. At the begimling the sample solution was stirred by the magnetic stirrer with lower speed (ca. 200 rpm). It was necessary to release the sticked magnetic stirrer from the bottom of the beaker. When this succeeded the speed of the mixing was increased up to 650 rpm. The solution was stirred by magnetic stirrer (650 rpm) for 60 minutes.

VEHICLE
- Lot/batch no. (if required): 5211201
- The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance solution in olive oil) was prepared daily just before administration.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The analyses were performed by spectrophotometric method developed by test facility. Spectrophotometric measurement conditions were set up according to test substance UVVIS spectrum.
Stability and homogeneity of Solvent Red 19E were determined by evaluation of absorbance measured at maximum of the test substance absorbance spectra.

Stability of the application form was checked by analyses of the application form in the interval of 120 min after it preparation (at the time 0 min, 30 min, 60 min 90 min and 120 min). The samples were taken from the middle of the beaker content after required time intervals.
The measurement was performed on both concentration levels (1 g/L and 100 g/L).

The homogeneity of the application form
Homogeneity of the application form was checked by determination of a concentration of the test substance in three places of solution (at the bottom, in the middle and at the surface). The measurement was performed on both concentration levels (1 g/L and 100g/L).

From the results of analyses (homogeneity and stability) follows that the test substance solution in vehicle (olive oil) at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes.
Duration of treatment / exposure:
Males and females: 2 weeks prior to the mating period and during the mating period.
Pregnant females: During pregnancy and to the 3rd day of lactation.
Males: After mating period; total 42 days
Non-pregnant females (mated females without parturition): For 25 days after the confirmed mating.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 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:
Basic groups: 12 males + 12 females (0, 160, 400 and 1000 mg/kg bw/day)
Satellite groups: 6 males + 6 females (0, 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study were chosen on the basis of the results of dose-range finding experiment.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes. 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 (12.00 - 14 p.m.) at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.
- Time schedule: The condition of the control was oberved daily during the acclimatisation and experimental part.
Morality control was observed daily.
Detailed clinical observations were made before the first application and then weekly, except the mating period.


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

BODY WEIGHT: Yes. 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 per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.
- Time schedule for examinations:
Males: Weekly
Females: Weekly in pre-mating and mating period
During pregnancy: 0, 7th, 14th and 20th day
During actation 0 or 1st, 3rd and 4th day
Satellite males and females: Weekly

FOOD CONSUMPTION: Yes. On a specified day, the remainder of pellets were 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 of 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 premating 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. Non-pregnant females (females without parturition) were not included in calculation of mean food consumption of pregnant females.
Males: Weekly (except the mating period)
Females: Weekly during the pre-mating period
Pregnancy and lactation: on the same days as body weight
Satellite males and females: Weekly

WATER CONSUMPTION: Yes. 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.
- Time schedule for examinations: Satellite males and females: Twice a week

OTHER:
Functional observations: 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.

Laboratory invenstigations:
Examination of Vaginal Smears: The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.

Urinlaysis: This examination was performed only in 6 males of each group and in satellite males. The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined:
Volume, colour, cloud, odour, glucose, protein, bilirubin, urobilinogen, pH, specific gravity, blood, ketones, nitrite, leucocytes.

Haematology: 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 systems.
Haematology analysers Coulter® Ac.T diffTM, Celltac alfa and Coagulometer ACL 200 were used for examination.

The following parameteres were determined:
Total erythrocyte count, Mean corpusuclar volume, Haematocrit, Harmoglobin concentration, total leucocyte count, total platelest count, partial thromboplastin time, prothrombin time, fibrinogen, granulocytes, lymphocytes, monocytes.

Biochemistry: 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.

The following parameters were determined:
Glucose, cholesterol (total), urea, bilirubin (total), aspartate aminotransferase, alanine aminotransferse, alkaline phosphatase, calcium, phosphorus, protein (total), protein (albumin), creatinine, sodium, potassium, chloride.
Sacrifice and pathology:
POST-MORTEM EXAMINATIONS: Yes. 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 (buffer 4 % formaldehyde). Testes and epididymides were fixed in modified Davidson's fixative.
Males and non-pregant females: At the end of the administration period.
Parental females: On the 4th day of lactation.
Satellite males and females: At the end of the recovery period.
Weight of organs: During necropsy.

Histological examination: After necropsy.
Samples of the following tissues and organs were collected at necropsy and fixed:
12 males and 12 females from each main group: Pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland, testes, all gross lesions.
6 males and 6 females from each main group and 6 males and 6 females from satellite groups: Adrenal galnds, aorta, brain (including cerebellum and med. oblongata), caecum, coagulating gland, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (thoracic), spleen, stomach, thymus, urinary bladder, female mammary gland, femur, heart, ileum (inc. Peyer's patches), jejunum (inc. Peyer's patches), kidneys, liver, lungs, lymph notes (mesenteric, para-aortal), oesophagus, all gross lesions.
The mentioned tissues and organs were collected from all 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. Organs demonstrating treatment­ related changes and reproductive organs: Testes epididymis, seminal vesicles, coagulation glands and pituitary gland in males, vagina, ovary, uterus and pituitary gland in females and organs with macroscopical changes were examined at the lowest and middle dose level groups.
Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated. Pathological changes were evaluated.
Other examinations:
Sperm observations: Observation of Sperm
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/45.

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, 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, 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.

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

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

Litter observations
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 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs. 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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Males
No clinical changes after application of the test substance were observed at the dose levels 160 and 400 mg/kg/day. Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected at the dose level 1000 mg/kg/day during the whole application period.

Satellite males
Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected in treated animals during the whole application period.

Females
No clinical changes after application of the test substance were observed at the dose levels 160 and 400 mg/kg/day. Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected at the dose level 1000 mg/kg/day from the 2nd to the 7th week of study. Salivation was detected sporadically at the dose level 1000 mg/kg/day in the 3rd week of application period.

Satellite females
Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected in treated females from the 2nd to the 6th week of application period.

Detailed clinical observation was performed in all 12 animals from each group and satellite animals and it is described in this part.

Males
The activity (poise, gait, reaction to handling) of all males of all treated groups was similar during the study and not different from the activity of males of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and secretion and respiration. Red coloured excrements and shavings and tail of animals contaminated by coloured excrements were detected at the dose level 1000 mg/kg/day from the 1st to the 6th week of application period.

Satellite males
The activity (poise, gait, reaction to handling) of all males of all treated groups was similar during the study and it was not different from the activity of males of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and secretion and respiration. Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected in treated males from the 1st to the 6th week of application period.

Females
The activity (poise, gait, reaction to handling) of all females of all treated groups was similar during the study and not different from the activity of females of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and secretion and respiration. Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected at the dose level 1000 mg/kg/day in the 5th and 6th week of application period.

Satellite females
The activity (poise, gait, reaction to handling) of all females of treated group was similar
during the study and not different from the activity of females of the control groups.
No significant changes were found at all dose levels during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane and secretion and respiration. Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements were detected in treated females from the 3rd to the 6th week of application period.
Mortality:
no mortality observed
Description (incidence):
Repeated dose section:
Males
There were no unscheduled deaths during the study in all animals.

Females
There were no unscheduled deaths during the study in all animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Repeated dose section:
Males
The statistical analysis was performed for body weight. Statistically significant differences were not found.
The body weight increment of treated groups and control group was similar (except the 1st week of application period at the dose level 1000 mg/kg/day, when it was decreased).

Satellite males Statistical analysis was performed for body weight. Statistically significant differences were not found.
The body weight increment of treated group and control group was similar.

Females
The statistical analysis was performed for body weight. The statistically significant differences were not found.
The body weight increment of treated groups and control group was similar.

Satellite females
The statistical analysis was performed for body weight. The statistically significant differences were not found.
The body weight increment of treated group and control group was similar (except the 1st week of recovery period in treated females, when it was slightly decreased).

Reproduction/Development Part of Study Observation of Parental Males
Body Weight and Body Weight Increment
The statistical analysis was performed for body weight. The statistically significant differences were not found.
The body weight increment was decreased at the dose level 1000 mg/kg/day in the 1st and 2nd week of application period and at the dose level 400 mg/kg/day in the 2nd week of application period.

Reproduction/Development Part of Study Observation of Parental Females
The statistical analysis was performed for body weight. The statistically significant differences were not found.

Before mating period
The body weight increment of treated females was relatively well-balanced with control group.

Pregnancy
Females without parturition (non-pregnant or aborted females) were not included in the evaluation of body weight increments during pregnancy.
The body weight increment of treated females was relatively well-balanced with control group.

Lactation
Only mothers (females with live pups) were included in the evaluation of body weight increment during lactation period.
The body weight increment was decreased at the dose level 1000 mg/kg/day in the 3rd day of lactation period. On the 4th day of lactation period the fall of body weight was recorded in all groups (this was caused by fasting before the biochemical examination of blood).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Repeated dose section:
Males
Slightly decreased mean food consumption at the treated groups was recorded during the whole application period.

Satellite males
Decreased mean food consumption at the treated group was recorded during the whole application and recovery period.

Females
The mean food consumption of all treated groups and control group was similar during premating period. During pregnancy and lactation period the mean food consumption was slightly decreased in all treated groups.

Satellite females
The mean food consumption of both groups was similar during the whole study (except the 3rd week of application period in treated females, when it was decreased).

Reproduction/Development Part of Study Observation of Parental Males
Food consumption of males at all treated dose levels was decreased during the whole study.

Reproduction/Development Part of Study Observation of Parental Females
Pre-mating period
The mean food consumption of all treated groups and control group was similar.

Pregnancy
Females without parturition (non-pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy.
The mean food consumption of mothers at all dose levels was lower than in control females.

Lactation
Only mothers (females with live pups) were included in evaluation of food consumption during lactation period.
Mean food consumption in females at the dose levels 160 and 1000 mg/kg/day was lower than in control females.
Food efficiency:
no effects observed
Description (incidence and severity):
Repeated dose section:
Males
Dis-balance of mean food conversion between treated groups and control group was recorded during the whole study.

Satellite males
Dis-balance of mean food conversion between treated group and control group was recorded during the whole study.

Females
Dis-balance of mean food conversion in all treated groups was recorded during pre-mating period. During pregnancy period the mean food conversion was relatively similar in treated and control females. During lactation period the mean food conversion at the dose levels 160 and 400 mg/kg/day was increased.

Satellite females
Dis-balance of mean food conversion between treated group and control group was recorded during the whole study (in the 1st week of recovery period, it was negative in treated group).
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Satellite males
Slightly decreased mean water consumption at the treated group was recorded during the whole application period. During recovery period the mean water consumption was similar.

Satellite females
Slightly decreased mean water consumption at the treated group was recorded during the whole study (except the 3rd week of application period, when it was similar).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Males
Statistically significantly decreased values of total erythrocyte counts, haematocrit, and haemoglobin in males at the dose levels 160 and 1000 mglkg /day were found out. The value of protrombin time was decreased significantly in all treated groups (at the dose levels 160 and 400 mg/kg/day it was under historical control limit). Significantly increased value of fibrinogen and insignificantly increased value of active partial tromboplastin time (APTT) was recorded at the dose levels 160 and 1000 mg/kg/day (value of APTT at the dose level 1000 mg/kg/day was above historical control limit). Dose-dependent increased value of total leucocyte count was recorded in all treated groups without statistical significance. The value of granulocytes was decreased in all treated groups.
Other measured parameters were similar to the control group.

Satellite males
In treated group the values of total erythrocyte count, haematocrit and haemoglobin were statistically significantly decreased. Insignificantly decreased values of total leucocyte count and granulocytes was found at the treated group. The value of activated partial tromboplastin time (APTT) was insignificantly increased.
Other measured parameters were similar to the control group.

Females
The value of protrombin time at the dose level 400 mg/kg/day was decreased with statistical significance. The value of fibrinogen was decreased insignificantly at the dose level 1000 mg/kg/day. Increased value of total leucocyte count at the dose levels 400 and 1000 mg/kg/day was recorded (at the dose level 1000 mg/kg/day with statistical significance). Insignificantly decreased value of monocytes was recorded in all treated groups. At the dose levels 400 and 1000 mg/kg/day value of granulocytes was decreased and value of lymphocytes was increased insignificantly.
Other measured parameters were similar to the control group.

Satellite females
Statistically significantly increased value of total leucocyte count was recorded in treated females. The value of granulocytes was decreased significantly and value of lymphocytes was increased insignificantly in treated females.
Other measured parameters were similar to the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Males
Statistically significantly increased value of glucose was recorded at the dose levels 160 and 400 mg/kg/day (at the dose level 160 mg/kg/day it was above historical control limit). The value of creatinine at the dose levels 400 and 1000 mg/kg/day was increased without statistical significance (at the dose level1000 mg/kg/day it was above historical control limit). Inorganic phosphorus was increased insignificantly at the dose level 1000 mg/kg/day but this value was above historical control limit. Insignificantly increased value of urea was recorded at the dose levels 160 and 1000 mg/kg/day. Decreased activity of AST at the dose levels 400 and 1000 mglkg/day and activity of ALT at the dose level 400 mg/kg/day were recorded (without statistical significance). Activity of ALP was increased at the dose levels 160 and 400 mg/kg/day.
All other measured parameters were similar to the control group. Satellite males
The value of bilirubin total and concentration of calcium ions were decreased in treated males
with statistical significance. Insignificantly decreased values of ALT and protein total were recorded in treated males. Activity of AST in treated males was increased.
All other measured parameters were similar to the control group.

Females
Statistically significantly decreased value of sodium ions at the dose level 400 mg/kg/day was recorded. The value of creatinine was increased insignificantly in all treated groups.
Increased value of glucose was recorded at the dose level 1000 mg/kg/day. Activity of ALT at the dose levels 400 and 1000 rng/kg/day was decreased and activity of ALP at the dose levels 160 and 1000 mg/kg/day was increased. The value of ALP at the dose level 1000 mg/kg/day was above historical control limit. Inorganic phosphorus was increased at the dose level 400 mg/kg/day (it was above historical control limit). The value of urea was decreased insignificantly at the dose level 400 mg/kg/day.
All other measured parameters were similar to the control group.

Satellite females
Statistically significantly decreased concentration of calcium ions and value of bilirubin total were recorded in the treated group. Activity of AST in treated females was significantly increased. Insignificantly decreased value of protein total was recorded in treated females.
All other measured parameters were similar to the control group
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Males
The statistical analysis was performed for urine volume and pH of urine. Statistically significant differences were not found.
The volume of urine was increased at the dose levels 160 and 1000 mg/kg/day. Change of colour of urine (from light yellow to dark yellow) was recorded at the dose levels 400 and 1000 mg/kg/day.
At the dose level 400 mg/kg/day presence of protein in two males was recorded.
Presence of leucocytes was recorded in all groups: One male in control group, three males at the dose level 160 mg/kg/day and four males at the dose levels 400 and 1000 mg/kg/day.

Satellite males
Statistical analysis was performed for urine volume and pH of urine. Statistically significant
differences were not found.
In the treated males presence of protein in two males and presence of leucocytes in two males were recorded.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Repeated dose section:
Males
Reactions to contact, to noise, to pain and pupillary reflex of treated males and satellite treated group were the same as in the control group.
The value of upstanding was decreased at the dose level 160 mg/kg/day. Results of emiction
and defecation in treated males were not the same as in control males but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in males did not show any difference between control and the treated dose levels.

Females
Reactions to contact, to noise, to pain and pupillary reflex of treated females and satellite treated group were the same as in the control group.
The value of upstanding was increased in satellite treated females. Results of emiction and defecation in treated females were not the same as in control females but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in females did not show any difference between control and the treated dose levels.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Absolute organ weight
Males
Increased absolute weight of liver in all treated groups was recorded (at the dose level 400 mg/kg/day with statistical significance). Dose-dependent increased absolute weight of spleen was recorded in all treated groups (without statistical significance). Slight decreased weight of heart was recorded in all treated groups. Absolute weight of epididermis at the dose level 160 mg/kg/day was increased. Absolute weight of prostate gland at the dose level 400 mg/kg/day was increased. At the dose level 1000 mg/kg/day absolute weight of thymus was decreased. The absolute weight of other organs was well-balanced with the control group.

Satellite males
Decreased absolute weight of thymus of treated group was recorded with statistical significance. Absolute weight of prostate gland was decreased in treated males without statistical significance. Insignificantly increased absolute weight of brain and pituitary gland was recorded in treated males.
The absolute weight of other organs was well-balanced with the control group.

Females
Decreased absolute weight of kidneys was recorded in all treated groups (at the dose level 160 mg/kg/day with statistical significance). Decreased absolute weight of adrenal glands was recorded in all treated groups (without statistical significance). Decreased absolute weight of ovaries was recorded in all treated groups (at the dose level 1000 mg/kg/day with statistical significance). Absolute weight of pituitarv gland was significantly decreased at the dose level 400 mg/kg/day and insignificantly at the dose level 160 mg/kg/day. Absolute weight of thymus was decreased in all treated groups. Increased absolute weight of spleen was found out at the dose levels 160 and 1000 mg/kg/day without statistical significance. Decreased absolute weight of heart was recorded at the dose level 400 mg/kg/day.
The absolute weight of other organs was relatively well-balanced with the control group.

Satellite females
Absolute weight of uterus was slightly decreased in treated females.
The absolute weight of other organs was relatively well-balanced with the control group.

Relative organ weight
Males
Dose-dependent increased relative weight of liver was recorded in all treated groups (at the dose levels 400 and 1000 mg/kg/day with statistical significance). Increased relative weight of spleen was recorded in all treated groups without statistical significance. Relative weight of prostate gland was increased at the dose levels 400 and 1000 mg/kg/day and relative weight of testes was decreased at the dose level 400 mg/kg/day without statistical significance. Decreased relative weight of heart was found out at the dose levels 160 and 400 mg/kg/day. The relative weight of other organs was well-balanced with the control group.

Satellite males
The statistical analysis of the data revealed significantly decreased weight of thymus in the treated group of males. Insignificantly increased relative weight ofliver, kidneys, epididymis, brain and heart were detected in treated animals.
The relative weight of other organs was well-balanced with the control group.

Females
Statistically significantly decreased relative weight of ovaries was recorded in all treated groups. Decreased weight of kidneys was recorded at the dose levels 160 and 1000 mg/kg/day with statistical significance. Significantly increased relative weight of spleen was recorded at the dose level 1000 mg/kg/day. Decreased relative weight of pituitary gland was recorded in all treated groups (at the dose level 400 mg/kg/day with statistical significance). Insignificantly increased weight of liver was recorded at the dose levels 160 and 400 mg/kg/day. Relative weight of heart was decreased at the dose levels 400 and 1000 mg/kg/day.
The relative weight of other organs was well-balanced with the control group.

Satellite females
Increased relative weight of ovaries and decreased relative weight of uterus and brain were
recorded in treated group.
The relative weight of other organs was well-balanced with the control group. Statistically significant differences were not found.

Reproduction/Development Part of Study Observation of Parental Males
The statistical analysis of the data revealed no significant intergroup difference.
Absolute and relative weight of prostate glands was insignificantly increased at the dose levels 400 and 1000 mg/kg/day. Increased absolute weight of epididymis at the dose level 400 mg/kg/day was found. Decreased absolute and relative weight of testes at the dose levels 160 and 400 mg/kg/day was detected and on the contrary at the dose level 1000 mg/kg/day it was increased.

Reproduction/Development Part of Study Observation of Parental Females
Non-pregnant females and females with abortion were not used for calculation of means and evaluation of organs weight.
Relative and absolute weight of ovaries and pituitary gland was decreased in all treated groups (absolute weight of pituitary gland at the dose level 400 mg/kg/day was decreased significantly). Absolute and relative weight of uterus at the dose level 1000 mg/kg/day was increased and at the dose level 400 mg/kg/day absolute weight of uterus was decreased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
The incidence in affected animals is expressed in numeric form and arranged in sequence in the dose levels 0-160-400-1000 in the main males groups (first-six males for group), 0-160- 400-1000 (first-six birth-giving females in group) in the main females groups and 0S-1000S in the satellite males and females group further in the text.

Macroscopic findings
Males
In 6-0-0-0 males no macroscopic changes were observed.
Red colouring of body fat (adipose tissue), GIT and lymph nodes was detected in 0-6-6-6 males. In liver red colouring and marked structure in 0-0-6-6 males was found out. Other changes were observed only sporadically.

Satellite males
In 6-0 males no macroscopic changes were observed during necropsy.
Red colouring of body fat (adipose tissue) and lymph nodes was detected in 0-6 males.

Females
In 6-0-0-0 females no macroscopic changes were observed.
Red colouring of body fat (adipose tissue) in 0-6-5-5 females, GIT in 0-4-6-6 females, lymph nodes in 0-5-6-6 females, ovaries in 0-6-6-6 females and liver in 0-5-6-5 females were detected. In stomach red colouring of mucous membrane in 0-2-0-0 females and yellowish coating on the mucous membrane in 0-0-0-2 females was found. Swollen mucous membrane of forestomach in 0-0-0-2 females was detected. Dark colouring of spleen in 0-0-0-5 females was observed.
Other changes were observed only sporadically.

Satellite females
In 6-0 females no macroscopic changes were observed.
Red colouring of body fat (adipose tissue), lymph nodes and ovaries were detected in 0-6 females. Dilatation of uterus (accompanied oestrus) in 3-3 females was recorded.
Other changes were observed only sporadically.

Reproduction/Development Part of Study Observation of Parental Males
In 12-11-12-12 males no macroscopic changes of reproductive organs and pituitary gland were observed.
Affections of genital organs were observed only sporadically (atrophy of testes and epididymis in one male at the dose level 160 mg/kg/day).
Red colouring of body fat (adipose tissue), GIT and lymph nodes was detected in 0-12-12-12 males. In liverred colouring in 0-0-12-11 and marked structure in 0-0-12-12 males was found out.
Other changes were observed only sporadically.


Reproduction/Development Part of Study Observation of Parental Females
In 12-0-0-0 females no macroscopic changes of reproductive organs and pituitary gland were observed.
Red colouring of ovaries in 0-12-12-11 females was detected. Dilatation of uterus (accompanied oestrus) in 0-1-1-1 females was recorded.
Red colouring of body fat (adipose tissue) in 0-12-11-11 females, GIT in 0-8-9-12 females, lymph nodes in 0-9-10-12 females and liver in 0-10-8-10 females were detected. In stomach mucous membrane coloured by test substance in 0-2-0-0 female, yellowish coating on the mucous membrane in 0-1-0-2 females and swollen mucous membrane in 0-2-0-0 females were detected. Dark colouring of spleen in 0-0-0-9 females was observed.
Other changes were only observed sporadically.

Reproduction/Development Part of Study Observation of Parental Females
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-160-400-1000 mglkg/day further in the text.
In the reproductive system pathological finding were recorded only sporadically.
In uterus, focal necrosis in mesometrium or myometrium in 1-2-0-0 females, necrotic tissue in lumen in 0-0-1-0 female, inflammation in myometrium in 1-0-0-0 female, chronic inflammation in 1-0-0-0 female and dilatation in 1-1-1-0 females were detected. Luteal cyst in ovaries in 0-1-0-2 females was found out.
In the reproductive system also findings which related with previous gravidity or proceeded oestrous cycle were recorded.
In the uterus, the following microscopical changes were found: Haemorrhage in 1-0-0-4 females, accumulation of lipophages and/or pigmentophages in 6-8-6-6 females, apoptosis in epithelium in 0-5-0-0 females and hydrometra in 0-0-1-1 females. Mucification of epithelium in 7-3-3-2 females, desquamation of epithelium in 0-0-2-2 females and apoptotic epithelium in 0-0-2-0 females in vagina were detected. In ovaries, degeneration of tertiary follicles in 3- 11-12-6 females, hyperplasia of interstitial glands in 3-10-4-5 females were found out. Extramedullary haematopoiesis in liver in 0-0-2-0 females was detected. Proliferation of mesangium in part of glomerules in kidneys in 1-/-/-2 females was recorded.
Other microscopical changes occurred in females only sporadically.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Repeated dose section:
Full histopathology of the preserved organs and tissues was performed for high dose and control animals. The following organs demonstrating treatment-related changes and reproductive organs: testes, epididymis, seminal vesicles, coagulation glands and pituitary gland in males and organs with macroscopical changes were examined at the lowest and middle dose level groups. The incidence in affected animals is expressed in numeric form and arranged in sequence in the dose levels 0-160-400-1000 in the main groups and 0S-1000S in the satellite groups further in the text.

Males
In 1-2-0-0 males no microscopical findings were recorded during histopathological examination. In the liver, the following microscopical changes were found: vacuolation of hepatocytes in 0-0-5-2 males and extramedullary haematopoiesis in 0-0-2-0 males. In kidneys thickening of membrane in part of glomerules and proteinuria in 0-/-/-2 males were found out. In lungs nonpurulent pneumonitis was recorded in 3-0-4-4 males. Eosinophil and/or neutrophil infiltration of submucosa in stomach in 0-0-0-2 males was detected. Focal mononuclear infiltration of interstitium and/or oedema of interstitium of prostate gland in 0-4- 4-0 males were detected.
Other microscopical changes in organs occurred in males sporadically.
Focal fatty changes (presence of frequent or diffuse liver steatosis) were detected by examination of specially stained cryotome sections: irregular (in irregular part of lobuli) in 0- 6-6-2 males.

Satellite males
Vacuolation of hepatocytes in liver was detected in 1-4 males. In lungs nonpurulent pneumonitis was recorded in 2-2 males. Hyperkeratosis in forestomach was found out in 2-0 males. In prostate gland oedema of interstitium in 0-3 males was detected.
Other microscopical changes in organs occurred in males sporadically.
The incidence of fatty droplets in liver of treated animals revealed no difference compared to the control group.

Only organs demonstrating treatment-related changes and reproductive organs: Testes. epididymis,
seminal vesicles, coagulation glands and pituitary gland in males and organs with macroscopical
changes were examined at the lowest and middle dose level groups.

Females
In the uterus, the following microscopical changes were found: haemorrhage in 0-0-0-4 females, focal necrosis in mesometrium in 0-2-0-0 females and accumulation of lipophages and/or pigmentophages in 2-5-6-4 females. Mucification of epithelium in 3-3-2-2 females, desquamation of epithelium in 0-0-2-1 females and apoptotic epithelium in 0-0-2-0 females in vagina were detected. In ovaries, degeneration of tertiary follicles in 1-5-6-3 females and hyperplasia of interstitial glands in 1-4-1-2 females were found out. Proliferation of mesangium in part of glomerules in kidneys in 0-/-/-2 females was recorded.
Other microscopical changes occurred in females only sporadically.
Focal fatty changes (presence of sporadic liver steatosis) were detected by examination of specially stained cryotome sections: irregular (in irregular part oflobuli) in 0-6-3-0 males.

Satellite females
In 4-3 females no microscopical findings were recorded during histopathological
examination. In the ovaries, the following microscopical changes were found: Degeneration of tertiary follicles in 4-5 females and hyperplasia of interstitial glands in 6-6 females. Hydrometra in uterus in 2-1 females was found out. Apoptotic cells in cortex in adrenal glands was recorded in 0-2 females.
Other microscopical changes occurred in females only sporadically.

The incidence of fatty droplets in liver of treated animals revealed no difference compared to the control group.
Note: Only organs demonstrating treatment-related changes and reproductive organs: Vagina, ovary, uterus and pituitary gland in females and organs with macroscopical changes were examined at the lowest and middle dose level groups.


Reproduction/Development Part of Study Observation of Parental Males
The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-160-400-1000 mg/kg/day further in the text.
In reproductive organs the microscopical changes were diagnosed sporadically: in the testes, atrophy of germ epithelium in 0-1-0-0 male, degeneration of germ cells in 0-0-1-0 male and atrophy of Leydic cells in 0-0-0-1 male were detected. Functional hyperplasia in 0-0-0-1 male and mononuclear infiltration of interstitium and/or oedema of interstitium in 1-5-5-1 males in prostate gland was detected.
Mononuclear infiltration of interstitium in 0-0-1-0 male and azoospermia and proliferation of interstitium in epididymis in 0-1-0-0 male were found out.
In the liver, the following microscopical changes were found: Vacuolation of hepatocytes in 0- 0-11-4 males and extramedullary haematopoiesis in 0-0-3-0 males. In kidneys thickening of membrane in part of glomerules and proteinnuria in 0-/-/-2 males were found out. In lungs nonpurulent pneumonitis was recorded in 3-0-4-4 males. Eosinophil and/or neutrophil infiltration of submucosa in stomach in 0-4-0-3 males and hyperkeratosis in forestomach in 0- 2-0-2 males were detected.
Other microscopical changes in organs occurred in males sporadically.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Repeated dose section:
Health Condition Control
Health Condition Control was performed in all 12 animals from each group and satellite animals and it is described in this part.

Males
In control males and treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
In treated males at the dose level 160 mg/kg/day no changes of health condition before, during and immediately after application of the test substance were observed. The red colouring of excrements was observed in males at the dose level 400 mg/kg/day in the 3rd, 4th, 5th and 6th week of application period. In males at the dose level 1000 mg/kg/day the following changes were observed: Red colouring of excrements and shavings and tail of animals contaminated by coloured excrements during the whole application period, immediately after application - disquiet after application, gait on toes and raised tail in the 2nd and 3rd week of application period and nuzzling in the shavings from the 2nd week to the end of application period.

Satellite males
In satellite control males and satellite treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
In satellite treated males the following changes were observed: red colouring of excrements and shavings and tail of animals contaminated by coloured excrements during whole application period and in the 1st week of recovery period, immediately after application - disquiet after application, gait on toes and raised tail in the znd and 3rd week of application period and nuzzling in the shavings from the znd week to the end of application period.

Females
In control females and treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
In treated females at the dose level 160 mg/kg/day no changes of health condition before,
during and immediately after application of the test substance were observed. Red colouring of excrements was observed in females at the dose level 400 mg/kg/day in the 4th, 5th, 6th and 7th week of study. In females at the dose level 1000 mg/kg/day the following changes were observed: red colouring of excrements and shavings, tail of animals contaminated by coloured excrements and innnediately after application - nuzzling in the shavings from the 2nd to the 7th week of application period, disquiet after application, gait on toes and raised tail in the 2nd and 3rd week of application period.

Satellite females
In satellite control females and satellite treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period.
In satellite treated females the following changes were observed: Red colouring of excrements and shavings, tail of animals contaminated by coloured excrements and immediately after application - nuzzling in the shavings from the 2nd to the 6th week of application period and in the 1st day of recovery period, disquiet after application, and gait on toes and raised tail in the 2nd and 3trd week of application period.


Reproduction/Development Part of Study Observation of Parental Males
Observation of Sperm
Sperm motility and sperm morphology in treated males and control group was similar.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for repeated dose toxicity was established as 160 mg/kg body weight/day for both males and females. The value was established on the basis of haematology (decreased values of RBC, haematocrit, haemoglobin and prothrombin time, increased values of CPTT and fibrinogen) and biochemistry blood parameters (increased activity of ALP in both sexes) and microscopical examination of organs (vacuolation of hepatocytes in males).
Executive summary:

The test substance, Solvent Red 19E, 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 March 22nd 1996.

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in olive oil. 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 160, 400, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding study phase.

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 3rd day of lactation, males - after mating period- totally for 42 days, non-pregnant females (mated females without parturition) - for 25 days after the confirmed mating.

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 control 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. Functional observation was performed at the end of application and recovery period. Vaginal smears were prepared daily during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) 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 were removed for weighing and histopathological examination.

 

Repeated Dose Toxicity Part of the Study:

Repeated oral administration of Solvent Red 19E to rats by gavage at the dose levels 160,400 and 1000 mg/kg/day did not cause any mortality.

At 160 mg/kg/day, no effect on growth of animals was detected in both sexes only slight changes of food conversion and consumption were detected. The animals’ heath condition was good during the whole study. During functional examination slight decrease of upstanding in males were recorded. Changes of haematological parameters (significantly decreased value of RBC, with the haematocrit, haemoglobin and prothrombin time, significantly increased value of fibrinogen in males), biochemical parameter (significantly increased value of glucose - above historical control limits in males), urinalysis in males (presence of leucocytes) and biometry of organs (significantly decreased absolute and relative weight of kidneys and significantly decreased relative weight of ovaries in females) were detected.

Occurrence of macroscopic changes in males and females (mainly reddish colouring of some organs and/or tissues in both sexes) and microscopic changes in prostate gland in males and ovaries, vagina, mammary gland and uterus in females were detected at the dose level 160 mg/kg/day. These microscopic changes in reproductive organs did not relate to the test substance treatment.

 

At 400 mg/kg/day, no effect on growth of animals was detected in both sexes only slight changes of food conversion and consumption were detected. Changes of clinical status in animals (red colouring of excrement), haematological parameters (significantly decreased value of prothrombin time in males and females, in males it was under historical control limits), biochemical parameters (significantly increased value of glucose in males and significantly decreased value of sodium ions in females), urinalysis in males (change of colour, presence of leucocytes) and biometry of organs (significantly increased absolute and relative weight of liver in males and significantly decreased absolute and relative weight of pituitary gland and significantly decreased relative weight of ovaries in females) were detected.

Occurrence of macroscopic changes in males and females (mainly red colouring of some organs and/or tissues by the test substance in both sexes and marked structure in liver in males) and microscopic changes of liver, lungs and prostate gland in males and ovaries, vagina and uterus in females were detected at the dose level 400 mg/kg/day. These microscopic changes in reproductive organs did not relate to the test substance treatment.

 

At 1000 mg/kg/day, no effect on growth of animals was detected in both sexes only slight changes of food conversion and consumption were detected. Clinical status of animals after application was influenced by the test substance treatment (red colouring of excrement and shavings, gait on toes, raised tail, nuzzling in the shavings and disquiet after application). Tail of animals was contaminated by coloured excrement. During functional examination increase of upstanding in satellite females were recorded.

Changes of haematological parameters (irreversible significantly decreased value of RBC, haematocrit and haemoglobin, significantly decreased value of prothrombin time, significantly increased value of fibrinogen in males and irreversible significantly increased value of WBC, delayed significantly decreased value of granulocytes in females), blood biochemical parameters (delayed significantly decreased value of bilirubin total and calcium ions in both sexes and delayed significantly increased activity of AST in females), urinalysis in males (change of colour, presence of leucocytes), biometry of organs (delayed significantly decreased absolute and relative weight of thymus, significantly increased relative weight of liver in males and significantly decreased absolute and relative weight of ovaries, significantly decreased relative weight of kidneys and significantly increased relative weight of spleen in females), gross examination of organs and tissues (mainly irreversible red colouring of some organs and/or tissues in males and females, marked structure in liver in males and dark colouring of spleen in females) and histological examination of organs and tissues (mainly vacuolation of hepatocytes in liver, nonpurulent pneumonitis in lungs in males and accumulation of lipophages and/or pigmentophages in uterus, haemorrhage in uterus and degeneration of tertiary follicles in ovaries in females) at the dose level 1000 mg/kg/day were recorded. These microscopic changes in reproductive organs did not relate to the test substance treatment.

 

Reproduction

The course of mating, pregnancy and lactation of parental animals, weights of reproductive organs and pituitary gland, spermatogenesis and sperm parameters, microscopic structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation, post­ implantation and post-natal losses of mothers and number, weight, sex ratio and development of pups were not adversely affected by the test substance treatment at the lowest dose level. The number of females achieving pregnancy, total number of pups, mean weight of litters and fertility index were slightly decreased.

Occurrence of macroscopic changes in females (red colouring of ovaries in females) and microscopic changes of prostate gland in males and ovaries, vagina, mammary gland and uterus in females were detected at the dose level 160 mg/kg/day. These microscopic changes in reproductive organs did not relate to the test substance treatment.

The course of mating, pregnancy and lactation, weights of reproductive organs, spermatogenesis, microscopic structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation losses of mothers and pathological examination of pups were not adversely influenced by the test substance treatment at the dose level 400 and 1000 mg/kg/day.

The number of females achieving pregnancy was decreased at the middle dose level. Calculation of reproduction parameters (significantly decreased number of corpora lutea and decreased number of uterus implantations) and fertility parameters (decreased fertility and gestation index) in animals showed mild changes at the middle dose level.

Occurrence of macroscopic changes in females (red colouring of ovaries in females) and microscopic changes of prostate gland in males and ovaries, vagina and uterus in females were detected at the dose level 400 mg/kg/day. These microscopic changes in reproductive organs did not relate to the test substance treatment.

The number of females achieving pregnancy was decreased at the highest dose level. Examination of number and weight of pups (decrease of mean number of pups per litter and significant decrease of mean weight of litters), calculation of fertility parameters (decreased fertility index) in animals found out changes at the highest dose level.

Occurrence of macroscopic changes in females (red colouring of ovaries in females) and microscopic changes of ovaries, vagina and uterus in females were detected at the dose level 1000 mg/kg/day. These microscopic changes in reproductive organs did not relate to the test substance treatment.

 

The test substance administration had no adverse effect on mortality, growth of animals and some reproduction parameters - course of mating, pregnancy and lactation, spermatogenesis and microscopic structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation losses of mothers and pathological examination of pups.

 

The test substance, Solvent Red 19E, during Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test showed effect on clinical status (red colouring of excrements and shavings, gait on toes, raised tail, nuzzling in the shavings, disquiet after application), haematological parameters (decreased values of RBC, haematocrit, haemoglobin and prothrombin time, increased value of APTT and fibrinogen), biochemical parameters (changes of bilirubin, glucose in males, bilirubin, calcium ions, sodium ions in females and ALT, ALP and AST activity), biometry of organs (decreased weight of thymus, kidneys and ovaries and increased weight of liver and spleen) and urine parameters (change of colour urine and occurrence of leucocytes) mainly at the middle and highest dose level.

The test substance had an influence on macroscopic structure of some organs and tissues (red colouring of liver, lymph nodes, the whole GIT, body fat in both sexes and ovaries in females) and microscopic structure of liver in males (vacuolation of hepatocytes).

The test substance treatment affected the number of pups (decrease of mean number of pups and mean weight of litters, decreased number of corpora lutea and uterus implantations). The mean weight of pups was unchanged.

 

The value of NOAEL (No Observed Adverse Effect Level) for repeated dose toxicity was established as lower than 160 mg/kg body weight/day both for males and females. The value was established on the basis of haematology (decreased values of RBC, haematocrit, haemoglobin and prothrombin time, increased value of APTT and fibrinogen) and biochemistry blood parameters (increased activity of ALP in both sexes) and microscopic examination of organs (vacuolation of hepatocytes in males).

 

The value of NOAEL (No Observed Adverse Effect Level) for the reproduction and for development of pups was established as 160 mg/kg body weight/day. The value was established on the basis decreased number of pups per litter, decreased number of corpora lutea, decreased number of implantations, decreased fertility index in males and females and gestation index. In males no effect on reproduction was observed.