Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 30, 2012 to December 14, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Acid Brown 75
IUPAC Name:
Acid Brown 75
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna Czech Republic
- Age at study initiation: 10 weeks
- Weight at study initiation: 356.38 ± 32.41 g

- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. 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: Complete pelleted diet for rats in SPF breeding - ST 1 BERGMAN, manufacturer; Diet was sterilised before using.
Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate , Vitamin and Mineral complex. Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7 %, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Water: Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry.
- Acclimation period: 11 days
- health check: daily during acclimatation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod: 12 hours cycle dark/light

OTHER:
The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriological contaminants every 2 months on a regular basis.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The application form was prepared by mixing the substance with aqua pro injection. Two concentrations of application form were prepared (50 mg/10 mL and 1000 mg/10 mL).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of Acid Brown 75 was performed by high-performance liquid chromatography based on a method developed at the test facility.
Duration of treatment / exposure:
56 days
Frequency of treatment:
7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 70, 250, 630
Basis:
actual ingested
No. of animals per sex per dose:
Basic groups:
1. Control 0 12 males + 12 females
2. Low dose 70 mg/kg/day 12 males + 12 females
3. Intermediate dose 250 mg/kg/day 12 males + 12 females
4. High dose 630 mg/kg/day 12 males + 12 females

Satellite groups:
5. Control – vehicle – satellite: 0 6 males + 6 females
6. High dose – satellite: 630 mg/kg/day 6 males + 6 females

Control animals:
yes, concurrent no treatment
Details on study design:
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 aqua pro injectione in the same volume. The application form (test substance solution in aqua pro injectione) was prepared daily just before administration.




Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes , daily during the acclimatization and the experimental part.
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.
Detailed clinical observation: before the first application and then weekly (except the mating period)
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, 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.
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.

BODY WEIGHT: Yes
males - weekly
females - weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st, 3rd and 4th day;
pups (litters) – 0. or 1st, 3rd and 4th day;
satellite males and females - weekly
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.

FOOD CONSUMPTION yes
males - weekly (except the mating period)
females - weekly during premating period during pregnancy and lactation – on the same days as body weight
satellite males and females – weekly
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 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. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption of pregnant females.


FOOD EFFICIENCY:yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain

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.

HAEMATOLOGY: Yes , Haematology and necropsies:
parental males – 43th day of study
satellite males – 57th day of study
parental females – 4th day of lactation
satellite females – 57th day of study
non-pregnant females – 55th day of study or 26th day after confirmed mating
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 and the following parameters were determined.
Total erythrocyte count
Mean corpuscular volume
Haematocrit
Haemoglobin concentration
Total leucocyte count
Total platelets count
Partial thromboplastin time
Prothrombin time
Granulocytes
Lymphocytes
Monocytes

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.
The following parameters were determined by automatic biochemical analysers SPOTCHEMTM EZ SP-4430 and SPOTCHEMTM EL SE-1520 (Arkray, Inc., Japan).
Glucose
Cholesterol, total
Urea
Bilirubin, total
Aspartate aminotransferase
Alanine amonitransferase
Alkaline phosphatase
Calcium
Phosphorus
Protein, total
Protein, albumin
Creatinine
Sodium
Potassium
Chloride

URINALYSIS: only males – 42nd and 56th day of study.
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 by analyser PocketChem PU-4210 (Arkray, Inc., Japan).
Volume
Colour
Cloud
Odour
Glucose
Protein
Bilirubin
Urobilinogen
pH
Specific gravity
Blood
Ketones
Nitrite
Leucocytes

OTHER:
Mortality control: daily
Laboratory examinations:
- vaginal smears: daily in mating period
- biochemistry: at the end of administration/observation period
- haematology: at the end of administration/observation period
- pathological examination: males and nonpregnant females – at the end of administration period parental females and pups - on the 4th day of lactation satellite males and females - at the end of observation period
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: after necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
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.


HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected at necropsy and fixed:
Reproduction part of study (12 males and 12 females from each main group)
Pituitary gland
Ovaries
Uterus
Cervix of uterus
Vagina
Epididymis
Prostate gland
Testes
All gross lesions

Repeated dose toxicity part of study (6 males and 6 females from each main group + 6 males and 6 females from satellite groups)
Adrenal glands
Aorta
Brain (incl. 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
Thyroid gland incl. parathyroid
Trachea
Urinary bladder
Female mammary gland area
Femur
Heart
Ileum (incl. Peyer´s patches)
Jejunum (incl. Peyer´s patches)
Kidneys
Liver
Lungs
Lymph nodes – mesenteric, paraaortal
Oesophagus
All gross lesions

The mentioned tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde solution (v/v) for further histopathological evaluation.
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: liver, kidneys, heart, spleen, lymph-nodes, stomach, intestines, rectum in males and females and testes and epididymides in males and vagina, ovary, uterus in females and organs with macroscopical changes were examined at the lowest and middle dose level groups and satellite groups.
Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).
Other examinations:
Mortality control: all rats during the treatment periods were examined for vitality or mortality changes daily.

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 and selected reproduction parameters. 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):
Excrements and tail in all treated groups were coloured. . After the end of application of the test substance theseis clinical symptoms disappeared. No other effects were recorded.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
at the highest dose level
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was observed only in satellite animals.

Satellite males
Increased 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
Increased mean water consumption at the treated group was recorded during the whole application period. During recovery period the mean water consumption was similar.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
avobe 250 mg/kg bw (primarily on the red blood cells - decreased value of total erythrocyte count, haematocrit and haemoglobin, increased value of mean corpuscular volume)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg bw for parametrs connected to spleen and kidney
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg bw for urine volume, colours, specific weight, leucocyte, proteins and ketones presence (only at the highest dose).
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg for spleen, brain (males), kideny, heart (females) and uterus (only at the highest dose).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
above 250 mg/kg for intestine and rectum (ulcer or erosion, focal inflammation of mucosa and/or submucosa); Extramedullar haemopoiesis and hemosiderin in spleen and/or liver
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
General Clinical Observation
General Clinical Observation was performed in all 12 animals from each group and satellite animals and it is described in this part. General Clinical Observation was not performed in satellite animals during recovery period (it was performed only after application).
Males
Excrements and tail of males in all treated groups and satellite treated group were coloured by the test substance during the whole study. Other symptoms of clinical status were not observed.
Females
Excrements and tail of females in all treated groups and satellite treated group were coloured by the test substance during the whole study. Other symptoms of changed clinical status were not observed.
Mortality Control
Males
There were no unscheduled deaths during the study in all animals.
Females
There were no unscheduled deaths during the study in all animals.
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
Excrements and tail of males in all treated groups were coloured by the test substance during the whole study. Other changes of health condition were not observed.
Satellite males
Excrements and tail of males in treated group were coloured by the test substance during application and recovery period. Other changes of health condition were not observed.
Females
Excrements and tail of females in all treated groups were coloured by the test substance during the whole study. Other changes of health condition were not observed.
Satellite females
Excrements and tail of females in treated group were coloured by the test substance during application and recovery period. Other changes of health condition were not observed.
Detailed Clinical Observation
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 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, secretion and respiration. Only excrements and tail of males in all treated groups were coloured by the test substance during the whole study.
Satellite males
The activity (poise, gait, reaction to handling) of all males of treated group was similar during the study 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, secretion and respiration. Excrements and tail of males in treated group were coloured by the test substance during application period. During recovery period only tail of males in treated group was coloured by the test substance.
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, secretion and respiration. Only excrements and tail of females in all treated groups were coloured by the test substance during the whole study.
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, secretion and respiration. Excrements and tail of females in treated group were coloured by the test substance during application period. During recovery period only tail of females in treated group was coloured by the test substance.
Functional Observation
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.
Results of upstanding were similar in males of all dose level in comparison with control. 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
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.
Results of upstanding were similar in females of all dose level in comparison with control. 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.


BODY WEIGHT AND WEIGHT GAIN:
Mean body weight
Males
Slight decreased body weight was recorded at the dose level 250 mg/kg/day from the 2nd week of application to the end of study. The body weight at the dose levels 70 and 630 mg/kg/day and control animals was similar during the whole application period. The statistical analysis was performed for body weights. Statistically significant differences were not found.
Satellite males
The body weight of satellite treated males was decreased from the 2nd week of application to the end of application and during recovery period.
Statistical analysis was performed for body weights. Statistically significant differences were not found.
Females
The body weight of treated groups females was slightly decreased in comparison with control group during the whole study (including the week before application).
The statistical analysis was performed for body weights. The statistically significant differences were not found.
Satellite females
The body weight of satellite females was well-balanced during application period. During recovery period body weight of treated females was slightly decreased.
The statistical analysis was performed for body weights. The statistically significant differences were not found.
Body weight increment
Males
The body weight increment of animals at all dose levels was similar to the body weight increment of animals in the control group during the whole application period.
Satellite males
The body weight increment of satellite treated males during application period was similar to satellite control (except the 6th week of application period in treated group, when it was decreased). During recovery period the body weight increment in treated group was increased.
Females
The evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study.
The body weight increment at the dose levels 70 and 250 mg/kg/day was slightly decreased during the first two weeks of exposure with respect to control (the 1st week at the dose level of 250 mg/kg/day was even negative). The body weight increment at the dose level 630 mg/kg/day was decreased during the first week of exposure and slightly increased during the second week.
Satellite females
The body weight increment of treated females in the 1st, 4th and 6th week of application period and in the 2nd week of recovery period was decreased or negative, in the 2nd, 3rd, 5th week of application period and in the 1st week of recovery period was similar to satellite control.

FOOD CONSUMPTION AND CONVERSION
Males
The mean food consumption of all treated groups and control group were similar.
Satellite males
The mean food consumption of both groups was similar during the whole study.
Females
The mean food consumption of all treated groups and control group were similar.
Satellite females
The mean food consumption of both groups was similar during whole study.
Food conversion
Males
The mean food conversion of treated males during application period was similar to control males (except the 1st week of application period at the dose levels 250 and 630 mg/kg/day, when it was decreased).
Satellite males
The mean food conversion of treated group was markedly decreased in the 1st and 6th week of application period and during recovery period the mean food conversion was increased.
Females
Unbalanced mean food conversion at the dose levels 250 and 630 mg/kg/day was recorded in pre-mating period. During pregnancy period the mean food conversion was similar in treated and control females. During lactation period the mean food conversion at the dose level 630 mg/kg/day was negative (the animals grew slim).
Satellite females
The mean food conversion of treated group was negative (the animals grew slim) in the 1st and 6th week of application period and in the 2nd week of recovery period the mean food conversion was zero.

WATER CONSUMPTION
Water consumption was observed only in satellite animals.
Satellite males
Increased 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
Increased mean water consumption at the treated group was recorded during the whole application period. During recovery period the mean water consumption was similar.

HAEMATOLOGY
Males
Decreased value of total erythrocyte counts in males from the 250 and 630 mg/kg/day dose levels was found out (under the historical control). Value of mean corpuscular volume at the dose level 630 mg/kg/day was increased with statistical significance (above historical control). Significantly decreased values of haematocrit and haemoglobin at the dose level 250 and 630 mg/kg/day were recorded (the value of haematocrit was under historical control at the dose level 630 mg/kg/day). Value of activated partial tromboplastin time (APTT) at the dose level 70 and 250 mg/kg/day was significantly increased, though values as were within historical control of ours laboratory.
Other measured parameters were similar to the control group.
Satellite males
In treated group the value of total erythrocyte count was statistically significantly decreased and the value of MCV was significantly increased (both the values were out of historical control range).
SignificantlySignificant increased platelet count was found at in the treated group (above historical control). Other parameters were similar to the control group.
Females
The values of total erythrocyte count and haematocrit at the dose level 630 mg/kg/day were significantly decreased (the value of total erythrocyte count was under historical control). Significantly increased value of mean corpuscular volume at the dose levels 250 and 630 mg/kg/day was recorded (above historical control). At the dose level 250 mg/kg/day the value of protrombin time was significantly decreased.
Other measured parameters were similar to the control group.
Satellite females
Statistically significant increase of haematocrit, haemoglobin and mean corpuscular volume were recorded in treated females (the values of haematocrit and mean corpuscular volume were above historical control).
Other measured parameters were similar to the control group.


CLINICAL CHEMISTRY
Males
Dose-dependent increased value of creatinine was recorded in all treated groups (at the dose level 250 and 630 mg/kg/day modulation was significant). Inorganic phosphorus at the dose level 250 and 630 mg/kg/day was significantly increased . Significantly increased value of protein total was recorded at the dose level of 630 mg/kg/day. Value of bilirubin total at the dose level 250 and 630 mg/kg/day was increased (at the dose level 630 mg/kg/day the data were significant). Increased value of glucose at the dose level 70 (significant) and 630 mg/kg/day (slight) was recorded.
Slight increased activity of ALT was recorded at the dose level 630 mg/kg/day.
All other measured parameters were similar to the control group.
Satellite males
Significant modulations of the Cconcentration of sodium ions and the value of creatinine were significanceobserved.
All other measured parameters were similar to the control group.
Females
Significant increased value of creatinine at the dose level of 250 and 630 mg/kg/day was recorded. At the dose level of 630 mg/kg/day value of bilirubin total was significantly increased and concentration of potassium ions was significantly decreased. Slight decreased value of glucose and urea at the dose level of 250 and 630 mg/kg/day was recorded. In all treated groups activity of AST was increased and activity of ALT and ALP was decreased (significant modulation on the activity of ALT at the dose level 250 mg/kg/day).
All other measured parameters were similar to the control group.
Satellite females Significantly decreased concentration of potassium ions and value of glucose were recorded in the treated group. Values of bilirubin total, creatinine and activity of AST were significantly increased.
All other measured parameters were similar to the control group.

URINALYSIS
Males
The statistical analysis was performed for urine volume and pH of urine. The volume of urine was statistically decreased at the dose level of 250 and 630 mg/kg/day. The number of males with higher value of specific weight of urine was recorded at the dose level 250 and 630 mg/kg/day. Change of colour of urine (from yellow to dark orange) was recorded in all males of treated groups.
At the dose level of 630 mg/kg/day presence of glucose in one male, presence of protein and ketones in four males, presence of bilirubin and urobilinogen in one male and presence of leucocytes in all six males were recorded.
At the dose level of 250 mg/kg/day presence of protein in one male, presence of nitrites in one male and presence of leucocytes in all six males were recorded.
At the dose level of 70 mg/kg/day presence of glucose in one male, presence of blood in one male and presence of leucocytes in two males were recorded.
In control group presence of leucocytes in two males was recorded.
Satellite males
Change of colour of urine (yellow – orange - glaringly orange), presence of protein in one male, presence of urobilinogen in one male and presence of leucocytes in four males were recorded in treated group.


GROSS PATHOLOGY
Biometry of Organs
Absolute organ weight
Males
Dose-dependent increased absolute weight of spleen was recorded in all treated groups (at the dose level of 250 and 630 mg/kg/day the modulations were significant.
Slight
Iincrease of weight of kidneys was recorded at the dose levels of 250 and 630 mg/kg/day. Absolute weight of thymus and brain was decreased at the dose level of 630 mg/kg/day, though not significant.
The absolute weight of other organs was well-balanced with the control group.
Satellite males
Significant Ddecreased absolute weight of brain of treated group was recorded. The absolute weight of other organs was well-balanced with the control group.
Females
Slight, Ddose-dependent increased absolute weight of spleen was recorded at the dose level 70 and 250 mg/kg/day. The same modulation was significant at the dose of 630 mg/kg/day. At the dose level 630 mg/kg/day significant increased absolute weight of uterus was recorded ) as well as
slight decrease of absolute weight of liver. Absolute weight of thymus was slightly modulated for all treatments without statistical significance.
The absolute weight of other organs was relatively well-balanced with the control group.
Satellite females
The absolute weight of all organs was relatively well-balanced with the control group.
Relative organ weight
Males
Dose-dependent increased relative weight of spleen was recorded in all treated groups (at the significant for the dose level of 250 and 630 mg/kg/day
Increased relative weight of kidneys was recorded at the dose level 250 and 630 mg/kg/day
without statistical significance. Relative weight of prostate gland was increased and relative weight of thymus was decreased at the dose levels 630 mg/kg/day without statistical significance.
Relative weight of other organs was well-balanced with the control group.
Satellite males
The statistical analysis of the data revealed significantly increased weight of kidneys in the treated group of males.
Significant
dose-dependent increased relative weight of spleen was recorded
at the dose level of 250 and 630 mg/kg/day
). Significant
increased weight of heart
was recorded (at the dose level of 250 and 630
mg/kg/day
). Significant increase of relative weight of kidneys and uterus at the dose level 630 mg/kg/day was recorded. Significantly increased weight of thymus was recorded at the dose level 250 mg/kg/day.
The relative weight of other organs was well-balanced with the control group.
Satellite females
Significant increased relative weight of kidneys and heart in treated group was recorded
. Mild increase of relative weight of spleen in treated group was recorded.
The relative weight of other organs was well-balanced with the control group.

Pathology
The incidence in affected animals is expressed in numeric form and arranged in sequence in the dose levels 0-70-250-630 in the main males groups (first-six males for group), 0-70-250-630 (first-six birth-giving females in group) in the main females groups and 0S-630S in the satellite males and females group further in the text.

Macroscopic findings
Males
In 6-2-0-0 males no macroscopic changes were observed. Brown colour of tail and anus in 0-0-6-6 males was observed. In stomach congested mucous membrane in 0-2-4-4 males and dark colour of chymus in 0-0-4-5 males was found out. Dark colour of spleen in 0-0-5-6 males and enlargement of spleen in 0-0-2-3 males was observed. Other changes were observed only sporadically.
Satellite males
In 6-5 males no macroscopic changes were observed during necropsy. Dark colour of spleen in 0-1 male was observed.
Females
In 6-4-1-0 females no macroscopic changes were observed. In stomach congested mucous membrane in 0-2-3-5 females and dark colour of chymus in 0-0-0-5 females was found. Dark colour of chymus in GIT of 0-0-2-4 females was observed. Dark colour of spleen in 0-0-2-6 females and enlargement of spleen (defined as intumescence, and increase of organ size) in 0-0-1-6 females was observed. Light colour of liver in 0-0-1-2 females was observed. In three females at the dose level 630 mg/kg/day dark colour of kidneys was observed.
Satellite females
In 6-2 females no macroscopic changes were observed. Cyst in ovary in 0-2 females was observed. Dilatation of uterus (accompanied oestrus) was recorded in 2-4 satellite females. Changes in stomach and spleen were observed only sporadically.

Histopathological findings
Full histopathology of the preserved organs and tissues was performed for all high dose and control animals in the following organs (organs demonstrating treatment-related changes): liver, kidneys, heart, spleen, lymph-nodes, stomach, intestines, rectum in males and females and testes and epididymides in males and vagina, ovary, uterus in females and organs with macroscopical changes were examined at the lowest and middle dose level groups and satellite groups. The incidence in affected animals is expressed in numeric form and arranged in sequence in the dose levels 0-70-250-630 in the main groups and 0S-630S in the satellite groups further in the text.
Males
In the spleen, the following microscopical changes were found: venostasis in 0-0-0-5 males, extramedullary haematopoiesis in 0-1-5-5 males and hemosiderin in 0-1-1-1 males. In kidneys brown pigment in 0-1-4-5 males, hyaline droplets in tubular epithelium in 0-1-2-2 males and
focal dystrophy or fibrosis in cortex in 0-0-1-2 males were found out. In liver extramedullary haematopoiesis in 0-1-1-2 males and focal mononuclear infiltration in 4-4-2-3 males were detected. Presence of brown pigment in mucosa and/or submucosa in 0-0-4-5 males, erosion or ulcer in 0-0-1-3 males and focal inflammation of mucosa and/or submucosa in 0-0-4-4 males in large intestine were recorded. Erosion or ulcer in rectum in 0-0-1-2 males was found out. Focal mononuclear infiltration and/or oedema of interstitium in prostate gland in 6-0-0-2 males and exfoliation of germ cells into the lumen of tubules in testes in 6-2-2-2 males were detected.
Other microscopical changes in organs occurred in males sporadically.
Satellite males
Brown pigment in kidneys was recorded in 0-6 males. Focal infiltration in liver was detected in 3-5 males. Hemosiderin in spleen was found out in 2-5 males. In large intestine presence of brown pigment in mucosa and/or submucosa in 0-5 males and focal inflammation of mucosa and/or submucosa in 0-5 males were detected. Hyperplasia in lymph-nodes (paraaortal) was found out in 2-1 males.
Focal mononuclear infiltration and/or oedema of interstitium in 1-3 males, cell detritus in lumen in 4-2 males in prostate gland and exfoliation of germ cells into the lumen of tubules in testes in 4-2 males were detected.
Other microscopical changes in organs occurred in males sporadically.
Females
In the spleen, the following microscopical changes were found: venostasis in 0-0-3-1 females, extramedullary haematopoiesis in 1-0-3-6 females and hemosiderion and/or pigmentation in 0-0-4-6 females. Brown pigment in kidneys was found out in 0-0-0-2 females. In liver hemosiderin in 0-0-2-3 females and extramedullary haematopoiesis in 1-0-4-6 females were recorded. Ulcer in large intestine or caecum in 0-0-0-2 females was found out. Focal inflammation in submucosa and/or pus floccule in lumen in large intestine in 0-0-0-2 was detected. Lobular hyperplasia in mammary gland in 6-6-6-6 females was recorded. In genital tract focal accumulation of lipophages and sideriphages in mesometrium in uterus in 2-3-4-4 females was detected. Other microscopical changes occurred in females only sporadically.
Satellite females
In 2-0 females no microscopical findings were recorded during histopathological examination. In the kidneys, the following microscopical changes were found: brown pigment in 0-5 females and hyaline droplets in 0-2 females. Hemosiderin in liver was recorded in 0-3 females. In spleen hemosiderin and/or pigmentation in 0-6 females was found out. Bursal cyst in ovary in 0-2 females and hydrometra in uterus in 3-5 females were found out. Other microscopical changes occurred in females only sporadically.





Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The oral administration of Acid Brown 75 to rats by gavage at the dose levels of 70, 250 and 630 mg/kg/day did not cause mortality or overt sign of toxicity on functional observations.

No effect of the test substance on growth of animals was observed up to the highest dose level. Modulation of body weight and/or food conversion was recorded in both sexes, especially at the highest dose treatment.

Clinical observations revealed influence of the test substance on clinical status of treated animals. Excrement and tails in all treated animals were coloured by the test substance. After the end of application of the test substance theseis clinical symptoms disappeared.

Weight differences between controls and treated animals observed during biometry of organs at the middle and highest dose levels: absolute and relative weight of spleen in both sexes was increased. The increased weight of spleen might be possibly explained with the increased incidence of venostasis observed in those animals . Delayed decreased absolute weight of brain and increased relative weight of kidneys were recorded in males. In females irreversible increased relative weight of kidneys was also recorded. The modulation observed

weight of kidneys in females could be related with increased microscopical presence of pigment in kidneys. Increased relative weight of heart was observed in female of the middle and highest dose level and in satellite females. Eventually increased absolute and relative weight of uterus was recorded in females at the highest dose level.

A decreased number of red blood cells and decrease in haemocoagulation parameters were observed during the haematological examination. The value of activated partial tromboplastin time was increased at the lowest and middle dose level in males and females and the value of protrombin time was decreased at the middle dose level. In satellite males also delayed increased value of platelet count was recorded. These changes were statistical significant.

In males the following changes were recorded: significant decreased values of total erythrocyte count, haematocrit and haemoglobin at the middle and high dose level, significant increased value of mean corpuscular volume in males of the highest dose level (values of RBC and MCV were changed irreversibly). In females at the highest dose level values of total erythrocyte count and haematocrit was significantly decreased and related value of mean corpuscular volume was increased. The value of MCV was also increased at the middle dose level and satellite treated females. Increased values of haematocrit and haemoglobin were observed in females. The modulations discussed above might be consistent with the microscopical findings in the spleen recorded during the histopathological examination such as occurrence of extramedullar haemopoiesis, hemosiderin and venostasis, suggesting the haematopoietic tissue as a possible target of Acid Brown 075 exposure.

Biochemical examination suggests that exposure of the test substance might also be consistent with possible activity of the test substance on kidneys and liver. In fact, . Enhanced formation of hyaline droplets in tubules of treated males and two survived females and dystrophy or fibrosis in cortex in males at the middle and highest dose level were also recorded in kidneys. These findings together with the increased concentration of brown pigments in kidneys' tubular epithelium and fibrosis might suggest kidneys as possible sensitive organ for Acid Brown exposure

. In females at the highest dose level value of potassium ions was also decreased as well as it was recorded a significant increased value of creatinine in males and females at the middle and highest dose level. These changes were irreversible. Increased value of phosphorus at the middle and highest dose level and delayed increased value of sodium ions in males. The increased value of bilirubin total in both sexes was observed along with modulation in the activity of AST and ALT in females. In males at the highest dose level increased value of protein total was recorded.

Significant changes of urine properties were recorded during urinalysis in males: decrease of urine volume was detected at the middle and highest dose level. The number of males with

higher value of specific weight of urine was recorded at the middle and highest dose level. Occurrence of leucocytes in urine was increased at the middle and highest dose level, occurrence of protein and ketones at the highest dose level in all treated males and changes of colour of urine was detected in all treated males. Water consumption in satellite males and females was reversibly increased.

The presence of brown pigment was observed during microscopical examination in males and females at the highest dose level and in satellite treated animals in rectum, intestines, kidneys and stomach. Administration of the test substance caused microscopical damage of intestines and rectum (ulcer or erosion, focal inflammation of mucosa and/or submucosa) in females at the highest dose level and markedly in males at the middle and highest dose level. The other parts of GIT were without any pathological lesions (except presence of brown pigment in mucosa of small intestine of one male at the highest dose level).

Extramedullar haemopoiesis and hemosiderin in spleen and/or liver was detected in males and females at the middle and highest dose level and satellite animals. These modulations along with modulation observed in the haematological examination as already discussed

andmight eventually lead to inflammatory response and haematopoietic disorders.

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 70 mg/kg body weight/day both for MALES and FEMALES. This value was established on the basis of haematology parameters (mainly - decreased value of total erythrocyte count, haematocrite and haemoglobin, increased value of mean corpuscular volume) and biochemistry findings (mainly - increased value of sodium ions, phosphorus, bilirubin, total protein and creatinine, decreased value of potassium ions) Histopathological evaluation revealed possible specific target organ toxicity effect on the spleen, kidneys and intestines.
Executive summary:

The substance was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd 1996.

Methods

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 70, 250, 630 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 (630 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,

nonpregnant 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 observation 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.

Results

Repeated dose toxicity part of study:

Repeated oral administration of Acid brown 75 to rats by gavage at the dose levels 70, 250 and 630 mg/kg/day did not cause mortality. 

Slight changes of body weight and body weight increment in males and females, clinical status of animals, haematological and biochemical blood parameters in males, biometry of organs (spleen) in males and females, macroscopical and microscopical structure of organs in males were detected even at the dose level 70 mg/kg/day. However, these modulations were considered incidental, without toxicological relevance.

Body weight, body weight increment, food conversion and clinical status of animals were slightly influenced by the test substance treatment at the dose level 250 mg/kg/day. Influence of the test substance on the haematopoietic tissue (significantly decreased value of RBC, HCT and HGB in males and increased value of MCV in females, significantly increased value of APTT in males and significantly decreased value of PT in females), some biochemical parameters (significantly increased value of phosphorus in males, significantly decreased value of creatinine in males and females and significantly decreased activity of ALT in females), urinalysis in males (urine volume, orange yellow colour, presence of leucocytes) and biometry of organs (mainly in spleen) were detected. Increased occurrence of macroscopical changes of spleen, in males and females (change of colour and enlargement) and microscopical changes of spleen, intestines, and kidneys (increased occurrence of extramedular haemopoiesis, hemosiderin and venostasis in spleen, increased occurrence of extramedular haemopoiesis, hemosiderin and focal infiltration in kidneys and increased occurrence of eros ion or ulcer, inflammation of mucosa and presence of pigment in large intestines) in males and/or females were detected at the dose level 250 mg/kg/day.

Growth of animals at the dose level 630 mg/kg/day was influenced by the test substance treatment (decreased body weight, imbalance of body weight increments and food conversion in females). Clinical status of animals after application was also influenced by the test substance treatment (excrements and tail coloured by the test substance). Haematological examination (irreversible significant decrease of RBC value in males and reversible in females, reversible significant decrease of HCT value in males and females, reversible significant decrease of HGB value in males, irreversible significant increase of MCV value in males and females and insignificant decreased activity of ALT in females) and blood biochemical examination (reversible significant increase of bilirubin total value in males and irreversible in females, irreversible significant increase of creatinine value in males and females and irreversible significant increase of potassium ions value in females), urinalysis (significant increase of urine volume and change of colour – glaringly orange, presence of protein, ketones and leucocytes), biometry of organs (reversible significant absolute and relative weight elevation in spleen of males and females, delayed significantly decreased absolute weight in males, delayed significantly increased relative weight in males, irreversible increased of relative weight of kidneys and heart in females, significantly decreased weight of uterus), gross examination of organs and tissues (reversible change of spleen, stomach, kidneys and chymus in GIT of males and/or females) and histological examination of organs and tissues (mainly partly irreversible occurrence of pigment in kidneys and intestines in males and females, reversible increased occurrence of extramedular haemopoiesis and venostasis in spleen and irreversible increased occurrence of hemosiderin in spleen of males and females, increased occurrence of ulcers or erosions in large intestines or rectum of males and females) at the dose level 630 mg/kg/day revealed significant changes attributable to the test substance administration.    

Conclusion

The test substance, Acid Brown 75, during Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test caused changes in clinical status (excrement and tail were coloured by the test substance), haematological parameters (primarily on the red blood cells - decreased value of total erythrocyte count, haematocrit and haemoglobin, increased value of mean corpuscular volume), biochemical parameters (primarily increased value of sodium ions, phosphorus, bilirubin, total protein and creatinine, decreased value of potassium ions), biometry of organs (changes weight of spleen, kidneys, heart and brain weight) and urine parameters (urine volume, colour, specific weight, occurrence of leucocytes, protein and ketones) at the highest dose level. 

The test substance had influence on macroscopical and microscopical structure of some organs and tissues (occurrence of pigment in kidneys, rectum, intestines and stomach, reversible increased occurrence of ulcer or erosion in large intestines and rectum, extramedular haemopoiesis and hemosiderin in spleen and liver, hyaline droplets in kidneys).

The test substance treatment affected the number of pups (decrease of the total number of live pups and mean weight of litters and affected pups, pre-implantation losses (decreased number of corpora lutea, uterus implantations and pups).

The highest incidence of statistically or biologically significant effects was recorded at the middle and highest dose level while most of changes which were found at the lowest dose level had only mild intensity without adverse alteration of animal organism.

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 70 mg/kg body weight/day both for MALES and FEMALES. This value was established on the basis of haematology parameters (mainly - decreased value of total erythrocyte count, haematocrit and haemoglobin, increased value of mean corpuscular volume) and biochemistry findings (mainly - increased value of sodium ions, phosphorus, bilirubin, total protein and creatinine, decreased value of potassium ions) Histopathological evaluation revealed specific target organ toxicity effect on the spleen, kidneys and intestines.