Disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Endpoint summary

Description of key information

Oral NOAEL: 180 mg/kg bw/day (subacute, rats)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 12th to August 1st, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted by the Council on March 22nd 1996

Deviations:

yes

Remarks:

no impact on the results of the study (details below)

GLP compliance:

yes (incl. QA statement)

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., Únětice, Czech Republic, RČH CZ 21760118.
- Age at study initiation: males, females: sexually adult, 9 weeks on arrival; dose-range finding experiment: 9 weeks on arrival.
- Weight at study initiation: males 363-510 g and females 224 - 382 g.
- Housing: SPF conditions according to internal SOP No.12. 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage.
- Bedding: sterilized soft wood fibres Lignocel.
- Diet: complete pelleted diet for rats and mice in SPF breeding.
- Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law.
- Acclimation period: at least 5 days (DRFE – at least 5 days).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70 %
- Photoperiod: 12 hour light / 12 hour dark.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro injection

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The application form for analysis was prepared in the same manner as for application to animals – i.e. suspension in water for injection.
- Concentration Level 50 mg/10 ml: ca. 0.5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150 ml glass beaker calibrated to 100 ml and the beaker was replenished by the water for injection and dissolved in ultrasonic bath for 15 min. The suspension was stirred by magnetic stirrer (600 rpm) for 15 minutes.
- Concentration Level 1000 mg/10 ml: ca. 5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150 ml glass beaker calibrated to 50 ml and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for a 30 min. The suspension was stirred by magnetic stirrer (850 rpm) for 30 minutes.
- Stability of the application form: the samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.
- Homogeneity of the application form: the homogeneity of the application form was checked by determination of a concentration of the test substance in three places of suspension (at the bottom, in the middle and at the surface).
- Results of analysis: from the results of analyses (homogeneity and stability) follows that the suspension of the test substance in vehicle in concentration level 50 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of suspension by defined manner) is homogenous and stable at least for 120 minutes starting with preparation of the application form.

DIET PREPARATION
The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test suspension was dissolved in ultrasonic bath for a 20 minutes and then the suspension was stirred by magnetic stirrer (800 rpm) for 30 minutes. The concentrations of suspension at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight.
For each dose level concentration, the suspension was prepared separately. The application forms were prepared daily just before administration.
The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of suspensions continued during administration.

Duration of treatment / exposure:

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,
non-mated females – to the 54th day of study.

Frequency of treatment:

The animals were treated 7 days per week at the same time (8.00 – 10.00 am).

Remarks:

Doses / Concentrations:
80, 180 and 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females and 12 males per dose per group, 6 males and 6 females per satellite group.
Dose-range finding experiment: 5 males and 5 females per group.

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION of EXPERIMENTAL ANIMALS
During the acclimatisation period the health condition of all animals was controlled daily. Then the animals were randomly divided into the control and test groups and they were marked individually.

Observations and examinations performed and frequency:

HEALT CONDITION CONTROL
- Time schedule for examinations: daily - during the acclimatization and the experimental part.
- Method of investigation: all rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before, during application and immediately after application.

CLINICAL OBSERVATIONS
- Time schedule for examinations:
males and females: daily during the administration period.
pups: as soon as possible after delivery and then daily.
- Method of investigation: males and Females: all rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 – 13.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.
Pups: all pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

DETAILED CLINICAL OBSERVATIONS
- Time schedule for examinations: before the first application and then weekly (except the mating period).
- Method of investigation: 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.

MORTALITY
- Time schedule for examinations: twice daily.
- Method of investigation: all rats during the treatment periods were examined for vitality or mortality twice daily.

BODY WEIGHT
- Time schedule for examinations:
males: weekly.
females: weekly in premating and mating period; during pregnancy at the 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.
- Method of investigation: 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 an mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations:
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.
- Method of investigation: in a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food conversion in % (weight increment/food consumption x 100) was calculated for animals of Repeated Dose Toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: satellite males and females: twice a week.
- Method of investigation: 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.

FUNCTIONAL OBSERVATION
- Time schedule for examinations: at the end of administration/observation period.
- Method of investigation: 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.

URINALYSIS
- Time schedule for examinations: the last day of administration/observation period – only males.
- Method of investigation: this examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for long time). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 ml of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined: volume, colour, cloud, odour, glucose, protein, bilirubin, urobilinogen, pH, specific gravity, blood, ketones, nitrite, leukocytes.

HAEMATOLOGY
- Time schedule for examinations: at the end of administration/observation period.
- Method of investigation: 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.
The following parameters were determined: total erythrocyte count, mean corpuscular volume, haematocrit, haemoglobin concentration, total leukocyte count, total platelets count, partial thromboplastin time, prothrombin time, granulocytes, lymphocytes, monocytes.

BIOCHEMISTRY
- Time schedule for examinations: at the end of administration/observation period.
- Method of investigation: 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 amonitransferase, alkaline phosphatase, calcium, phosphorus, protein total, protein albumin, creatinine, sodium, potassium, chloride.

PATHOLOGICAL EXAMINATION
- Time schedule for examinations:
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.
- Method of investigation: during the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4 % formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Sacrifice and pathology:

ORGANS WEIGHT
- Time schedule for examinations: during necropsy.
- Method of investigation: 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.

HISTOPATHOLOGICAL EXAMINATION
- Time schedule for examinations: after necropsy.
- Samples of the following tissues and organs were collected at necropsy and fixed:
Reproduction part of study: pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland, seminal vesicles and coagulating gland, testes and all gross lesions.
Repeated Dose Toxicity part of study: adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord - thoracic, spleen, stomach, thymus, thyroid gland incl. parathyroid, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mesenteric, paraaortal, oesophagus, all gross lesions.
The mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals.
Organs demonstrating treatment-related changes: mesenteric lymph nodes, large intestine, caecum, liver, adrenal glands, kidneys, testes, epididymis, prostate gland, seminal vesicles, ovaries, uterus and organs with macroscopical changes were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.
Detailed histological examination was performed on testes of all males from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

substance-coloured excrements

Mortality:

mortality observed, treatment-related

Description (incidence):

substance-coloured excrements

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at 500 and 180 mg/kg bw/day increases in males and females; at 80 mg/kg bw/day increases in males

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 500 and 180 mg/kg bw/day increases in males and females

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

at 500 and 180 mg/kg bw/day increases in males and females

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

differences from the control recorded at all the doses tested and both, in males and females

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

differences from the control recorded at all the doses tested and both, in males and females

Urinalysis findings:

no effects observed

Description (incidence and severity):

statistically significant differences were not recorded

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

differences from the control recorded at all the doses tested and both, in males and females

Gross pathological findings:

no effects observed

Description (incidence and severity):

no macroscopical findings were recorded

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

differences from the control recorded at all the doses tested and both, in males and females

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the main study.

Health Condition Control
- 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, test-substance coloured excrements were recorded in different time-intervals: in males of the lowest dose level – during the last week of application; in males of the middle dose level - from the 5th week and in males of the highest dose level – from the 2nd week of study.
- 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 test-substance coloured excrements were observed in application period – from the second week of study till the end of application period (the 6th week). During the observation period no changes of health status were noted in satellite treated males.
- 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, test-substance coloured excrements were recorded in different time-intervals: in females of the lowest dose level – during the last week of application; in females of the middle dose level - from the 5th week and in females of the highest dose level – from the 2nd week of study.
- 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 test-substance coloured excrements were observed in application period – from the second week of study till the end of application period (the 6th week). During the observation period no changes of health status were noted in satellite treated females.

Clinical Observation
- Males: at all treated animals, only the test substance-coloured excrements in different weeks of application period were recorded.
- Satellite males: in satellite treated males test substance-coloured from the 2st week to the 6th week of application period were found out during clinical examination performed after application.
- Females: at all treated females, only the test substance-coloured excrements in different weeks of application period were recorded.
- Satellite females: in satellite treated females - test substance-coloured excrements were observed after application from the 2nd week of study.

BODY WEIGHT AND WEIGHT GAIN
- Males: different body weight before application was caused by sequential putting animals on the study. Body weight of treated males was within whole application period increased and quite well-balanced in comparison with control males. Statistically significant differences were not found in treated males. Weight increments in treated males were increased compared to control males within the application period. Only in the 6th week of study, decreased weight increment in males of the lowest dose levels was detected.
- Satellite males: body weight of satellite treated males was decreased in comparison with satellite control males for the whole time of application and during recovery period. Statistically significant differences were not found in satellite treated males. Weight increments of satellite treated males in application period were quite balanced. In recovery period weight increments of satellite treated males were slightly increased against satellite control.
- Females: body weight of control females and females of the lowest dose level was quite balanced during the pre-mating period and pregnancy period. Increased body weight (without statistical significance) of females of the lowest dose level was recorded during the lactation period. Increased body weight against the control animals was recorded in females of the middle and the highest dose level during the pre-mating, pregnancy and lactation period. Statistically significantly increased body weight was found in females of the middle dose level in the 7th 14th and 20th day of pregnancy and in the 1st and 4th day of lactation. Statistically significantly increased body weight was found in females of the highest dose level during the day 0, in the 7th and 14th day of pregnancy period and in the 1st and 4th day of lactation.
Increased weight increments in treated females were recorded. The evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study.
- Satellite females: body weight of satellite treated females was increased (without statistical significance) in comparison with control animals for the whole time of application and recovery period. Weight increments of satellite treated females were very variable in comparison with control: increased in the 1st – 4th week, similar in the 5th week and then variable.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Males: the food consumption of treated males was slightly increased and quite balanced in comparison with control animals.
- Satellite males: the food consumption of satellite treated males was slightly increased till the 3rd week of study and then similar to the control group.
- Females: in pre-mating period the food consumption of treated females was higher compared to control females. During pregnancy period the food consumptions of treated females compared to control animals was higher. During lactation period the food consumption of treated females was lower in comparison with control animals.
- Satellite females: the food consumption of satellite treated females was slightly increased for the whole time of application period except for the last week of recovery period.

FOOD EFFICIENCY
- Males: the food conversion of treated males was changed in a dose dependent manner in the 1st week of study. Increased food conversion was recorded in treated males in the 2nd and the 5th week of study. Decreased food conversion during the 6th week of application was noted in males of the lowest dose levels.
- Satellite males: the food conversion of satellite treated males was variable in comparison with control animals – similar in the 1st and 3rd week; increased in the 5th, 7th and 8th week of study and decreased in the 2nd, 4th and 6th week of study.
- Females: the food conversion of treated females in pre-mating period was obviously increased (except females of the lowest dose level in the 1st week) compared to control animals. In pregnancy period, food conversion of treated females was similar to control females till the 14th day of pregnancy. In the last week of pregnancy, lower food conversion was recorded in females of the dose levels 80 and 500; vice versa increased conversion was noted in females of the middle dose level.
Decreased food conversion was calculated in treated females in lactation period. Significant decreasing of conversion was reported in females of the middle dose level (this change was caused by one female, which had significantly negative food conversion).
- Satellite females: the food conversion of satellite treated females was very variable in comparison with control: decreased in the 4th - 6th week, 8th week of application and increased from the 1st till the 3rd week and in the 7th week of study .

WATER CONSUMPTION AND COMPOUND INTAKE
- Satellite males: the water consumption of satellite treated males was higher compared to satellite control during the whole study.
- Satellite females: in application period the water consumption of satellite treated females was higher compared to satellite control from the 1st to 5th week and in 7th week of study. Decreasing water consumption was recorded in treated animals in the 6th and 8th week of study.

DETAILED CLINICAL OBSERVATION
- Males: excrements coloured by the test substance were found out at all treated groups in different time interval of the study.
- Satellite males: excrements coloured by the test substance were observed in satellite treated males from the 2nd to the 6th week of the study.
- Females: excrements coloured by the test substance were found out at all treated groups in different time interval of the study.
- Satellite females: excrements coloured by the test substance were observed in satellite treated males from the 2nd to the 6th week of the study.

FUNCTIONAL OBSERVATION
- Males: reactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The activity – number of upstanding was quite well-balanced. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated males.
- Satellite males: no significant differences were detected in examined parameters.
- Females: reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control females. The activity – number of upstanding was quite well-balanced. The values of grip strength of pectoral and pelvic legs were without significant differences.
- Satellite females: no significant differences were detected in examined parameters.

HAEMATOLOGY
- Males: red blood component (RBC, MCV, Hct, Hgb) and platelet count were not influenced by administration of the test substance and were similar in control and treated males. Parameters of white blood component were influenced in treated males. Total leukocyte count (WBC) was increased in a dose dependant manner though the modulation was significant and out of historical value range only in treated males of the highest dose level. The percentage value of granulocytes was significantly decreased in males of the middle and the highest dose level (in the range of historical control). The percentage value of lymphocytes was significantly increased –in males of the middle dose level (in the range of historical control).
Haemocoagulation parameters (APTT, PT) were significantly influenced in treated males of the highest dose level. Decreased protrombin time and activated partial protrombin time were recorded
- Satellite males: only significant increase of MCV value was found out in satellite treated males. The percentage value of granulocytes was slightly decreased in treated males. Other parameters of red and white component and haemocoagulation were similar in satellite treated and control males.
- Females: statistically significant difference of RBC value was detected in treated females of the lowest dose level. Other parameters of red blood component were similar in control and treated females. Slight increase of total leukocyte cells count was recorded in treated females of middle and the highest dose levels. Statistically significant increase of proportional count of lymphocytes (in the range of historical control) in comparison with control animals was recorded in females of middle and the highest dose levels. On the contrary, slight (though dose related) decrease of proportion of granulocytes and monocytes were observed in females. Coagulations parameters were not influenced in treated groups of females.
- Satellite females: statistically significant decrease of Hct, Hgb value and APTT was noted in satellite treated females. Slight increased percentage count of lymphocytes in treated females was observed. Other parameters were similar in satellite control and treated females.

CLINICAL CHEMISTRY
- Males: the concentration of total cholesterol of 4-6-5-6 males was lower than 1.29 mmol/l (the limit of detection of analyser ). The activity of ALT of 5-3-3-1 male was lower than 0.17 µkat/l (limit of detection of analyser). Statistically significantly increased value of glucose, bilirubin, creatinine (out of the range of historical control) and ALP and calcium (calcium - out of range of historical control in the lowest and the middle dose levels) were recorded in males of the highest dose level. These changes were not dependent on dose level.
Value of ALP was statistically significantly increased also in males of middle dose level.
Value of calcium was statistically significantly increased in all treated groups of males, but without dose-dependence.
Values of other biochemical parameters of treated males were similar to the control group.
- Satellite males: the concentration of total cholesterol of 2-6 males was lower than 1.29 mmol/l (the measurable limit of analyser). The activity of ALT of 2-3 male was lower than 0.17 µkat/l (the measurable limit of analyser). Statistically significant decrease of glucose and chloride concentrations were recorded in satellite treated males. All these differences were within the limits of historic control. Values of other biochemical parameters of satellite treated males were similar to the satellite control group.
- Females: the concentration of total cholesterol of 5-3-5-6 females was lower than 1.29 mmol/l (the measurable limit of analyser). The activity of ALT of 6-4-2-3 female was lower than 0.17 81µkat/l (the measurable limit of analyser). Statistically significant differences were not found out in treated females, thus only slight modulation were observed such as: increase of glucose, bilirubin, ALP activity and creatinine at the highest dose level. These changes were not dependent on dose level. Increased value of ALP was also detected in females of the lowest and the middle dose level. Increased value of creatinine was also recorded in females of the lowest dose level. Values of other biochemical parameters of treated females were similar to the control group.
- Satellite females: differences in values of some biochemical parameters between females of basic and satellite group related to previous pregnancy and lactation of basic females. The concentration of total cholesterol of 3-3 females was lower than 1.29 mmol/l (the measurable limit of analyser). The activity of ALT of 6-4 satellite females was lower than 0.17 µkat/l (the measurable limit of analyser).
Statistically significant decrease of phosphorus concentration was recorded in satellite treated females. Values of all other parameters were slightly modulated and within the limits of historic control. Increase of glucose and creatinine concentration was detected at the satellite treated females. Values of other biochemical parameters of satellite treated females were similar to the satellite control females.

URINALYSIS
Urinalysis was performed only in males (six of each group) during the last week of the study. Statistical evaluation was performed for pH and volume of urine.
- Males: statistically significant differences were not recorded in treated males. Different specific gravity of urea was recorded in males of the middle and the highest dose levels. Presence of leucocytes in urine was noted in 2 males of the lowest dose level, in 2 males of the middle dose level and in one male of the highest dose level.
- Satellite males: statistically significant differences were not recorded in treated males. Presence of leucocytes in urine was noted in 2 males of the control dose level and in one male of the treated group of males.

ORGAN WEIGHTS
Absolute Organ Weight
- Males: statistically significantly increased thymus weight was detected only at the highest dose level. Slight increase of absolute weight of thymus was also recorded in males of the lowest and the middle dose levels. Increased weight of brain – statistically significant, was noted in males of the middle dose level. Dose dependent increasing of absolute weight of spleen, kidneys and liver though not significant was detected at treated males. On the contrary dose dependent decreasing of absolute prostate weight was recorded in treated males (still not significant). Decreased weight of testes was recorded in all treated males. Weight of other organs was similar in treated and control males.
- Satellite males: slight increase of absolute weight of thymus was noted in treated group of animals. Significant increased weight of adrenal glands and decreased prostate gland weight was noted in treated males. Weight of other organs was similar in satellite treated and satellite control males.
- Females: slight increase of weight of thymus was recorded in females of all treated groups. Statistically significant increased weight of adrenal glands, liver and kidneys were recorded in treated females of the highest dose level. Also increased weight of kidneys in females of the middle dose level with the statistical significance was recorded. Slight increasing dose-dependent weight of adrenal glands, heart, kidneys, liver was recorded in females of all treated groups. Weights of other organs were similar in treated and control females.
- Satellite females: the only significant increase observed was the weight of adrenal glands in treated females. Slight increased weight of spleen, heart, kidneys and liver was recorded. Weights of other organs were similar in satellite treated and control females.

Relative Organ Weight
- Males : significant increase in relative weight of thymus was reported in males of the highest dose level as well as decrease in relative weight of brain in males of the middle dose level was noted. Dose dependent increasing weight of kidneys without statistical significance was reported in treated males. Weight of liver was also significantly increased in males of the middle and the highest dose level. Slight decreased weight of testes in all treated groups was reported. Decreased relative weight of prostate gland was observed in all treated groups of males. Significant decrease weight of prostate gland was reported in males of the middle and the highest dose level. Relative weights of other organs of treated males were similar to control males.
- Satellite males: increased relative weight of thymus was reported in treated males. Significant increase of relative weights of adrenal glands and decrease of prostate gland weight were reported in satellite treated males. Slight increase weight of thymus, brain, heart, kidneys and liver was reported.
Relative weights of other organs were similar in satellite treated and control males.
- Females: slight increase relative weight of thymus of all treated groups of females was reported. Relative weight of adrenal glands of treated females was significant increase in females of the highest dose level. All the other modulation was slight and within historical range, such as decreased weight of brain at the highest dose level, and increased weight of liver at the dose levels of 180 and 500. Weights of other organs of treated females were quite well-balanced in treated and control animals.
- Satellite females: significant increase weight of adrenal glands and decreased weight of brain and pituitary gland were detected in satellite treated females. Relative weight of thymus was insignificantly decreased in satellite treated females. Relative weights of other organs were similar in satellite treated and control females.

GROSS PATHOLOGY
- Males: no macroscopical findings were recorded in 6-0-0-0 males. The major changes observed were related to increase pigmentation of organs and tissue given by the brownish colour of the test substance. The content of gastrointestinal tract was coloured by the test substance in all treated males, mesenteric lymph nodes was coloured in males of middle and the highest dose levels. An orange colour was recorded in the pancreas of animals treated at the highest dose level. Atrophy and soft testes were observed in two males of the middle dose level.
- Satellite males: in 6-6 satellite males no macroscopical findings were recorded.
-Females: no macroscopical findings were recorded in 6-0-0-0 females.
The major changes observed were related to increase pigmentation of organs and tissue given by the brownish colour of the test substance. – The content of gastrointestinal tract was coloured by the test substance in all treated females, mesenteric lymph nodes was coloured in females of middle and the highest dose levels. The same orange colour of pancreas was also observed in females treated at the highest dose level.
- Satellite females: no macroscopical findings were recorded in 6-6 females.

HISTOPATHOLOGY: NON-NEOPLASTIC
Full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. The following organs: mesenteric lymph nodes, large intestine, caecum, liver, adrenal glands, kidneys, testes, epididymis, prostate gland, seminal vesicles, ovaries, uterus and organs with macroscopical changes were examined at the lowest and middle dose level groups.
- Males: in 1-0-0-0 males no histological findings were diagnosed. Presence of pigmentophages with light-brown pigment was detected in mesenteric lymph nodes of 0-3-1-5 males. Light brown cytoplasm of enterocytes was registered in large intestine of 0-1-5-6 males and in caecum of 0-1-5-5 males.
Focal mononuclear infiltration was reported in liver of 0-2-2-3 males. Tubular degeneration/atrophy in testes was recorded in 0-0-1-2 males. Other modulations were observed both for controls and treated animals, such as acinar atrophy of prostate gland in 2-3-3-3 males , focal interstitial mononuclear infiltration or oedema of prostate gland in 2-1-3-. Other microscopical changes were observed for both control and treated groups or they occurred sporadically.
- Satellite males : in 0-0 satellite male no histological findings were diagnosed. Generally in satellite males modulations were observed both for control and treated animals. Acinar atrophy in 2-1 males and focal interstitial infiltration of prostate gland in 1-1 males were observed. Degeneration/atrophy of testes were observed in 1-4 males. Other histopathological changes were recorded for both satellite control and satellite treated groups or they were diagnosed only sporadically.
- Females: in 0-1-0-0 female no histological findings were diagnosed. During microscopical examination the following findings were recorded: focal mononuclear infiltration in liver in 0-1-2-0 females; focal dystrophy in cortex of kidneys in 0-0-3-0 and focal mononuclear infiltration in kidneys in 0-0-0-1 females; light brown cytoplasm of enterocytes in large intestine in 0-0-3-6 females and in caecum in 0-0-4-3 females. Other microscopical changes were observed both in controls and treated rats such as pigmentophages with light brown cytoplasm in mesenteric lymph nodes in 1-1-3-2 females or other modulations which occurred only sporadically or they did not relate to test substance treatment.
- Satellite females: in 3-0 females no histological findings were diagnosed. During microscopical examination of satellite treated females the following changes were recorded: pigmentophages with light brown cytoplasm in mesenteric lymph nodes in 0-2 females. Hydrometra of uterus was observed in control and treated females. Other microscopical changes were not connected with the test substance treatment or they were observed only sporadically.

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The value was determined mainly on the basis of haematology and biochemistry results.

Critical effects observed:

not specified

Conclusions:

NOAEL: 180 mg/kg bw/day. The value was determined mainly on the basis of haematology and biochemistry results.

Executive summary:

Method

The substance was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422. Wistar rats of SPF quality were used for testing and the test substance was administered in the form of suspension in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Main groups contained 3 treated groups (doses 80, 180, 500 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 (500 mg/kg/day).  

During the study clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. The functional observation was performed at the end of application and observation period.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals.

Results

Haematological examination showed negative effect of the test substance on white blood component. Dose-dependent increase of total leukocyte count (WBC) in males and statistically significantly increased total leukocyte count in males of the highest dose level was reported. This change was reversible. Increased but not statistically significant total leukocyte count was noted in females of the highest dose level and this change was irreversible. Haematological findings could be related with irreversible increased absolute and relative weight of thymus which was reported in all treated groups of males and females. Absolute and relative weight of thymus was statistically significantly increased in males of the highest dose level. Increasing of absolute and relative weight of thymus was still at the end of recovery period. Reversibly increased absolute weight of spleen in treated males and females at the middle and highest dose levels could be also connected with mentioned findings.

Red blood component was not significantly changed in treated animals.

Significant changes in clinical biochemistry were observed for all treated groups of males.

Bilirubin was significantly modulated – irreversibly increased in males of the highest dose level and reversibly in females of the highest dose level. The value of ALP was also significantly modulated in all treated groups of males. In females, the change of ALP value was slightly increased and reversible. These increased values could be related with increased weight of liver in treated males and females at the middle and highest dose levels. In females of the highest dose level the change of weight of liver was statistically significant.

Significant reversible increased value of calcium was observed in all treated males –and slightly increased in females. Also significantly increased value of creatinine was reported in males of the middle and highest dose levels (in males of the highest dose level – the value was out of range of historical control) and in females of all treated groups. This change could be related with reversibly increased weight of kidneys in treated males and females at the middle and highest dose levels. In females change of weight of kidneys was statistically significant.

Gross necropsy in all dosed animals demonstrated reversible changes of colour of the following organs: digestive tract, mesenteric lymph nodes and pancreas.

Microscopical evaluation found out findings in large intestine and caecum (brown cytoplasm of enterocytes) and pigmentophages with light-brown cytoplasm in mesenteric lymph nodes.

Conclusion

NOAEL: 180 mg/kg bw/day. The value was determined mainly on the basis of haematology and biochemistry results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422. Wistar rats of SPF quality were used for testing and the test substance was administered in the form of suspension in water for injection. Oral application by stomach tube was performed daily. The study includes doses-groups for 80, 180, 500 mg/kg of body weight /day (adjusted on the basis of the substance purity: 43.2, 97.2 and 270 mg/kg bw/day, respectively) and one control group (vehicle only).

Haematological examination showed negative effect of the test substance on white blood component. Dose-dependent increase of total leukocyte count (WBC) in males and statistically significantly increased total leukocyte count in males of the highest dose level was reported. This change was reversible. Increased but not statistically significant total leukocyte count was noted in females of the highest dose level and this change was irreversible. Haematological findings could be related with irreversible increased absolute and relative weight of thymus which was reported in all treated groups of males and females. Absolute and relative weight of thymus was statistically significantly increased in males of the highest dose level. Increasing of absolute and relative weight of thymus was still at the end of recovery period. Reversibly increased absolute weight of spleen in treated males and females at the middle and highest dose levels could be also connected with mentioned findings.

Red blood component was not significantly changed in treated animals.

Significant changes in clinical biochemistry were observed for all treated groups of males.

Bilirubin was significantly modulated – irreversibly increased in males of the highest dose level and reversibly in females of the highest dose level. The value of ALP was also significantly modulated in all treated groups of males. In females, the change of ALP value was slightly increased and reversible. These increased values could be related with increased weight of liver in treated males and females at the middle and highest dose levels. In females of the highest dose level the change of weight of liver was statistically significant.

Significant reversible increased value of calcium was observed in all treated males –and slightly increased in females. Also significantly increased value of creatinine was reported in males of the middle and highest dose levels (in males of the highest dose level – the value was out of range of historical control) and in females of all treated groups. This change could be related with reversibly increased weight of kidneys in treated males and females at the middle and highest dose levels. In females change of weight of kidneys was statistically significant.

Gross necropsy in all dosed animals demonstrated reversible changes of colour of the following organs: digestive tract, mesenteric lymph nodes and pancreas. Microscopical evaluation found out findings in large intestine and caecum (brown cytoplasm of enterocytes) and pigmentophages with light-brown cytoplasm in mesenteric lymph nodes.

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity was established as 180 mg/kg body weight/day. The value was determined mainly on the basis of haematology and biochemistry results. Nevertheless, the substance purity of the lot tested was about 54 %, thus the NOAEL can be adjusted at 97.2 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Study conducted according to internationally accepted testing guidelines and performed according to GLP.

The NOAEL recorded in the study was 180 mg/kg bw/day; the effect level corrected on the basis of the substance purity would be 97.2 mg/kg bw/day.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

 

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. Nevertheless, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for 28-day study the guidance values are increased by a factor of three.

In the case of Acid Brown 282, statistically significant effects were recorded at 500 mg/kg bw/day in females and males, which were treated for 54 days and 42 days, respectively. Taken into account the reference values indicated in the CLP Regulation, the substance effects were recorded at doses exceeding of the classification criteria.

In conclusion, the substance is not classified for repeated dose toxicity according to the CLP Regulation (EC 1272/2008).