Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

The NOAEL (No Observed Adverse Effect Level) for oral repeated dose toxicity was established as 1000 mg/kg body weight/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted on 28th July 2015

Deviations:

yes

Remarks:

not impacting the study results

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o.
- Age: males, females: sexually adult, 7-9 weeks on arrival.
- Weight at study initiation: males 383 - 3978 g; females 250 - 261 g.
- Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period; one male and one female in one cage, pregnant females; individually, offspring; with mother, satellite animals; 2 rats of the same sex in one cage.
- Bedding: sterilized soft wood fibers Lignocel.
- Diet: complete pelleted diet for rats and mice in SPF breeding - Altromin for Rats/Mice.
- Water: drinking water ad libitum.
- Acclimation period: at least 6 days. During the acclimatisation period the health condition of all animals was controlled daily. In all females cyclicity of oestrous cycle was monitored.
- Rationale for animal selection: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex did not exceed ± 20% of the mean weight.

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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro iniectione

Details on oral exposure:

PREPARATION OF APPLICATION FORM
The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test solution was dissolved in ultrasonic bath for a 30 minutes and then the solution was stirred by magnetic stirrer (800 rpm) for 40 minutes. The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY AND HOMOGENEITY
The stability and the homogeneity of application form were determined in testing laboratory analytical laboratories (Analytical Group I).
Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a high-performance liquid chromatography based on a method developed at the test facility.
Both application forms 10 mg and 1000 mg /10 ml of the test substance at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

Duration of treatment / exposure:

Parental males: totally 49 days of administration
Parental females: 13 days, pre-mating period
Non-pregnant females (with and without evidence of copulation): 13 days, pre-mating period
Satellite males and females: totally 49 days of administration + 14 days of observation

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

control and satellite groups

Dose / conc.:

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

Remarks:

tested group

Dose / conc.:

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

Remarks:

tested group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

tested and satellite groups

No. of animals per sex per dose:

12 females and 12 males per group
6 males and 6 females per satellite group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a Dose-Range Finding Experiment was performed. According to results of Dose-Range Finding Experiment the following dose levels 250, 500 and 1000 mg/kg/day were chosen for the main test.

Observations and examinations performed and frequency:

Functional observations, haematology, biochemistry, full biometry and full pathology for evaluation of repeated dose toxicity will be performed only in 6 males + 6 females of each dose level and in satellite animals.

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

HEALTH CONDITION CONTROL
Experimental data collection: daily - during the acclimatization and the experimental part.

CLINICAL OBSERVATIONS
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 probable maximal effect of the test substance. Animals were observed in natural conditions in their cages.

Experimental data collection: males and females daily during the administration period; pups as soon as possible after delivery and then daily.

DETAILED CLINICAL OBSERVATIONS
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

Experimental data collection: before the first application and then weekly (except the mating period).

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.

Experimental data collection: at the end of administration/observation period.

BODY WEIGHT
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too. Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

Experimental data collection:
males and satellite animals - the first day of administration and then weekly
females - the first day of administration and then weekly
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 1st, 4th day, 12th day and 13th day

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

Experimental data collection: weekly and on the same days as body weight (except the mating period); satellite males and females weekly.

WATER CONSUMPTION
The drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

Experimental data collection: satellite males and females twice a week.

HAEMATOLOGY
Parameters: Total erythrocyte count , Mean corpuscular volume, Haematocrit, Haemoglobin concentration, Total leucocyte count, Total platelets count, Partial thromboplastin time, Prothrombin time, Fibrinogen, Granulocytes, Lymphocytes, Monocytes.
Reticulocytes were examined by light microscope. Blood and reticulocyte staining solution e.g. New Methylene Blue were mixed and incubated briefly at room temperature. Smears were made on microscope slides, air dried and evaluated under oil immersion on a light microscope.

Blood collection for haematology and biochemistry:
parental males – 64th day of study
satellite males – 78th day of study
parental females - 13th day of lactation period
satellite females – 78th day of study

BIOCHEMICAL ANALYSIS
Parameters: Protein total, Alkaline phosphatase, Cholesterol total, Triglycerides, Alanine aminotranferase, Aspartate aminotransferase, Creatinine, Urea, Albumin, Bilirubin total, Glucose, Calcium, Phosphorus, Cholinesterase, Bile acids, Sodium, Potassium and Chloride.
Blood samples from the day 13 the parental males were assessed for serum levels of thyroid hormone thyroxine (T4) by ELISA kit.

URINALYSIS
This examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for long time). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 ml of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined: Colour, Cloud, Odour, Glucose, Protein, Bilirubin, Urobilinogen, pH, Specific gravity, Blood, Ketones, Nitrite and Leucocytes.

Experimental data collection: only males – 63rd and 77th day of study. Experimental data collection: the last day of administration/observation period.

PATHOLOGICAL EXAMINATION
Experimental data collection: males and nonpregnant females after the end of application period; parental females on the 13th day of lactation; satellite animals after the end of observation period.

Sacrifice and pathology:

NECROPSY
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4 % formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Experimental data collection:
parental males – 64th day of study
parental females - 13th day of lactation period
non-pregnant females – 26th day after the end of mating period or confirmed mating
satellite males and females – 78th day of study

BIOMETRY OF ORGANS
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected.
The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
From all adult males and females and one male and female day 13 thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

HISTOPATHOLOGY
The tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
The full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes only were examined at the lowest and middle dose level groups used.
Samples of the following tissues and organs were collected at necropsy and fixed: 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, Trachea, Urinary bladder, Female mammary gland area, Femur, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes – mesenteric, paraaortal, Oesophagus and All gross lesions.

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.
The results statistically significant on probability level 0.05 are indicated.

Clinical signs:

no effects observed

Description (incidence and severity):

Only changes related to the colour of the test substance.

Males: in control males and treated males of all dose levels no signs of diseases were recorded during the application period. Only changes related to the colour of the test substance – coloured faeces and bedding were recorded. No clinical changes were recorded in control and treated males during the application period.

Satellite males: in satellite control males and satellite treated males of all dose levels no signs of diseases were recorded during application period. Only changes related to the colour of the test substance – coloured faeces and bedding were recorded in application period. During the observation (recovery) period no changes of health status were noted in satellite treated males. No clinical changes were recorded in control and treated males.

Females: in control females and treated females of all dose levels no signs of diseases were recorded during the application period. Only changes related to the colour of the test substance – coloured faeces and bedding were recorded in application period. At all control and treated females, no clinical changes were recorded during the whole study.

Satellite females: in satellite control females and satellite treated females of all dose levels no signs of diseases were recorded during the application period. Only changes related to the colour of the test substance – coloured faeces and bedding were recorded. During the observation (recovery) period no changes of health status were noted in satellite treated females. No clinical changes were recorded in control and treated females.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males: there were no unscheduled deaths during the main study.

Females: female No.142 (the dose level 500 mg/kg/day) died on the sixteenth day of application due to intubation error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant differences were not found.

Males: body weight of treated males at the dose level 250 and 1000 mg/kg was slightly lower in comparison with the control males within the whole study. Statistically significant differences in necropsy body weight were not found in treated males. Weight increments in treated males were balanced with the control males within three weeks of application. Next four weeks the body weight increments were variable.

Satellite males: body weight of satellite treated males was slightly lower 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 and recovery period were variable and not adversely influenced by the test substance administration.

Females: body weight of all groups of treated females was quite balanced in comparison with the control group of females during the pre-mating period and pregnancy period. Slightly decreased body weight of treated females compared to control females was noted during the lactation period (without dose dependence). Statistically significant differences in necropsy body weight were not found in treated females. Body weight increments were variable within the 1st and 2nd week of application. Low or negative body weight increment after the 1st week of application can be considered to be adaptive process to the test substance administration.

Satellite females: body weight of satellite treated females was comparable with control animals for the whole time of application and recovery period. Statistically significant differences in necropsy body weight were not found in satellite treated females. Weight increments of satellite treated females were variable in comparison with the control group and not adversely influenced by the test substance administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: the food consumption of treated males was quite balanced in comparison with control animals.

Satellite males: the food consumption of satellite treated males was slightly increased compared to the control group in application an recovery period.

Females: in pre-mating period the food consumption of treated females was well balanced with control females. During pregnancy and lactation period the food consumptions of treated females was similar compared to control animals.

Satellite females: the food consumption of satellite treated females was slightly increased in comparison with the control group for the whole time of application and recovery period.

Food efficiency:

no effects observed

Description (incidence and severity):

Males: the food conversion of treated males compared to control animals was similar during the pre-mating period. Different food conversion was recorded in the period after mating, however was not affected by the application of the test substance.

Satellite males: the food conversion of satellite treated males was variable in comparison with control animals – most of time increased and not influenced by the test substance application. In recovery period, increased food conversion of satellite treated males was recorded.

Females: the food conversion of treated females within the 1st week in pre-mating period was decreased compared to control group. It corresponds to low or negative body weight increments of treated females. This finding can be considered as adaptive change to the test substance administration because the food conversion of treated females within the 2nd week of application was similar or slightly increased in comparison with the control females (except females at the dose level 500 mg/kg/day). In pregnancy period, food conversion of treated females no adversely influenced by the test substance treatment.

Satellite females: the food conversion of satellite treated females within application and recovery period was variable in comparison with the control group.

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

no effects observed

Description (incidence and severity):

Satellite males: the water consumption of satellite treated males was higher compared to satellite control group during the whole study.

Satellite females: the water consumption of satellite treated females was higher compared to satellite control group during the whole study.

Haematological findings:

no effects observed

Description (incidence and severity):

Findings considered of no toxicological significance.

Males
Red blood components (RBC, MCV, Hct, Hgb) of treated males were not adversely influenced by administration of the test substance. The platelet count only was statistically significantly increased in the males at the highest dose level, but still in range of historical control. This change was reversible.
The value of reticulocytes was statistically significantly changed (at the dose levels 250 and 1000 mg/kg/day out of historical range) in all treated groups of males. This change was reversible, so it can be considered as adaptive response of organism to chemical stress.
Parameters of white blood component were quite well-balanced in treated males compared to control males. Only differential percentage counts of granulocytes and lymphocytes were changed (in range of historical control), but without dose dependency.
Haemocoagulation parameters (APTT, PT) were influenced by administration of the test substance in males at the highest dose level – statistically significantly increased value were recorded. The concentration of fibrinogen (FIB) was decreased in males of all treated groups in comparison with the control males; at the dose level 500 and 1000 mg/kg/day statistically significantly. The value of PT and FIB were out of historical range, but all changes were reversible.
All findings observed in males were in absence of a treatment-related clinical signs of toxicity can be considered to be of no toxicological significance.

Satellite males
Only significant decrease of total leucocyte count (in a historical range) was found out in satellite treated males. Higher percentage count of granulocytes was found out in treated males. Other parameters of red and white component and haemocoagulation were not adversely affected by the test substance administration.

Females
Parameters of red blood component were quite well-balanced in treated females compared to control females. The value of erythrocytes was slightly decreased in females at the highest dose level and with that relates decreased value of haemoglobin and haematocrite (statistically significantly decreased haematocrite). The value of reticulocytes was statistically significantly changed in females at the highest dose level (out of historical range). This change was irreversible. Parameters of white blood component were quite well-balanced in treated females compared to control females. Coagulation parameters of treated animals were quite well-balanced with the control animals, only the value of fibrinogen at the highest dose level was statistically significantly decreased (in a historical range). This change was reversible.

Satellite females
Statistically significantly changed value of reticulocytes was recorded in satellite treated females. The value at the end of recovery period was still significantly increased, but there was an evident reduction in value into the range of historical control. Other parameters were similar in satellite control and treated females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Findings considered of no toxicological significance.

Males: all biochemical parameters of treated males were similar with the control males. Statistically significant differences were not found out in treated males.

Satellite males: statistically significantly decreased values of ALT, creatinine, BUN and concentration of chloride were recorded in satellite treated males. Statistically significantly increased values of BA and concentration of Ca and IP were recorded in satellite treated males. All changed values were still in the range f historical control values. Values of other biochemical parameters of satellite treated males were similar to the satellite control group.

Females: statistically significant differences were found out in treated females – total bilirubin concentration was decreased at the dose levels 250 and 500 mg/kg/day (in a historical range), phosphorus concentration was decreased at the dose levels 500 and 1000 mg/kg/day (out of historical range). All changed values were without dose dependency and reversible. Values of other biochemical parameters of treated females were quite well-balanced to the control group.

Satellite females: differences in values of some biochemical parameters between females of basic and satellite group did not show any significant differences.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Findings considered of no toxicological significance.

Males: statistically significant differences were recorded in volume of urine in males at the dose levels 250 and 500 mg/kg/day.
Presence of proteins, blood and leucocytes were recorded in treated males as well as in control males that is why these findings were not associated with the application of the test substance.

Satellite males: statistically significant differences were not recorded in treated males. Similar findings were recorded in treated and control satellite males.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Only excreta coloured by the test substance were recorded

Males: the activity (poise, gait, reaction to handling) of all males of all treated groups was similar during the study and not different from the activity of males of the control group. Only excreta coloured by the test substance were recorded within the application period. 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: the activity (poise, gait, reaction to handling) of all males of treated group was similar during the study and not different from the activity of males of the control group. Only excreta coloured by the test substance were recorded within the application period. No significant differences were detected in examined parameters.

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 group. Only excreta coloured by the test substance were recorded within the application period. 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: 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 group. Only excreta coloured by the test substance were recorded within the application period. No significant differences were detected in examined parameters.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Statistically significant differences were not recorded.

Males: statistically significant differences were not recorded. Slight decrease of absolute weight of prostate gland+seminal vesicles was recorded in all treated males compared to control group of males. Weight of other organs was similar in treated and control males. Increase relative weight of adrenal gland was recorded in males at the dose level 1000 mg/kg/day. Increased relative weight of kidneys was found out in all treated males compared to the control males. Relative weights of other organs of treated males were similar to control males.

Satellite males: statistically significantly decreased absolute weight of epididymis and prostate gland+seminal vesicles were recorded in treated satellite males. Weight of other organs was similar in satellite treated and satellite control males. Slightly increased weight of kidneys was reported in treated males. Statistically significantly increased relative weight of testes was recorded in treated satellite males. Relative weights of other organs were similar in satellite treated and control males.

Females: statistically significant differences were not recorded. Slight decrease of weight of thymus in females at dose levels 500 and 1000 mg/kg/day and adrenal glands in females at the dose levels 250 and 1000 mg/kg/day was recorded. Increased weight of liver was found out in females at the dose level 1000 mg/kg/day.Weights of other organs were similar in treated and control females.
Slightly decreased relative weight of thymus and adrenal glands of females at the dose level 1000 mg/kg/day was reported. Increased relative weight of liver of all treated females was noted. Statistically significantly changed relative weight of spleen (increased) and ovaries (increased) was recorded in females at the dose level 250 mg/kg/day. Weights of other organs of treated females were quite well-balanced in treated and control animals.

Satellite females: statistically significant differences were not recorded. Absolute weights of organs were similar in satellite treated and control females. Relative weights of organs were similar in satellite treated and control females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Males
Control: no macroscopical findings were recorded in 6 males.
250 mg/kg/day: only findings related to the test substance administration – coloured content of the gastro-intestinal tract in 2 males were recorded.
500 mg/kg/day: reduced right seminal vesicle in one male and coloured content of the gastro-intestinal tract in all six males were recorded.
1000 mg/kg: coloured mucus on stomach mucosa in four males and coloured content of the gastro-intestinal tract in all six males were recorded.

Satellite males
Control satellite: no macroscopical findings were recorded.
1000 mg/kg/day satellite: no macroscopical findings were recorded.

Females
Control: no macroscopical findings were recorded in all six females.
250 mg/kg/day: no macroscopical findings were recorded in all six females.
500 mg/kg/day: no macroscopical findings were recorded in all six females.
1000 mg/kg: coloured content of the gastro-intestinal tract in 1 female only was recorded.

Satellite females
Control satellite: no macroscopical findings were recorded (dilatation of uterus in 3 female).
1000 mg/kg/day satellite: no macroscopical findings were recorded (dilatation of uterus in 3 female).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Full histopathology of the preserved organs and tissues was performed for high dose and control animals and satellite animals. No findings related to the test substance treatment were recorded, that is why only organs with macroscopical changes were examined at the other dose level groups.
Only one male at the dose level 500 mg/kg/day was examined – macroscopic finding: reduced right seminal vesicle. Histopathological examination did not record any finding.

Males: in 4-4 males no histological findings were diagnosed. Mild hydronephrosis was revealed in the kidneys of 1-1 males. Slight focal chronic inflammation of prostate gland was noted 0-1 male. Cyst on thymus was recorded in 1-0 male. All histopathological findings were incidental, with no relation to the substance treatment.

Satellite males: in 4-2 satellite male no histological findings were diagnosed. Slight focal chronic inflammation was recorded in epididymis of 1-0 male. Kidneys: hyaline casts in 1-0 male, hydronephrosis in 0-1 male, tubular basophilia in 0-1 male and venostasis in 1-0 male were recorded. Focal chronic inflammation of prostate gland was noted in 0-1 male. All histopathological findings were incidental, with no relation to the substance treatment.

Females: in 0-0 female no histological findings were diagnosed. Hydronephrosis of kidneys was recorded in 1-4 females. In uterus and vagina, the changes related to previous pregnancy were found in both controls and treated animals: accumulation of lipophages and siderophages in mesometrium in 6-5 females, hemosiderin in mucosa in 6-6 females and mild mucosal hypertrophy with mucification in 1-1females. Also lobular hyperplasia of mammary gland recorded in 6-6 females can be related to the previous pregnancy and lactation. Other microscopical changes observed in reproductive organs occurred only sporadically and they did not relate to test substance treatment.

Satellite females: in 3-3 females hydrometra of uterus (the finding related to the oestrous cycle) was recorded. Corticomedullary mineralization in kidneys was found out in 0-2 females. No treatment-related changes were found out in female genital tract. Other findings were observed either in control animals only, or in both groups of animals. They were of spontaneous character.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for repeated dose toxicity was established as 1000 mg/kg body weight/day.

Executive summary:

The substance was tested for reproduction and subacute toxicity using the OECD No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on 28th July 2015.

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day).

The treated groups were administered daily for the following periods: males and females 2 weeks prior to the mating period and during the mating period; pregnant females during pregnancy and till the 12th day of lactation; males after mating period (totally for 49 days); nonpregnant females (mated females without parturition) for 25 days after the confirmed mating; non-mated females for 25 days after the end of mating period. After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.   

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

The test substance treatment did not produce changes detected in health condition control, daily clinical observation after application (except coloured faeces) and functional observation of animals. The test substance did not interfere with normal growth of treated parental animals. Body weight and body weight increments of treated animals were not significantly affected by the test substance administration. 

Haematological examination of males showed effect of the test substance on coagulation parameters – APTT and PT values were statistically significantly increased and concentration of fibrinogen was statistically significantly decreased in males at the dose level 1000 mg/kg/day compared to the control animals. Values of PT and FIB were out of historical ranges. There was no dose dependence and changes were reversible (not observed in animals at the end of recovery period). Red blood components were not significantly influenced by administration of the test substance. Platelet number in males at the dose level 1000 mg/kg/day was statistically significantly increased (but in range of historical control).Also this change was without dose dependence and reversible. The value of reticulocytes was statistically significantly increased in all treated groups of males (at the dose levels 250 and 1000 mg/kg/day – out of historical range). This change was reversible, so it can be considered as adaptive response of organism to chemical stress. Parameters of white blood component were quite well-balanced in treated males compared to control males. Only differential percentage counts of granulocytes and lymphocytes were changed, but in historical control range and without dose dependency.

Haematological examination of females showed quite well-balanced red blood components in treated females compared to control females. The value of erythrocytes was slightly decreased in females at the highest dose level and with that relates decreased value of haemoglobin and haematocrite (statistically significantly decreased value of haematocrite; in historical control range). The value of reticulocytes was statistically significantly changed in females at the highest dose level (out of historical range). This change was irreversible, the value at the end of recovery period was still significantly increased, but there was an evident reduction in value into the range of historical control. Parameters of white blood component were quite well-balanced in treated females compared to control females. Coagulation parameters of treated animals were quite well-balanced with the control animals, only the value of fibrinogen at the highest dose level was statistically significantly decreased. This change was in range of historical control and reversible.

All findings observed in both sexes (but in absence of a treatment-related clinical signs of toxicity) were considered to be of no toxicological significance.

 

No significant changes in biochemical examination were observed in males at all treated groups. Statistically significantly changed parameters were recorded only in satellite treated males. Decreased values of ALT, creatinine, BUN and concentration of chloride and increased values of BA and concentration of Ca and IP were recorded. All changed values were still in the range of historical control values.

Statistically significant differences were found out in treated females – total bilirubin concentration was decreased at the dose levels 250 and 500 mg/kg/day (in historical control range), phosphorus concentration was decreased at the dose levels 500 and 1000 mg/kg/day (out of historical control range). All changed values were without dose dependency and reversible.

During biometry of organs, significant changes in absolute weights of organs related to test substance administration in treated males and females were not detected. Statistically significantly increased relative weight of spleen and ovaries was recorded in females at the dose level 250 mg/kg/day.

No macroscopical changes related to test substance administration were recorded.

Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs.

 

Conclusion

The NOAEL (No Observed Adverse Effect Level) for repeated dose toxicity was established as 1000 mg/kg body weight/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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 No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on 28^th July 2015.

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

The treated groups were administered daily for the following periods: males and females 2 weeks prior to the mating period and during the mating period; pregnant females during pregnancy and till the 12th day of lactation; males after mating period (totally for 49 days); nonpregnant females (mated females without parturition) for 25 days after the confirmed mating; non-mated females for 25 days after the end of mating period. After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.   

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

The test substance treatment did not produce changes detected in health condition control, daily clinical observation after application (except coloured faeces) and functional observation of animals. The test substance did not interfere with normal growth of treated parental animals. Body weight and body weight increments of treated animals were not significantly affected by the test substance administration. 

Haematological examination of males showed effect of the test substance on coagulation parameters – APTT and PT values were statistically significantly increased and concentration of fibrinogen was statistically significantly decreased in males at the dose level 1000 mg/kg/day compared to the control animals. Values of PT and FIB were out of historical ranges. There was no dose dependence and changes were reversible (not observed in animals at the end of recovery period). Red blood components were not significantly influenced by administration of the test substance. Platelet number in males at the dose level 1000 mg/kg/day was statistically significantly increased (but in range of historical control).Also this change was without dose dependence and reversible. The value of reticulocytes was statistically significantly increased in all treated groups of males (at the dose levels 250 and 1000 mg/kg/day – out of historical range). This change was reversible, so it can be considered as adaptive response of organism to chemical stress. Parameters of white blood component were quite well-balanced in treated males compared to control males. Only differential percentage counts of granulocytes and lymphocytes were changed, but in historical control range and without dose dependency.

Haematological examination of females showed quite well-balanced red blood components in treated females compared to control females. The value of erythrocytes was slightly decreased in females at the highest dose level and with that relates decreased value of haemoglobin and haematocrite (statistically significantly decreased value of haematocrite; in historical control range). The value of reticulocytes was statistically significantly changed in females at the highest dose level (out of historical range). This change was irreversible, the value at the end of recovery period was still significantly increased, but there was an evident reduction in value into the range of historical control. Parameters of white blood component were quite well-balanced in treated females compared to control females. Coagulation parameters of treated animals were quite well-balanced with the control animals, only the value of fibrinogen at the highest dose level was statistically significantly decreased. This change was in range of historical control and reversible.

All findings observed in both sexes (but in absence of a treatment-related clinical signs of toxicity) were considered to be of no toxicological significance.

 

No significant changes in biochemical examination were observed in males at all treated groups. Statistically significantly changed parameters were recorded only in satellite treated males. Decreased values of ALT, creatinine, BUN and concentration of chloride and increased values of BA and concentration of Ca and IP were recorded. All changed values were still in the range of historical control values.

Statistically significant differences were found out in treated females – total bilirubin concentration was decreased at the dose levels 250 and 500 mg/kg/day (in historical control range), phosphorus concentration was decreased at the dose levels 500 and 1000 mg/kg/day (out of historical control range). All changed values were without dose dependency and reversible.

During biometry of organs, significant changes in absolute weights of organs related to test substance administration in treated males and females were not detected. Statistically significantly increased relative weight of spleen and ovaries was recorded in females at the dose level 250 mg/kg/day.

No macroscopical changes related to test substance administration were recorded.

Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs.

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.

 

The No Observed Adverse Effect Level was established at 1000 mg/kg bw/day, on the basis of the results from the subacute study of 28 days, on rats.

 

In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for repeated dose toxicity according to the CLP Regulation (EC 1272/2008).