Registration Dossier

Administrative data

Description of key information

NOAEL (49d) (males and females): 125 mg/kg bw/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
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 30th, 2015 to July 29th, 2016
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 28th July 2015
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Laboratory rat has been chosen because our testing laboratory has long experience with this species.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500.
- Age at study initiation: males, females: sexually adult, 7-9 weeks on arrival.
- 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 (Altromin Spezialfutter GmbH & Co.).
- Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law.
- Acclimation period: at least 6 days. During the acclimatisation period the health condition of all animals was controlled daily. Normal course of the oestrus cycle of all females was controlled during 14 days before start of application. Females with abnormal oestrus cycle were removed from mating.

ENVIRONMENTAL CONDITIONS
- Temperature:22 ± 3 °C
- Relative humidity: 30-70 %
- Photoperiod: 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
APPLICATION FORM
- Stability and homogeneity: determined by means of measuring of a peak area of the test substance by a liquid chromatography based on a method developed at the test facility. Test item in vehicle at defined laboratory conditions resulted to be homogenous and stable at least for 120 minutes from the finalization of application form preparation.
- Preparation: the application form for analysis was prepared in the same manner as for application to animals – i.e.solution in water for injection. Concentration Level 10 mg/10ml: ca 0.1 g of the test substance was weighed into a 250 ml glass beaker calibrated to 100 ml and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for a 30 min. The solution was stirred by magnetic stirrer (750 rpm) for 20 minutes. Concentration Level 1000 mg/10 ml: ca10 g of the test substance was weighed into a 250 ml glass beaker calibrated to 100 ml and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for a 30 min. The solution was stirred by magnetic stirrer (750 rpm) for 40 minutes.
- Concentration: 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.
Duration of treatment / exposure:
Totally 49 days of administration.
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
two groups: one common group and one satellite group
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
Details on study design:
DOSE-RANGE FINDING EXPERIMENT
- Age at study initiation: 9 weeks on arrival.
- Number of animals: 5 males and 5 females per group.
- Acclimatization: at least 5 days
- Dose selection: according to results of Dose-Range Finding Experiment the following dose levels – 125, 500 and 1000 mg/kg/day were chosenfor the main test. The dose levels were approved by Sponsor.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS
- Data Collection: health condition control daily during the acclimatization and the experimental part.
- Data Collection: males and females daily during the administration period.
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 application, during application and immediately after application. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 – 13.00 p.m.). Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATIONS
- Data Collection: before the first application and then weekly (except the mating period).
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, 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
- Data Collection: twice daily.
All rats during the treatment periods were examined for vitality or mortality twice daily.

BODY WEIGHT
- 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 at 0, 7th, 14th, 20th day; during lactation at 1st, 4th day, 12th day and 13th day.
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too. Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION
- Data Collection: weekly and on the same days as body weight (except the mating period). Satellite males and females weekly.
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values were calculated for each week of the study (except 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. In pre-mating period the food consumption and conversion of females was calculated from values of all females.

WATER CONSUMPTION
- Data Collection: satellite males and females twice a week.
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
- Data Collection: at the end of administration/observation period.
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.

HAEMATOLOGY
- Data Collection: males and nonpregnant females after the end of application period; parental females on the 13th dy of lactation; satellite animals after the end of observation period.
- 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.
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation system. Reticulocytes count was examined according to the internal SOP No. M/2. Haematology analysers Celltac alfa and Coagulometer ACL were used for examination and the following parameters were determined.

CLINICAL CHEMISTRY
- 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.
- Parameters: protein total, alkaline phosphatise, cholesterol total, triglycerides, alanine aminotranferase, aspartate aminotransferase, creatinine, urea, albumin, bilirubin total, glucose, calcium, phosphorus, cholinesterase, bile acids, sodium, potassium, chloride.
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. Blood samples from the day 13 the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) by ELISA kit (Biovendor, Brno, Czech republic).
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
Total protein, total bilirubin, urea, creatinine, glucose, transaminases (AST, ALT), cholesterol, albumin, alkaline phosphatase (ALP), phosphorus and calcium, triglycerides, cholinesterase, bile acidswill be measured by automatic clinical chemistry analyser ACCENT-200 (Cormay, Poland); sodium, potassium and chloride ions will be measured by automatic electrolyte analyser with ion-selective electrodes SPOTCHEMTM EL SE-1520 (Arkray, Inc.).

URINALYSIS
- Data Collection: the last day of administration/observation period, only males.
- Parameter: volume, colour, cloud, odour, glucose, protein, bilirubin, urobilinogen, pH, specific gravity, blood, ketones, nitrite, leucocytes.
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 by 2 ml of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined by analyser PocketChem PU-4210 and 4010 (Arkray, Inc., Japan).
Sacrifice and pathology:
SACRIFICE
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla.

PATHOLOGY
Full pathology was performed only in 6 males + 6 females of each dose level and in satellite animals.
- Data collection: males and non-pregnant females after the end of application period. Parental females on the 13th day of lactation; satellite animals after the end of observation period.

ORGAN WEIGHTS
- Data collection: during necropsy.
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 oruterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded (6 males and females from each group + satellite groups). 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 thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

HISTOPATHOLOGICAL EXAMINATION
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.
- Data collection: after necropsy
- Organs for histopathological examination: 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, 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.
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 and kidneys, small and large intestines, stomach, forestomach and lymph nodes (mesenteric and/or paraaortal and/or submadibular) were examined at the lowest and middle dose level groups.
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):
The males and females were not distributed into the groups during the check-in, acclimatisation and the pre-exposure period due to oestrous cycle monitoring in females. No signs of diseases were recorded during that time.

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 were recorded.

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 were recorded.
During the observation (recovery) 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 application period. Only changes related to the colour of the test substance – coloured faeces were recorded.

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 were recorded. During the observation (recovery) period no changes of health status were noted in satellite treated females.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the main study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Different body weight before application was caused by sequential putting animals on the study. Body weight of treated males at the dose level 125 mg/kg/day was slightly higher in comparison with the other males within the whole study. Body weight of middle and high dose groups was comparable with control group during 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. Only during the 6th week the increment in males at the 1000 mg/kg dose level was slightly lower.

Satellite males
Body weight of satellite treated males was similar 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 similar with satellite control males and were not adversely influenced by the test substance administration.

Females
Different body weight before application was caused by sequential putting animals to the study. Body weight of all groups of treated females was quite balanced in comparison with the control group of females during the pre-mating period. At the end of pregnancy period slightly decreased weight of treated females at the dose level 1000 mg/kg was recorded.
Body weight of treated females of all groups was quite balanced in comparison with the control group of females during the lactation period.
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.

Satellite females
Body weight of satellite treated females was comparable with satellite 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 satellite control group and not adversely influenced by the test substance administration.
Low or negative body weight increment was recorded in control as well as in treated animals.
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 or slightly increased in comparison with control animals.

Satellite males
The food consumption of satellite treated males was slightly increased compared to the control group in application and 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 more variable but still similar compared to control animals.

Satellite females
The food consumption of satellite treated females was similar in comparison with the control group for the whole time of application and recovery period.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The food conversion of treated males compared to control animals was variable during the pre-mating period. Different food conversion was recorded also 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 more similar but still variable in comparison with control animals and not influenced by the test substance application.

Females
The food conversion of treated females in the pre-mating period was variable compared to control group.
In pregnancy period, the food conversion of treated females was more similar with control group and was not adversely influenced by the test substance treatment.

Satellite females
The food conversion of satellite treated females was variable in comparison with the control group and not influenced by test substance treatment.
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 slightly decreased compared to satellite control group during the whole study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Red blood parameters (RBC, MCV, Hct, Hgb) of treated males were not adversely influenced by a dministration of the test substance.
Parameters of white blood component were quite well-balanced in treated males compared to control males. Only differential percentage counts of granulocytes and monocytes were changed, but without dose dependency.
Haemocoagulation parameters (APTT, PT) were influenced by administration of the test substance –statistically significantly increased value of PT was recorded at the dose level 500 and 1000 mg/kg/day.
APTT was dose-dependently increased without statistical significance. The concentration of fibrinogen (FIB) was quite well-balanced in males of all treated groups in comparison with the control males. The platelet count was without any changes in treated males in comparison with control males.
The value of reticulocytes was well-balanced and not statistically significantly changed in treated groups of males.
All findings observed in males were without of a treatment-related clinical signs of toxicity and were in range of historical control, so it could be considered there was no toxicological significance.

Satellite males
Only the significant increase of differential percentage counts of granulocytes was found out in satellite treated males. Count of lymphocytes was decreased without statistical significance. Other parameters of red and white component were not adversely affected by the test substance administration.
Haemocoagulation parameters (APTT, PT) were still slightly increased but without statistical significance.
The haematological parameters except granulocytes at the highest dose level were in range of historical control.

Females
Some changes of parameters of red blood component were recorded. Total erythrocyte count (RBC) was statistically significantly decreased at the dose level 500 and 1000 mg/kg/day and Mean corpuscular volume (MCV) was statistically significantly increased at the dose level 1000 mg/kg/day. Both of these values were changed dose-dependently and together with haematocrit value were at the dose level 500 mg/kg/day out of range of historical control. Values of haemoglobin and haematocrite were also slightly dose-dependently lower in comparison with control females.
Parameters of white blood component were quite well-balanced in treated females compared to control females. Only differential percentage counts of monocytes were changed, but without dose dependency.
Haemocoagulation parameters (APTT, PT) were not influenced by administration of the test substance – statistically significant changes were not recorded. Only value of APTT was dose-dependently increased without statistical significance. The concentration of fibrinogen (FIB) was quite well-balanced in females of all treated groups in comparison with the control females. The platelet count was without any changes in treated females in comparison with control females.
The value of reticulocytes was not statistically significantly changed in females and was quite well-balanced in comparison with control females.
All findings observed in females except red blood parameters were without of a treatment-related clinical signs of toxicity and were in range of historical control.

Satellite females
Statistically significantly increased value of MCV was recorded in satellite treated females. This increase was irreversible. Other parameters of red blood components were similar with satellite control females.
Parameters of white blood components were without statistically significant changes, but increased value of granulocytes was recorded.
Haemocoagulation parameters (APTT, PT) were not influenced by administration of the test substance – statistically significant changes were not recorded. The concentration of fibrinogen (FIB) was statistically significantly decreased in treated females.
Other parameters were similar in satellite control and treated females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Statistically significantly decreased concentration of total bilirubin (without dose dependence) was detected in treated males at the dose levels 500 and 1000 mg/kg/day and decreased activity of ALP was recorded in males at the dose level 1000 mg/kg/day.
Value of triglycerides (TG) was dose-dependently decreased but without statistical significance. Values of bile acids (BA) were also changed mostly at the dose level 500 and 1000 mg/kg/day but without statistical significance and dose dependence.
Values of other biochemical parameters of treated males were quite well-balanced to the control group.
Blood samples from the all adult parental males were assessed for serum levels for thyroid hormone thyroxine (T4 total).
Mean concentration of T4 hormone at the dose level 125 mg/kg/day was insignificantly increased in comparison with other test groups. Concentration of others dosed groups were similar to the control group.

Satellite males
Statistically significant decrease of values of glucose, and concentration of chloride was recorded in satellite treated males. Values of bile acids (BA) were also changed but without statistical significance.
Values of other biochemical parameters of satellite treated males were similar to the satellite control group.

Females
Statistically significant difference was found out in treated females just in value of glucose at the dose level 500 mg/kg/day. Value of total bilirubin was insignificantly decreased at all treated animals but without dose dependence.
Values of other biochemical parameters of treated females were more or less well-balanced to the control group.

Satellite females
Statistically significant difference was found out in treated satellite females just in activity of ALT which was statistically significantly decreased. Value of bile acids was increased in comparison with satellite control females but without significance.
Differences in values of other biochemical parameters between satellite control and satellite treated females were not significant.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis was performed only in males (six of each group) during the last week of the study. Statistical evaluation was performed for pH and volume of urine.

Males
Statistically significant differences were recorded in volume of urine in males at the dose level 1000 mg/kg/day. The volume was decreased dose-dependently. Presence of proteins 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):
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.
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 application and observation period and not different from the activity of males of the control group. 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.
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 between control and treated females.

Satellite females
The activity (poise, gait, reaction to handling) of all females of treated group was similar during the application and observation period and not different from the activity of females of the control group.
No significant differences were detected in examined parameters.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Statistically significant difference was recorded in lower weight of brain in all treated males in comparison with control males (without dose dependence).
Weight of other organs was similar in treated and control males.
Relative weights of all organs of treated males were quite similar to control males. Just very slight increase of weight of spleen and liver was recorded.
No statistical significance was recorded.

Satellite males
Weight of all organs was similar in satellite treated and satellite control males. No significant changes were found out.
Relative weights of all organs were similar in satellite treated and control males. No statistical significance was recorded.

Females
Statistically significant differences were not recorded. Slight decrease of weight of brain and slight increase weight of spleen was recorded in treated females but without statistical significance. Absolute weight of liver in females at the dose level 500 and 1000 mg/kg/day was also increased but without statistical significance and dose dependence.
Weight of other organs was similar in treated and control females.
Increased weight of pituitary gland was recorded in females at the dose level 500 mg/kg/day. This increase was statistically significant but not dependent on dose level. Then dose-dependent increase of spleen and liver was recorded but without statistical significance.
Relative weights of other organs of treated females were relatively similar to control females.

Satellite females
Statistically significant differences were not recorded.
Absolute weights of organs were similar in satellite treated and control females. Just weight of heart and uterus was lower in treated females.
Relative weight of heart of treated females was statistically significantly decreased. Relative weight of uterus was also decreased but without statistical significance. Relative weights of other organs were similar in satellite treated and control females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Control: no macroscopical findings were recorded in 6 males.
125 mg/kg/day: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract and coloured kidneys in all males were recorded.
500 mg/kg/day: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract and coloured kidneys in all males were recorded. Colouration of submandibular lymph nodes was recorded in one male.
1000 mg/kg: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract and coloured kidneys in all males were recorded. Colouration of mesenteric lymph nodes was recorded in all six males and also colouration of paraaortal and submandibular lymph nodes was observed in two males. Urine in urinary bladder coloured by the test substance was recorded in three males.

Satellite males
Control satellite: no macroscopical findings were recorded.
1000 mg/kg/day satellite: only findings related to the test substance administration – coloured intestinal and stomach wall, coloured intestinal lymphoid tissue and coloured kidneys in all males were recorded.
Colouration of paraaortal lymph nodes was recorded in all six males.

Females
Control: no macroscopical findings were recorded in all six females.
125 mg/kg/day: findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract and coloured kidneys in all females were recorded. Colouration of submandibular lymph nodes was recorded in three females. Haemorhage in right uterine horn was observed in one female.
500 mg/kg/day: mostly findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract and coloured kidneys in all females were recorded.
Colouration of submandibular lymph nodes was recorded in five females and colouration of liver was also observed in one female.
Congestion of mucous membrane of stomach was observed in one female.
1000 mg/kg: only findings related to the test substance administration – coloured content and mucous membrane of the gastro-intestinal tract and coloured kidneys in all females were recorded. Colouration of liver was observed in two females and colouration of uterus in three females.

Satellite females
Control satellite: no macroscopical findings were recorded (except dilatation of uterus in one female).
1000 mg/kg/day satellite: only findings related to the test substance administration – coloured intestinal and stomach wall, coloured intestinal lymphoid tissue, coloured kidneys and paraaortal lymph nodes in all females were recorded. Colouration of submandibular lymph nodes was observed in five females.
Green colour of urine in urinary bladder was also recorded in one female.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Full histopathology of the preserved organs and tissues was performed mainly for high dose and control animals and satellite animals. Because of macroscopically findings related with administration of the test substance in animals at the lower and middle dose level were recorded, the histopathological examination of selected macroscopically changed organs and then of kidneys, small and large intestine, stomach, forestomach and lymph nodes was performed.
The test substance orally administered to rats caused pathological changes connected with colouration of test substance practically in all treated animals mostly at the middle and the highest dose level.
The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-125-500-1000 mg/kg/day and 0S-1000S in satellite groups further in the text.
For the finding “blue pigment in epithelium of cortical tubules” the following grading was used: very sporadic – sporadic – mild – medium intensity.

Males
In 2-0-0-0 males no histological findings were diagnosed.
The test substance orally administered to rats caused pathological changes connected with colouration of test substance practically in all treated males mostly at the middle and the highest dose level. Blue pigment in epithelium of cortical tubules in kidneys (very sporadic intensity 0-0-4-0, sporadic 0-0-0-5, mild 0-0-0-1), macrophages containing blue pigment in mesenteric lymph nodes (0-2-6-6), in submandibular lymph nodes (0-0-0-1), blue pigment in enterocytes of large intestines (0-6-6-3) and lamina propria of small intestinal villi (0-2-6-6) and blue pigment in enterocytes and lymphoid tissue of caecum (0-0-0-1) was detected.
Presence of blue pigment was not accompanied by inflammatory or degenerative changes. The function of organs was not affected by presence of pigment.
Then only sporadic findings which were not connected with test substance treatment were found out.
These organs were examined only in control and the highest dose groups. The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-1000 mg/kg/day further in the text.
In reproductive organs these findings were observed: tubular degeneration or atrophy of testes (mostly in sporadic tubules) in 2-1 males, acinar atrophy of prostate gland in 1-0 males and focal interstitial oedema of prostate gland in 0-2 males, desquamation of epithelium of seminal vesicles in 0-1 male.
In other organs these sporadic findings were detected: cysts on thymus (1-0), basophilic tubules in kidneys (0-1), chronic inflammation of valve in heart (0-1), focal mononuclear infiltration (1-1) and focal extramedullary haemopoiesis (0-1) in liver and basophilic corpuscle in salivary gland (0-1).

Satellite males
In 0-0 satellite male no histological findings were diagnosed.
Findings connected with colouration of test substance were observed in treated satellite animals. Blue pigment in epithelium of cortical tubules in kidneys (sporadic intensity 0-4, mild intensity 0-2), macrophages containing blue pigment in mesenteric lymph nodes (0-6), presence of macrophages containing blue pigment in lymphoid tissue in caecum (0-1), blue pigment in lamina propria of intestinal villi in small intestines (0-6) and in enterocytes of large intestines (0-3) was detected.
Presence of blue pigment was not accompanied by inflammatory or degenerative changes. Blue pigment in renal epithelium was observed in a slightly increased intensity in satellite males. There was an irreversible effect of the test substance treatment caused by its accumulation.
Then only sporadic findings which were not connected with test substance treatment were found out. In reproductive organs these findings were observed: tubular degeneration or atrophy of testes (mostly in sporadic tubules) in 4-2 males, germ cells in epidydimidal tubules in 3-0 males and azoospermia of epidydimis in one control male.
In other organs these sporadic findings were detected: brown pigment in epithelium of cortical tubules in kidneys (1-0), accessory cortical nodule of adrenal glands (1-0), focal mononuclear infiltration of liver (4-3) and focal vacuolation of cells of exocrine part of pancreas (0-1).
Blue pigment in epithelium of cortical tubules in kidneys was observed in the higher intensity in satellite animals. There was a delayed effect of the test substance treatment caused by its accumulation.

Females
In 0-5-0-0 female no histological findings were diagnosed.
The test substance orally administered to rats caused pathological changes connected with colouration of test substance practically in all treated females mostly at the middle and the highest dose level. Blue pigment in epithelium of cortical tubules in kidneys (very sporadic intensity 0-0-2-0, sporadic 0-0-4-2, mild 0-0-0-4), macrophages containing blue pigment in mesenteric lymph nodes (0-0-4-5), blue pigment in enterocytes of large intestines (0-0-1-3) and lamina propria of small intestinal villi (0-0-3-4) and blue pigment in enterocytes of caecum (0-0-0-1) was detected.
Presence of blue pigment was not accompanied by inflammatory or degenerative changes.
Then only sporadic findings which were not connected with test substance treatment were found out. These organs were examined only in control and the highest dose groups. The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-1000 mg/kg/day further in the text.
In reproductive organs findings related with previous pregnancy and delivery of pups were observed: accumulation of lipophages and/or siderophages in mesometrium and/or endometrium of uterus (6-4) and hemosiderin in endometrium of uterus (0-4).
In other organs these sporadically findings were detected: chronic focal inflammation of pancreas (0-1), focal haemorrhages of thymus (1-0), erythrocytosis of sinus of mesenteric lymph nodes (1-0), plasmacytosis of paraaortal lymph nodes (0-1) and focal mononuclear infiltration of liver (1-0).

Satellite females
In 2-0 satellite male no histological findings were diagnosed.
Findings connected with colouration of test substance were observed in treated satellite animals. Blue pigment in epithelium of cortical tubules in kidneys (mild intensity 0-3, medium 0-3), macrophages containing blue pigment in mesenteric (0-6) and in submandibular (0-1) lymph nodes, blue pigment in lamina propria of intestinal villi in small intestines (0-6) and in enterocytes of large intestines (0-1) was detected.
Presence of blue pigment was not accompanied by inflammatory or degenerative changes. Blue pigment in renal epithelium was observed in the higher intensity in satellite females. There was an delayed effect of the test substance treatment caused by its accumulation.
Then only sporadic findings which were not connected with test substance treatment were found out. In reproductive organs only hydrometra (2-1) were observed.
In other organs these sporadic findings were detected: metastatic calcification (1-0), pyelonephritis (1-0) and vacuolation of tubular epithelium of kidneys (0-1), cysts on thymus (2-2), pigmentation of spleen (0-1), focal mononuclear infiltration of liver (1-2) and focal histiocytosis of lungs (1-0).
Blue pigment in epithelium of cortical tubules in kidneys was observed in the higher intensity in satellite animals. There was a delayed effect of the test substance treatment caused by its accumulation. But due to water solubility it is possible that pigment deposits will gradually disappear from kidneys.
Details on results:
The NOAEL (No Observed Adverse Effect Level) was established as 125 mg/kg body weight/day; the frequency of pigment presence was negligible at this dose level and it was found only in organs directly related to the route of the test substance administration (digestive system and in regional lymph nodes). Accumulation of pigment was found in epithelium of renal cortical tubules and lymph nodes of males and females at the dose levels 500 and 1000 mg/kg/day; at the higher dose, the accumulation resulted to be irreversible.
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: accumulation of pigment
Critical effects observed:
no
Conclusions:
NOAEL (49d) (males and females): 125 mg/kg bw/day
Executive summary:

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

The test substance was administered to rats 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 125, 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).

Repeated oral administration of the test substance to rats by gavage at the dose levels of 125, 500 and 1000 mg/kg/day did not cause any mortality. The test substance did not interfere with normal growth of treated animals. Body weight and body weight increment, food consumption and conversion and water consumption of treated animals at all dose levels were not affected by the test substance administration.

The test substance treatment did not produce changes detected in functional observation of animals. Changes connected with colour of test substance (colouration of faeces) were found out during health condition control and clinical observations in animals at all dose levels.

Urinalysis of treated males did not manifest negative effect of the test substance treatment at all dose levels.

Haematological and biochemical examinations were without any statistically significant changes at the dose level 125 mg/kg/day.

Statistically significantly decreased absolute weight of brain was recorded in males of this dose level nevertheless the relative weight of this organ was similar to the control group of animals and without statistical significance. During pathological examination changes connected with colour of the test substance were found out. Coloured content and mucous membrane of the gastro-intestinal tract and changed colour of kidneys were recorded in all males and females at the dose level 125 mg/kg/day. Effect of the test substance treatment was observed during histopathological examination just in males at the dose level 125 mg/kg/day: presence of blue pigment in enterocytes of large intestines, in lamina propria of villi of small intestines and in mesenteric lymph nodes. The frequency of pigment presence was negligible at this dose level and it was found only in organs directly related to the route of the test substance administration (digestive system and in regional lymph nodes).

The following statistically significant changes of haematological parameters were recorded dose level 500 mg/kg/day: increased value of prothrombin time in males and dose dependent decrease of total erythrocyte count in females (the value of RBC was on the lower limit of historical control).

Only few significant changes were recorded during biochemistry examination of animals at the dose level 500 mg/kg/day (decreased concentration of total bilirubin in males and increased value of glucose in females). These changes are probably not connected with the test substance treatment because dose-independent.

Biometry of organs manifested significant decrease of absolute weight of brain in males at this dose level nevertheless the relative weight of this organ was similar to the control group. Statistically significant increase of relative weight of pituitary gland (without dose dependence) was observed in females.

During pathological examination changes connected with colour of the test substance were found out. Coloured content and mucous membrane of the gastro-intestinal tract, changed colour of kidneys and changed colour of submandibular lymph nodes were recorded in males and females at the dose level 500 mg/kg/day.

Effect connected with the colour of the test substance was observed also during histopathological examination of males and females at this dose level: presence of blue pigment in enterocytes of large intestines, in lamina propria of villi of small intestines, in macrophages of mesenteric lymph nodes and in epithelium of renal cortical tubules (very sporadic or sporadic intensity). Presence of blue pigment was not accompanied by inflammatory or degenerative changes. Dysfunction of intestines, kidneys or lymph nodes caused by the presence of pigment was not demonstrated. The number of affected animals as well as intensity of above mentioned microscopical changes were lower than at the dose level 1000 mg/kg/day.

Examinations of animals at the dose level 1000 mg/kg/day revealed the following changes:

Statistically significantly changed haematological parameters were recorded in both sexes: increased value of prothrombin time in males, increased percentage of granulocytes in satellite males, decreased value of total erythrocyte count and simultaneously increased value of mean corpuscular volume in females. Increase of mean corpuscular volume persisted in recovery period in females and decrease of fibrinogen was also observed in satellite females. All changed values were in range of historical control.

Significant changes probably not connected with treatment of test substance were recorded during biochemistry examination of 1000 mg/kg/day animals: decreased concentration of total bilirubin and activity of ALP in males, decreased values of glucose and chloride ions concentration satellite males and just decreased activity of ALT in satellite females. All changed values were in range of historical control.

Statistically significantly decreased absolute weight of brain of males was recorded at the dose level 1000 mg/kg/day nevertheless the relative weight of this organ was similar to the control group of animals and without statistical significance. No significant changes were recorded in satellite males. Statistically significantly decreased relative weight of heart was recorded in satellite females (probably not connected with the test substance treatment).

During the pathological examination changes connected with colouration of test substance were found out. Coloured content and mucous membrane of the gastro-intestinal tract and changed colour of kidneys were recorded in males and females at the dose level 1000 mg/kg/day at the end of administration and also at the end of recovery period. Lymph nodes (mesenteric, paraaortal and submandibular) were also coloured in some males and females even in satellite animals. Change of colour of liver was observed in few females.

Effect connected with the colour of the test substance was observed also during histopathological examination of animals at the dose level 1000 mg/kg/day. Presence of blue pigment in enterocytes or lymphoid tissue of large intestines, in lamina propria of villi of small intestines was observed in both males and females (incl. satellite animals). Blue pigment was then recorded in macrophages of mesenteric lymph nodes, in submandibular lymph nodes of males or females (incl. satellite animals) and in epithelium of cortical tubules of kidneys (sporadic or mild intensity) in all examined males and females. Presence of blue pigment was not accompanied by inflammatory or degenerative changes. The function of organs was not affected by the presence of pigment. Presence of blue pigment in renal epithelium was observed also at the end of recovery period even in higher intensity (mild or medium intensity) compared to the end of administration period. There was an irreversible effect of the test substance treatment caused by accumulation of the test substance.

Conclusions

The NOAEL (No Observed Adverse Effect Level) was established as 125 mg/kg body weight/day; the frequency of pigment presence was negligible at this dose level and it was found only in organs directly related to the route of the test substance administration (digestive system and in regional lymph nodes). Accumulation of pigment was found in epithelium of renal cortical tubules and lymph nodes of males and females at the dose levels 500 and 1000 mg/kg/day; at the higher dose, the accumulation resulted to be irreversible..

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 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

Direct Blue 199 was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422. The test substance was administered to rats in the form of solution in water for injection. The study includes four main groups and two satellite groups of animals: 3 treated groups, dosed at 125, 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 at 1000 mg/kg/day.

 

Repeated oral administration of the test substance did not cause any mortality. The test substance did not interfere with normal growth of treated animals. The test substance treatment did not produce changes detected in functional observation of animals. Changes connected with colour of test substance (colouration of faeces) were found out during health condition control and clinical observations in animals at all dose levels.

The test substance treatment did not produce changes detected in health condition control, daily clinical observation after application and functional observation of animals. The substance did not interfere with normal growth of treated parental animals.

Haematological examination showed an effect on haemostatic parameters in males and changes of red blood parameters in females which partly persisted even in recovery period. Biochemical examination did not show significant effect of the test substance treatment. In males effect of the test substance on haemocoagulation parameters was recorded: the value of PT was statistically significantly increased at the middle and the highest dose level and APTT was increased with dose dependency but without statistical significance. These changes were reversible. Red and white blood components were not significantly influenced by administration of the test substance in males. In satellite males only significant increase of granulocytes was recorded. The difference of red blood parameters caused by test substance treatment was detected in treated females. Total erythrocyte count (RBC) was statistically significantly and reversibly decreased at the middle and highest dose level and Mean corpuscular volume (MCV) was statistically significantly and irreversibly increased only at the highest dose level. Both of these values were changed dose-dependently. Values of white blood components were variable but without significant and dose-dependent changes. In satellite females significant increase of MCV persisted and also significant decrease of fibrinogen was recorded. Most of changed values of haematological parameters were in range of historical control. Only value of RBC at the middle dose level females was on the lower limit of historical control).

Some significant changes in clinical biochemistry parameters were observed in treated groups of males but always without dose-dependency. Statistically significantly decreased value of bilirubin was recorded in males at the middle and highest dose levels and decreased activity of ALP at the highest dose level. Both changes were reversible and without dose-dependency. Value of triglycerides was dose-dependently decreased and values of bile acids were lower at the middle and the highest dose level. Both parameters were changed without statistical significance. Statistically significant decrease of values of glucose, and concentration of chloride was recorded in satellite treated males. Biochemistry parameters in females were without toxicologically significant changes. Statistically significant difference was found out in treated females just in value of glucose at the middle dose level. Parameters of satellite females were also well-balanced. Only statistically significantly decreased activity of ALT was recorded.

Changes of biochemistry parameters in both males and females could be considered as no toxicological importance. All values were in range of historical control. Simultaneously no changes were observed in urinalysis parameters.

Biometry of organs showed statistically significantly decreased absolute weight of brain in all groups of treated males. Nevertheless the relative weight of this organ was similar to the control group of animals. Weight of other organs was similar or just slightly changed in treated and control males. No significant changes were found out also in satellite males.

No statistically significantly changed absolute weights of organs were recorded in treated females. The relative weight of pituitary gland at the middle dose level was statistically significantly increased. Dose-dependent but insignificant increase of relative weight of spleen and liver can be considered as adaptive response to chemical stress. At the end of recovery period, this weight was decreased or similar in comparison with the control animals, so the change was reversible. Relative weight of heart was statistically significantly decreased in treated satellite females. Weight of other organs was without any changes.

Gross necropsy in all dosed animals demonstrated effect of the test substance treatment on organs and tissues due to colouration of the test substance. Colouration of mucosa membrane of digestive system, kidneys and lymph nodes (mainly mesenteric) was observed practically in all animals (males and females) across all dose levels.

Microscopical evaluation revealed that the test substance orally administered to rats caused pathological changes connected with colouration of the test substance practically in all treated animals mostly at the middle and the highest dose level. Blue pigment in epithelium of cortical tubules in kidneys, macrophages containing blue pigment in mesenteric or submandibular lymph nodes, blue pigment in enterocytes or lymphoid tissue of large intestines and lamina propria of small intestinal villi was detected. Presence of blue pigment in above mentioned organs was not accompanied by inflammatory or degenerative changes. The function of above mentioned organs was not affected by presence of pigment.

Then only sporadic findings which were not connected with test substance treatment were found out.

Blue pigment in epithelium of cortical tubules in kidneys was observed in the higher intensity in satellite animals. This was an irreversible effect of the test substance treatment caused by its accumulation. But due to water solubility it is possible that pigment will gradually disappear from kidneys.

 

The NOAEL (No Observed Adverse Effect Level) was established as 125 mg/kg body weight/day; the frequency of pigment presence was negligible at this dose level and it was found only in organs directly related to the route of the test substance administration (digestive system and in regional lymph nodes).

Accumulation of pigment was found in epithelium of renal cortical tubules and lymph nodes of males and females at the dose levels 500 and 1000 mg/kg/day; at the higher dose, the accumulation resulted to be irreversible.

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, thus the limit for sub-acute studies should be 300 mg/kg bw/day.

 

In the specific case, the No Observed Adverse Effect Level was established at 125 mg/kg bw/day, on the basis of the results from the subacute study on rats. It should be taken into account that 125 mg/kg bw/day was indicated as NOAEL only because of the lacking of recovery information at 500 mg/kg bw/day and not on the basis of the effects observed at this concentration. Actually, none of the concentrations tested showed really “Adverse Effects”. The coloration was the main effect observed that was not accompanied by inflammatory, neither degenerative reactions.

 

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