Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1.3.-2016-1.3.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The study was performed according to the updated OECD guideline No.422:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on the 29th July 2016 instead of version given in Study Plan.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

EC Number:

219-708-8

EC Name:

Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

Cas Number:

2503-73-3

Molecular formula:

C42H29N7O13S4.4Na

IUPAC Name:

tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Direct Blue 78- Physical state: solid, powder- Analytical purity: 95% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Lot/batch No.: 7013/207- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

Test animals

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., Czech Republic, RČH CZ 11760500- Age at study initiation: sexually adult (9 weeks on arrival)- Fasting period before study: no- Housing: All the study proceeded in SPF (Specified Pathogen Free) animal house of CETA in SPFconditions (according to internal SOP No. 12).- Animal per cage: 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, satelliteanimals - 2 rats of the same sex in one cage- Diet: ad libitum, maintenance pelleted diet for rats and mice - Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany- Water: ad libitum- Acclimation period: 6 days, During the acclimatisation period the health condition of all animals wascontrolled daily.Then the animals were randomly divided into the control and test groups and they weremarked individually. - Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight - Identification of animals: the animals have been identified by the colour marks on their fur, each cage was marked with the number of animals, sex, number of cage, name and dose level of the test substanceENVIRONMENTAL CONDITIONSTemperature (°C): 22±3°C- Humidity (%): 30-70%- Air changes (per hr): 15 air- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycleSTUDY TIME SCHEDULETest substance delivery:09. 12. 2015Determination of stability and homogeneity: 01. 03. – 04. 03. 2016Dose-range finding experiment: 13. 04. – 10. 05. 2016Main Study:Animal arrival: 17. 08. 2016Acclimatisation: 6 daysPre-exposure period: 23. 08. – 05. 09. 2016Administration: from 06. 09. 2016Administration Parental males (totally 49 days of administration):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 43rd day – 63rd day (administration period) → 64th day (necropsy) Satellite males (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)Parental females:1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration)→ gestation → lactation → day 12 post partum Satellite females (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25 days after the end of mating period Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25th day after confirmed mating Urine collection: only males – 63rd and 77th day of studyBlood collection for haematology and biochemistry: parental males – 64th day of study satellite males – 78th day of studyparental females – 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation2 pups per litter – 13th day of lactationsatellite females – 78th day of studyNecropsy: parental males – 64th day of study satellite males – 78th day of studyparental females – 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation, other pups - 13th day of lactationsatellite females – 78th day of studynon-pregnant females – 26th day after the end of mating period or confirmed matingHistopathological examination: 02. 01. – 01. 03. 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:The application form for analysis was prepared in the same manner as for application to animals – i.e. solution in water for injection.Concentration Level 10 mg/10mLCa 0.1 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer (500 rpm) for 10 minutes. Concentration Level 1000 mg/10 mLCa 10 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150 mL glass beaker calibrated to 100 mL, mixed using glass rod during of vehicle adding and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (600 rpm) for 15 minutes, let to stand for min. 17 hours without mixing and then stirred by magnetic stirrer (900 rpm) for 45 minutes again. VEHICLE -Aqua pro iniectione (water for injection) Producer: Ardeapharma Ševětín a.s., Czech Republic- Batch No. and expiration: 1508310535, exp. 08/2017 (DRFE) 1511090667, exp. 11/2017 (DRFE) 1603110144, exp. 03/2018 1605100264, exp. 05/2018 1606130339, exp. 06/2018

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and the homogeneity of application form were determined in CETA 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.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Basic groups: 1. Control012 males + 12 females2. Low dose250 mg/kg/day12 males + 12 females3. Intermediate dose500 mg/kg/day 12 males + 12 females4. High dose1000 mg/kg/day12 males + 12 femalesSatellite groups:5. Control – vehicle – satellite:06 males + 6 females 6. High dose – satellite:1000 mg/kg/day6 males + 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

- The Dose-Range Finding Experiment was performed as it is described in Annex 2 of this final report. 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. The dose levels were approved by Sponsor. - Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

MORTALITY CONTROL- Time schedule: twice dailyHEALTH CONDITION CONTROL- Time schedule: daily - during the acclimatization and the experimental partCAGE SIDE OBSERVATIONS: Yes- Time schedule: males and females - daily during the administration periodThis observation was made in order to record possible clinical effects after application and all changesin behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cagesDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: males and females 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 ofskin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.BODY WEIGHT: Yes-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 daypups (litters) - 1st , 4th day, 12th day and 13th daypups – individually – 4th day of lactationFOOD CONSUMPTION- Time schedule: parental males - weekly (except the mating period)parental females - weekly during premating periodduring pregnancy and lactation – on the same days as body weightsatellite males and females – weeklyIn a specified day the remainder of pellets was weighed in each cage, the new food was weighed outand the food consumption for the previous week was computed.In males mean values were calculated for each week of the study (except of mating period in parentalmales). Food consumption for animal/day was calculated from mean values of each group.The same way of calculation of mean food consumption was used for females in premating period and for satellite females. In pregnancy and lactation period mean individual values (grams/animal/day)were calculated for each week of the study. Mean food consumption of parental females for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption of pregnant females.FOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: YesWATER CONSUMPTION: Yes- Time schedule for examinations: : satellite males and females – twice or three times a week (the 1stand 2nd week – twice a week and from 3rd week to the end of study a week)- The mean values in groups (water consumption per animal and per day) were calculated for eachweek of the study.OPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: at the end of administration/recovery period- Anaesthetic used for blood collection: Yes, light ether narcosis- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females.- Parameters checked in table [No.3] were examined.BIOCHEMICAL EXAMINATION: Yes- Time schedule for collection of blood: at the end of administration/recovery period- Anaesthetic used for blood collection: Yes, light ether narcosis- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females. Theanimals starved approximately for 18 hours before blood collection but they were supplied by drinkingwater ad libitum.- Parameters checked in table [No.4] were examined.URINALYSIS: Yes- Time schedule for collection of urine: last day of administration/recovery period – only males- Metabolism cages used for collection of urine: Yes, for 2 hours- Immediately before entering metabolic cages the animals were administered 2 mL of drinking waterfor 100 g of body weight by gavage to the stomach.- Animals fasted: No- How many animals: 6 males of each group and in satellite males- Parameters checked in table [No.2] were examined.Sacrifice and pathologyDuring 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 withfixative (buffered 4% formaldehyde).Testes and epididymides were fixed in modified Davidson’s fixative.At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spineand medulla.After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing andfurther histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary glandand heart were recorded (repeated dose toxicity part of study – 6 males and females from each group+ satellite groupsHISTOPATHOLOGY: Yes (see table No. 5)StatisticsFor 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 parametric tests were used for statistical evaluation of• body weight• mean pups weight• litter weight• selected haematology parameters• blood biochemistry parameters• data from urinalysis• data from biometry of organs

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males No clinical changes were recorded in control and treated males during the application period. Only changes related to the colour of the test substance – coloured faeces were recorded (at the dose level 500 mg/kg/day: from the 8th day of study, at the dose level 1000 mg/kg/day: from the 3rd day of study).The behaviour, clinical status and activity of all males of all treated groups were similar during the study and not different from males of the control group. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of treated males. Satellite malesNo clinical changes were recorded in control and treated males in application period. Only changes related to the colour of the test substance – coloured faeces were recorded (from the 3rd day of study).The behaviour, clinical status and activity of satellite treated males were similar during the application and observation period of the study and not different from satellite control males. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of satellite treated males. Females At all control and treated females, no clinical changes were recorded during the whole study. Only changes related to the colour of the test substance – coloured faeces were recorded (at the dose level 500 mg/kg/day: from the 8th day of study, at the dose level 1000 mg/kg/day: from the 3rd day of study).The behaviour, clinical status and activity of all females of all treated groups were similar during the study and not different from females of the control group. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of treated females. Satellite femalesNo clinical changes were recorded in control and treated females in application period. Only changes related to the colour of the test substance – coloured faeces were recorded (from the 3rd day of study).The behaviour, clinical status and activity of satellite treated females were similar during the application and observation period of the study and not different from satellite control females. No serious changes were found out during examination of skin, hair, eye, visible mucous membranes, respiration, poise, gait, tonic movements, clonic movements, reaction to handling and other activities of satellite treated females.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

MalesThere were no unscheduled deaths during the main study.FemalesOne female from the highest dose level (No. 161) died on the 14th day of pregnancy. It was considered as probably accidental death without treatment relationship

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males Different body weight on the 1st day of administration was caused by sequential including of animal groups to the study. Body weight of treated males of the dose levels 250 and 1000 mg/kg/day at the end of the study was comparable with control group. Body weight of males of the dose level 500 mg/kg/day was slightly decreased compared to control males. Statistically significant differences in necropsy body weight were not found in treated males.Weight increments in treated males were quite balanced with the control males exc. the 6th week at the dose level 500 mg/kg/day. Satellite malesDifferent body weight on the 1st day of administration was caused by sequential including of satellite groups to the study. Body weights of satellite treated males and satellite control males were quite balanced at the end of application as well as recovery period. Statistically significant differences in necropsy body weight 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. FemalesDifferent body weight on the 1st day of administration was caused by sequential including of animal groups to the study. Body weights of treated females and control females were quite balanced during pre-mating period, pregnancy period. In lactation period slightly decreased weight of treated females at the dose level 250 mg/kg was recorded. Statistically significant differences in necropsy body weight were not found in treated females.Weight increments in treated females were quite balanced with the control females within the 1st and 2nd week of application. The evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study.Satellite femalesBody weight of satellite treated females was comparable with satellite control animals for the nearly whole time of application (exc. the 6th week) and recovery period. Statistically significant differences in necropsy body weight were not found in satellite treated females.No marked differences in weight increments were recorded between satellite treated females and satellite control females. 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):

MalesThe food consumption of treated males was similar or slightly increased compared to consumption of control males during the whole study. Satellite malesThe food consumption of satellite treated males was similar or slightly increased compared to the control group in application and recovery period. FemalesIn 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 were more variable compared to control animals but adverse influence of the test substance administration was not evident. Satellite femalesThe food consumption of satellite treated females was similar compared to control group for the whole time of application and recovery period.

Food efficiency:

no effects observed

Description (incidence and severity):

MalesThe food conversion of treated males compared to control animals was variable during the pre-mating period. Differences in food conversion of control and treated males were recorded also in the period after mating, however it was not affected by the application of the test substance.Satellite malesThe food conversion of satellite treated males was quite similar to satellite control males exc. the 2nd and the 4th week.FemalesThe food conversion of treated females in the pre-mating period was decreased compared to control group (without dose dependence). In pregnancy period the food conversion of treated females was also decreased against control group, dose dependence was not recorded. In lactation period the food conversion of treated females was similar to control.Satellite femalesThe food conversion of satellite treated females was quite similar to satellite control males exc. the 3rd and the 6th week.

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

effects observed, treatment-related

Description (incidence and severity):

Satellite malesThe water consumption of satellite treated males was higher compared to satellite control group during the application period. In recovery period the difference was not so evident.Satellite femalesThe water consumption of satellite treated females was higher compared to satellite control group during the application period. In recovery period the difference was not so evident

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Males Statistically significant decrease (without dose dependence) of haemoglobin concentration was detected in males of all dose levels. Other red blood parameters (RBC, MCV, Hct, reticulocytes) of treated males were not influenced by administration of the test substance. Parameters of white blood component (total and differential leucocyte count) were quite balanced in treated males and control males. The following changes of haemocoagulation parameters were recorded in treated males: statistically significantly decreased value of PT at the dose level 1000 mg/kg/day, statistically significant increase of fibrinogen concentration at the dose level 250 mg/kg/day and decrease at the dose level 500 mg/kg/day, nonsignificant decrease of platelet count at the dose level 1000 mg/kg/day. All differences observed in treated males were in range of historical control. Satellite malesRed and white blood components of satellite treated males were not changed. The following changes of haemocoagulation parameters were recorded in satellite treated males: statistically significant increase of fibrinogen concentration and nonsignificant increase of PT value. The haematological parameters were in range of historical control.Females No statistically significant differences were recorded in treated females.No treatment related changes were observed in red blood component. Total leucocyte count in treated females was similar to control, differential leucocyte count (portion of granulocytes, lymphocytes and monocytes) was changed without dependence on dose level. The values of haemocoagulation parameters (APTT, PT, fibrinogen, platelet count) were quite balanced in treated and control females. All values of haematological parameters were in range of historical control.Satellite femalesStatistically significantly increased value of MCV was recorded in satellite treated females. Other values of red blood components were similar with satellite control females. Parameters of white blood component were not significantly changed. The following statistically significant changes of haemocoagulation parameters were noted in satellite treated females: prolonged prothrombin time and decreased concentration of fibrinogen. All values of haematological parameters were in range of historical control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

MalesStatistically significantly increased concentration of creatinine (Crea) and decreased concentration of albumin (ALB) were detected in treated males at the dose levels 250 and 1000 mg/kg/day. The differences were not depend on dose level. The concentration of kalium ions was increased significantly only at the dose level 1000 mg/kg/day vice versa the value was slightly decreased at the dose levels 250 and 500 mg/kg/day compared to control. The values of cholinesterase (CHE) and bile acids (BA) were dose-independently increased in treated males (without statistical significance). Values of other biochemical parameters of treated males were quite well-balanced compared to control.All values of biochemical parameters were in range of historical control.Satellite malesStatistically significant decrease of total cholesterol concentration (T-Chol) was recorded in satellite treated males. Values of other biochemical parameters of satellite treated males were similar to the satellite control group. All values of biochemical parameters were in range of historical control.FemalesStatistically significant differences were not found in treated females. Value of cholinesterase (CHE) was insignificantly increased in females of the lowest dose level. The values of bile acids (BA) were insignificantly changed in a dose-independent manner at treated groups. Values of other biochemical parameters of treated females were quite balanced to the control group. All values of biochemical parameters were in range of historical control.Satellite femalesThe following statistically significant differences were found out in treated satellite females: decreased value of cholinesterase (CHE) and increased total bilirubin (T-Bil). Value of bile acids (BA) was increased in comparison with satellite control females but without statistical significance. Values of other biochemical parameters of treated females were quite balanced to the control group. All values of biochemical parameters were in range of historical control.

Urinalysis findings:

no effects observed

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 for pH of urine in males at the dose level 1000 mg/kg/day. The pH was decreased dose-dependently.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 malesStatistically significant differences were not recorded in satellite treated males. Value of urine pH was insignificantly decreased in satellite treated males.Presence of proteins, blood and leucocytes were recorded in satellite treated males as well as in satellite control males.

Description (incidence and severity):

MalesReactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The activity – number of upstanding in treated males was quite well-balanced with control except slight decrease at the lowest dose level. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated males.Satellite males No significant differences were detected in examined parameters. FemalesReactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control females. The activity – number of upstanding in treated females was quite well-balanced with control. The values of grip strength of pectoral and pelvic legs were without significant differences between control and treated females. Satellite females No significant differences were detected in examined parameters.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute Organ WeightMales Absolute weight of kidneys was statistically significantly decreased at all dose levels in comparison with control males (without dose dependence). Absolute weight of prostate gland with seminal vesicles was statistically significantly decreased at the dose levels 250 and 500 mg/kg/day (without dose dependence). Absolute weight of thyroid gland was decreased with statistical significance and dose dependence at the dose levels 500 and 1000 mg/kg/day. Weight of other organs was similar or insignificantly decreased in treated males. Satellite malesStatistically significant differences were not detected in satellite treated males. Weight of organs of satellite treated males was similar or insignificantly decreased compared to satellite control males. Females Statistically significant differences were observed only in absolute weight of thyroid gland: increase at the dose level 250 mg/kg/day and decrease at the dose level 1000 mg/kg/day. Increased variability was recorded in pituitary gland and uterus weight (insignificant and without dose dependence). Weight of other organs was similar in treated and control females. Satellite femalesStatistically significant differences were not recorded. Differences in uterine weight probably related to oestrous cycle. Absolute weights of other organs were insignificantly changed in satellite treated females. Relative Organ WeightMales Only relative weight of kidneys was statistically significantly decreased: at the dose levels 250 and 1000 mg/kg/day (without dose dependence). Relative weights of spleen, prostate gland with seminal vesicles and thyroid gland were insignificantly decreased in treated males (without dose dependence). Relative weight of other organs were similar in treated and control males.Satellite malesThe following statistically significant differences were detected in satellite treated males: increase of adrenal glands weight and decrease of spleen and liver weight. Relative weight of other organs were similar in satellite treated and satellite control males.Females Statistically significant differences were not recorded in treated females. Increased variability was observed in thymus, uterus and pituitary gland weight (insignificant without dose dependence). Relative weights of other organs of treated females were relatively similar to control females. Satellite femalesRelative weight of ovaries of treated females was statistically significantly increased. Relative weight of thymus and uterus was decreased in satellite treated females but without statistical significance. Relative weight of adrenal glands was insignificantly increased in satellite treated females. 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 5 males. In one male flabby consistence of right testis, reduced size of right epididymis and red focus on right kidney capsule were noted.250 mg/kg/day: only findings related to the test substance administration – slightly coloured content of stomach and intestines were recorded in 2 males.500 mg/kg/day: only findings related to the test substance administration – slightly coloured content of stomach and intestines were recorded in 4 males. Mucous membrane of stomach was coloured by the test substance in 3 males.1000 mg/kg/day: only findings related to the test substance administration – coloured content of stomach and intestines were recorded in 6 males. Mucous membrane of stomach was coloured by the test substance in 5 males.Satellite malesControl satellite: no macroscopical findings were recorded.1000 mg/kg/day satellite: no macroscopical findings were recorded. Females Control: no macroscopical findings were recorded in 5 females. In one female cyst on left ovary was noted.250 mg/kg/day: findings related to the test substance administration – slightly coloured content of stomach and intestines were recorded in 1 female. Cyst on right ovary was observed in one female.500 mg/kg/day: only findings related to the test substance administration – slightly coloured content of stomach and intestines were recorded in 2 females. 1000 mg/kg/day: findings related to the test substance administration – coloured content of stomach and intestines were recorded in 4 females. Mucous membrane of stomach was coloured by the test substance in 1 female.Satellite femalesControl satellite: no macroscopical findings were recorded (except dilatation of uterus in 3 females).1000 mg/kg/day satellite: no macroscopical findings were recorded.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Full histopathology of the preserved organs and tissues was performed for high dose and control animals and satellite animals. Because of probably treatment related changes at the highest dose level (kidneys) and some macroscopical findings at the lowest and the middle dose level the histopathological examination of macroscopically changed organs and then of kidneys was performed at the middle and the lowest dose level.The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-250-500-1000 mg/kg/day and 0S-1000S in satellite groups further in the text. MalesIn kidneys of treated males microscopical changes probably related to the test substance treatment were observed: very early stage of chronic progressive nephropathy in 0-3-1-1 males and early stage of chronic progressive nephropathy in 0-1-2-3 males. In kidneys of control as well as of treated groups hydronephrosis (unilateral or bilateral) was sporadically noted: in 1-2-1-0 males.Then mostly sporadic findings without connection to the test substance treatment were found out. Tubular degeneration or/and atrophy was diagnosed in testes of 2-/-/-3 males. In one control male degeneration was bilateral with affection of 1 % of tubules and in one control male 100 % of tubules of one testis was degenerated. In males of the highest dose level degeneration or atrophy was detected maximum in 4 % of tubules. Germ cells in epididymal tubules occurred in 1-/-/-1 males and sporadic acinar atrophy was observed in prostate gland of 2-/-/-1 males. In digestive system the following microscopical findings were found out: focal mononuclear infiltration in liver of 1-/-/-2 males and focal vacuolation of cells in exocrine gland of pancreas in 2-/-/-1 males. Incidence of other microscopical findings was very sporadic and these findings are mentioned only in individual tables (Annex 3).Satellite males In urinary system of satellite treated males some probably treatment related changes were observed: very early stage of chronic progressive nephropathy in kidneys of 0-2 satellite males, early stage of chronic progressive nephropathy in kidneys of 0-1 satellite males and focal chronic inflammation in urinary bladder in 0-3 satellite males. In kidneys of satellite control as well as of satellite treated males hydronephrosis was sporadically noted: in 1-1 satellite males.Other findings did not relate to the test substance treatment. Tubular degeneration or atrophy (max. 10 % of affected tubules) were observed only in testes of satellite control: 4-0 satellite males. In prostate gland the following microscopical changes were detected: sporadic acinar atrophy in 3-2 satellite males and focal chronic inflammation in 1-3 satellite males. In liver of 2-3 satellite males foci of mononuclear infiltration were recorded. Focal vacuolation of exocrine cells occurred in pancreas of 0-2 satellite males.Females In urinary system very sporadic microscopical findings were diagnosed: chronic progressive nephropathy (early stage) in kidneys of 0-0-0-1 female and hydronephrosis in kidneys of 0-0-1-0 female. Other sporadic changes not related to the test substance treatment were observed in treated females: oedema of forestomach submucosa in 0-/-/-1 female, cysts in thymus of 0-/-/-1 female and follicular hyperplasia in mesenteric lymph nodes of 1-/-/-2 females. In reproductive organs findings related to previous pregnancy were observed: accumulation of siderophages in mesometrium and/or endometrium of uterus in 5-/-/-5 females. Incidence of other microscopical findings was very sporadic and these findings are mentioned only in individual tables (Annex 3).Satellite femalesIn urinary system very sporadic microscopical findings were diagnosed: chronic progressive nephropathy (early stage) in kidneys of 0-1 satellite female, chronic progressive nephropathy (very early stage) in kidneys of 0-1 satellite female and focal chronic inflammation in urinary bladder of 0-1 satellite female. Other sporadic changes not related to the test substance treatment were observed in satellite treated females: focal oedema of stomach submucosa in 0-1 satellite female and cysts in thymus of 4-3 satellite females.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES was established as lower than 250 mg/kg body weight/day (changes of microscopical structure of kidneys accompanied by significant decrease of weights of kidneys at the dose levels 250, 500 and 1000 mg/kg/day). The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in FEMALES was established as 1000 mg/kg body weight/day for females (microscopical changes of kidneys observed very sporadically at the dose level 1000 mg/kg/day and renal weights without significant changes at any dose levels).

Executive summary:

Introduction

The test substance,Direct Blue 78, 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 the 29^thJuly 2016.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 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 adose-range finding experiment (see the Annex 2) and approved by Sponsor.

 

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 12^thday 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.   

 

During the study clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. The functional observation was performed at the end of application and observation period. Vaginal smears were prepared daily, 2 weeks before start of administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy day. Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups, measurement of anogenital distance, nipple retention) were also recorded.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from adult animals and pups were removed for weighing and histopathological examination.

 

 

Results

  

Repeated oral administration of the test substance,Direct Blue 78,to rats by gavage at the dose levels of 250 and500 mg/kg/daydid not cause any mortality. One female from the dose 1000 mg/kg/day died on the 14^thday of pregnancy.It was considered as probably accidental death without treatment relationship.

 

Repeated Dose Toxicity part of study:

The test substance did not affect growth of treated animals. Body weight and body weight increment, food consumption and conversion 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 (faeces coloured by the test substance) were found out during health condition control, clinical observations and detailed clinical examination in animals at the dose levels 500 and 1000 mg/kg/day. At the dose level 250 mg/kg/day no changes were observed during health condition control, clinical observation and detailed clinical examination.

 

Water consumption was irreversibly increased at the dose level 1000 mg/kg/day in both sexes. Urinalysis manifested the influence of the test substance treatment on value of male urine pH at the dose level 1000 mg/kg/day. The difference was partly irreversible.

Haematological examination of males revealed reversible statistically significant decrease of haemoglobin concentration at the dose levels 250, 500 and 1000 mg/kg/day. Haemocoagulation parameters were changed significantly in the following way: decrease of prothrombin time at the dose level 1000 mg/kg/day, increase of fibrinogen concentration at the dose level 250 mg/kg/day and in satellite treated males and decrease of fibrinogen concentration at the dose level 500 mg/kg/day. The above mentioned haematological parameters were changed in a non-dependent manner and values of them were in historical control range. These changes were probably not connected with the test substance treatment. Haematological examination of females demonstrated only some statistically significant changes at the end of observation period: increase of mean corpuscular volume and prothrombin time and decrease of fibrinogen concentration. All values were in range of historical control.

 

During biochemical examination of males statistically significant but dose independent changes of creatinine (reversible increase at the dose levels 250 and 1000 mg/kg/day), albumin (reversible decrease at the dose levels 250 and 1000 mg/kg/day) and kalium ions concentrations (reversible increase at the dose level 1000 mg/kg/day) were detected. In satellite treated males only the concentration of total cholesterol was decreased significantly. Only some delayed statistically significant differences were observed in females: increase of cholinesterase and total bilirubin in satellite treated females.  All values of changed parameters were in range of historical control. Ascertained differences were not considered as the test substance treatment related.

 

Partly reversible decrease of absolute and relative weight of kidneys was found out in males of the dose levels 250, 500 and 1000 mg/kg/day. Except relative weight at the dose level 500 mg/kg/day other differences of kidneys weight were statistically significant.This change was probably treatment related even if the dose dependence was not confirmed but the lowest weights were observed at the dose level 1000 mg/kg/day.Significant changes of other organ weights probably did not relate to the test substance treatment: decrease of absolute weight of prostate gland with seminal vesicle at the dose levels 250 and 500 mg/kg/day, decrease of absolute weight of thyroid gland at the dose levels 500 and 1000 mg/kg/day, increase of relative weight of adrenal glands and decrease of relative weight of spleen and liver in satellite treated males. In females only sporadic statistically significant changes without relation to the test substance treatment were noted: increase of absolute weight of thyroid gland at the dose level 250 mg/kg/day, decrease of absolute weight of thyroid gland at the dose level 1000 mg/kg/dayand increase of relative weight of ovaries in satellite treated females. Weights of kidneys in treated females were quite balanced with control.

 

During pathological examination only non-adverse changes connected with colouration of test substance were found out. Coloured content of stomach and intestines were observed in males and females at the dose levels 250, 500 and 1000 mg/kg/day at the end of administration period. Gastric mucosa was coloured by the test substance in males of the dose level 500 and 1000 mg/kg/day and sporadically in females of the dose level 1000 mg/kg/day at the end of administration period. In satellite animals no macroscopic findings were noted.

 

Histopathological examination of males showed microscopical changes probably related to the test substance treatment: chronic progressive nephropathy (early or very early stage) was observed in kidneys of males at the dose levels 250, 500 and 1000 mg/kg/day. In satellite males the finding persisted and it was accompanied by focal chronic inflammation in urinary bladder.In females of the dose level 1000 mg/kg/day chronic progressive nephropathy occurred only very sporadically at the end of administration and observation period.