Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016 - 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)

Version / remarks:

adopted by the Council on the 29th July 2016

Deviations:

yes

Remarks:

see Any other information on materials and methods

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

EC Number:

274-700-1

EC Name:

Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

Cas Number:

70616-89-6

Molecular formula:

C24H18ClN7O10S3.3Na

IUPAC Name:

trisodium 2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl)amino]-1-hydroxy-3-sulfonato-2-naphthyl}azo)naphthalene-1,5-disulfonate

impurity 1

Reference substance name:

Unknown constituents

IUPAC Name:

Unknown constituents

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 7002/2007

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

laboratory rat has been chosen because the testing laboratory has long experience with this species and because rat is recommended according to the test guideline

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, - Age at study initiation: 9 - 11 weeks- Weight at study initiation: cca 400 g (males), cca 252 g (females)- 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- Diet (e.g. ad libitum): maintenance pelleted diet for rats and mice (Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany)- Water (e.g. ad libitum): drinking water ad libitum- Acclimation period: 5 days (dose-range finding experiment), 13 days (main study)ENVIRONMENTAL CONDITIONS- Temperature: 22 ± 3°C- Humidity: 30 - 70 %- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:The test substance was weighted on analytical balances into glass beaker and the beaker was gradually replenished by water for injections. During that the sample was intensively stirred with a glass rod. The test substance was dissolved in ultrasonic bath for 10 minutes together with occasional mixing with glass rod. The solution was then stirred by magnetic stirrer (400 rpm) for 10 minutes. The stirring of solutions continued during administration. The application forms were prepared daily just before administration.The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The administration of the test substance to animals was performed during one hour after preparation of application form. The test substance was administered to the stomach by gavage. Oral way of administration was chosen according to the guideline and it was approved by Sponsor. The animals were treated 7 days per week at the same time (7.00 – 10.00 am). The vehicle control group was administered by water for injection in the same volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL METHOD: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. PREPARATION OF APPLICATION FORM: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/10mLCca 0.1 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150 mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The test substance was dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer (400 rpm) for 5 minutes. Concentration Level 1000 mg/10 mLCca 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 and the beaker was replenished by the vehicle. The test substance was dissolved in ultrasonic bath for 10 min together with occasional mixing with glass rod. The solution was stirred by magnetic stirrer (400 rpm) for 10 minutes.STABILITY OF THE APPLICATION FORM:The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at each time interval.Conc. level 10 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 5 minutes of ultrasonication and 5 minutes of mixing by magnetic stirrer at 400 rpm.Conc. level 1000 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 10 minutes of ultrasonication together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer at 400 rpm.HOMOGENEITY OF THE APPLICATION FORM:Conc. level 10 mg/ 10 mL: The samples were taken after 5 minutes in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (400 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.Conc. level 1000 mg/ 10 mL: The samples were taken after 10 minutes in ultrasonic bath together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer (400 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.RESULTS OF ANALYSIS:It follows from the results of analyses (homogeneity and stability) that the both application forms (10 mg and 1000 mg/10 mL) of the test substance, Reactive Orange 13, at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

Duration of treatment / exposure:

Males: 49 days; 63 days in satellite groupFemales: according to mating, gestation and lactation period; 63 days in satellite group

Frequency of treatment:

The animals were treated 7 days per week at the same time (7.00 – 10.00 am).

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:

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

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF EXPERIMENTAL ANIMALS:During the acclimatisation period, the health condition of all animals was controlled daily. 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. Then the animals were randomly divided into the control and test groups and they were marked individually. MATING PROCEDURE:Animals were mated from the 29th day of study. Mating 1:1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.EXPERIMENTAL DATA COLLECTIONHealth condition control: daily - during the acclimatization and the experimental part Body weight: 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, 12th and 13th day pups (litters) - 1st, 4th, 12th and 13th day pups – individually – 4th day of lactationFood consumption: weekly and on the same days as body weight (except the mating period) satellite males and females – weeklyWater consumption: satellite males and females – twice a weekClinical observations: males and females - daily during the administration period pups - as soon as possible after delivery and then dailyMortality control: twice dailyDetailed clinical observation: before the first application and then weekly (except the mating period) Functional observation: at the end of administration/observation period Laboratory examinations: - vaginal smears: daily – 1st – 14th day of study; daily in mating period (max. 14 days); on necropsy day- urinalysis: the last day of administration/observation period – only males- haematology: males – after the end of application period parental females – on the 13th day of lactation satellite animals – after the end of observation period- biochemistry: males and nonpregnant females – after the end of application period 2 pups per litter - on the 4th day of lactation parental females and pups – on the 13th day of lactation satellite animals – after the end of observation period- anogenital distance measurement: pups – 4th day of lactation- pathological examination: males and nonpregnant females – after the end of application period 2 pups per litter - on the 4th day of lactation parental females and pups – on the 13th day of lactation satellite animals – after the end of observation period- weight of organs: during necropsy- sperm observation: parental males – during and after necropsy (not performed in satellite males) - histopathological examination: after necropsyMETHODS OF INVESTIGATION - Body WeightThe 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 ConsumptionIn 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 Food conversion in % (weight increment/food consumption x 100) was calculated for animals of Repeated Dose Toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females. - Water Consumption 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. - Mortality ControlAll rats during the treatment periods were examined for vitality or mortality twice daily. - Health Condition Control 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. - Clinical Observations of Males and FemalesAll rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.m.). Animals were observed in natural conditions in their cages. - Clinical Observation of PupsAll pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded. - Detailed Clinical Observation 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. - Functional Observation This observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover, the individual observations of grip strength were performed using grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons. - Laboratory examinations Examination of Vaginal SmearsVaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of oestrous cycle of females. Only females with regular cyclicity were put into the study. Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa. Vaginal smears have been made also on necropsy day to determine the stage of oestrous cycle. UrinalysisThis 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. Haematological ExaminationThis 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. Biochemical ExaminationThis examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.Blood samples from the day 13 pups and the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total). Treatment related changes of T4 total blood serum levels, of thyroid gland weight and microscopical structure were not observed in the day 13 pups therefore assessment of blood serum level of T4 total was not performed in day 4 pups. Anogenital Distance (AGD) MeasurementThe AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalised to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight. Nipples ExaminationThe presence and number of nipples in male pups were counted on day 13 of lactation. Pathological ExaminationDuring the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative. Observation of SpermIn all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm MotilitySperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm. Sperm MorphologySperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck ¬– were recorded. Biometry of OrgansAt the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation. Histopathological Examination Samples of the following tissues and organs were collected at necropsy and fixed:pituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland + seminal vesicles, testes, all gross lesions, thyroid gland, 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, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lLymph nodes – mesenteric, paraaortal, oesophagus, eyeThe mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used. In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopic changes and kidneys, intestines, forestomach, stomach, mesenteric lymph nodes and rectum were examined at the lowest and middle dose level groups. Treatment-related changes were not observed at the high dose group therefore detailed histological examination of testes was performed only for all high dose and control males from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).

Examinations

Observations and examinations performed and frequency:

Health condition control: daily - during the acclimatization and the experimental part Body weight: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, 12th and 13th daypups (litters) - 1st, 4th, 12th and 13th daypups – individually – 4th day of lactationFood consumption: weekly and on the same days as body weight (except the mating period) satellite males and females – weeklyWater consumption: satellite males and females – twice a weekClinical observations:males and females - daily during the administration periodpups - as soon as possible after delivery and then dailyMortality control: twice dailyDetailed clinical observation: before the first application and then weekly (except the mating period) Functional observation: at the end of administration/observation period Laboratory examinations: - vaginal smears:daily – 1st – 14th day of study daily in mating period (max. 14 days)on necropsy day - urinalysis: the last day of administration/observation period – only males - haematology:males – after the end of application period parental females – on the 13th day of lactation satellite animals – after the end of observation period - biochemistry:males and nonpregnant females – after the end of application period 2 pups per litter - on the 4th day of lactation parental females and pups – on the 13th day of lactation satellite animals – after the end of observation period - anogenital distance measurement: pups – 4th day of lactation - pathological examination:males and nonpregnant females – after the end of application period 2 pups per litter - on the 4th day of lactation parental females and pups – on the 13th day of lactation satellite animals – after the end of observation period - weight of organs: during necropsy - sperm observation: parental males – during and after necropsy (not performed in satellite males)

Sacrifice and pathology:

GROSS PATHOLOGY: YesHISTOPATHOLOGY: Yes

Statistics:

see Any other information on materials and methods

Results and discussion

Results of examinations

Clinical signs:

no effects observed

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 29th day of study and at the dose level 1000 mg/kg/day from the 2nd day of application to the end of administration period.The behaviour 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. Only coloured excrements were recorded in males of the dose levels 500 mg/kg/day from the 5th – 7th week and 1000 mg/kg/day from 1st – 7th week of study.Satellite malesNo 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 1000 mg/kg/day (2rd – 49th day of study).The behaviour 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. Only coloured excrements were recorded in satellite males of the dose level 1000 mg/kg/day from 1st – 7th week of study.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 29th day of study and at the dose level 1000 mg/kg/day from the 2nd day of application to the end of administration period.The behaviour 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. Only coloured excrements were recorded in females of the dose levels 500 mg/kg/day from the 5th – 7th week and 1000 mg/kg/day from 1st – 7th week of study.Satellite femalesAt 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 1000 mg/kg/day (2nd – 49th day of study).The behaviour 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. Only coloured excrements were recorded in satellite females of the dose levels 1000 mg/kg/day from 1st – 7th week of study.

Mortality:

no mortality observed

Description (incidence):

MalesThe male No. 93 (the dose level 1000 mg/kg/day - satellite group) died on the 43rd day of study due to intubation error.FemalesThere were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males Body weight of all treated animals was comparable with control group during the whole study. Statistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Weight increments in treated males were balanced with the control males. Only during the 7th week the body weight increments in males at all dose levels were slightly decreased in comparison with control.Satellite malesBody weight of satellite treated males was similar in comparison with satellite control males for the whole time of application and 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.FemalesBody weight of all groups of treated females was quite balanced in comparison with the control group of females during the pre-mating, pregnancy and lactation period. Statistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Body weight increments were variable within the 1st and 2nd week of application. Low or negative body weight increment after the 1st week of application was recorded in control as well in treated females. 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 whole time of application and recovery period. Statistically significant differences in necropsy body weight were not found in satellite treated females.Weight increments of satellite treated females were variable in comparison with the control group and not adversely influenced by the test substance administration.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

MalesThe food consumption of treated males was quite similar to consumption of control males practically during the whole study. Satellite malesThe food consumption of satellite treated males was quite similar to consumption of satellite control males during the whole administration and recovery period. FemalesIn pre-mating period and during pregnancy period the food consumption of treated females was similar with control females. The food consumption of treated females was lower during the first four days of lactation period.Satellite femalesThe food consumption of satellite treated females was quite similar to consumption of satellite control females during the whole administration and recovery period.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

MalesThe food conversion of treated males compared to control animals was variable during the pre-mating period. During the 1st week was the food conversion in treated males increased compared to the control group (without treatment relation) but during the 2nd week it was the opposite. In the weeks after the mating period the food conversion of treated males was decreased compared to control males (exc. 6th week). Satellite malesThe food conversion of satellite treated females within application and recovery period was variable in comparison with the control group.FemalesThe food conversion of treated females in the pre-mating period was variable compared to control – most of time increased and not influenced by the test substance application.In pregnancy period the food conversion of treated females was similar or slightly decreased against control group, dose dependence was not recorded.In lactation period the food conversion of treated females was variable in comparison with the control group and it was not adversely influenced by the test substance treatment. Satellite femalesThe food conversion of satellite treated females within application and recovery period was variable in comparison with the control group.

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

effects observed, treatment-related

Description (incidence and severity):

Satellite malesThe water consumption of satellite treated males was slightly lower compared to satellite control group during the whole study exc. the 9th week in the recovery period.Satellite femalesThe water consumption of satellite treated females was lower compared to satellite control group during the whole study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males Red blood parameters (total erythrocyte count, mean corpuscular volume, haematocrit, haemoglobin) of treated males were not adversely influenced by administration of the test substance. Parameters of white blood component were quite well-balanced in treated males compared to control males. Haemocoagulation parameters (activated partial thromboplastin time, prothrombin time, fibrinogen, platelet count) were not adversely influenced by administration of the test substance. Relative count of reticulocytes was dose-independently increased in treated groups, the value at the dose level 1000 mg/kg/day was statistically significantly changed but in range of historical control. All haematological parameters were in range of historical control.Satellite malesStatistically significant differences were not found in satellite treated males. None of haematological parameters of satellite treated males was influenced by the test substance treatment. The haematological parameters were in range of historical control.Females Any changes of parameters of red blood components (total erythrocyte count, mean corpuscular volume, haemoglobin, haematocrit) were not recorded. Parameters of white blood component were quite well-balanced in treated females compared to control females. Haemocoagulation parameters (prothrombin time, fibrinogen, platelet count) were not influenced by administration of the test substance – statistically significant changes were not recorded. Only the values of APTT (activated partial thromboplastin time) in all treated females were decreased with dose dependency compared to the control females (but in range of historical control). The value of APTT was statistically significantly changed in females at dose levels 500 and 1000 mg/kg/day. The value of reticulocytes was not statistically significantly changed in females and was quite well-balanced in comparison with control females. The haematological parameters were in range of historical control.Satellite femalesNo statistically significant differences were recorded in satellite treated females. None of haematological parameters of satellite treated females was influenced by the test substance treatment. All values of haematological parameters were in range of historical control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

MalesOnly one statistically significant difference was found out in treated males – total bilirubin concentration was decreased at the dose level 1000 mg/kg/day (in a range of historical range). All other biochemical parameters of treated males were similar with the control males. The values of biochemical parameters were in range of historical control.Satellite malesValues of biochemical parameters of satellite treated males were quite balanced to the satellite control group. No statistically significant differences were found out in treated satellite males. All values of biochemical parameters were in range of historical control.FemalesThe values of total bilirubin were dose-dependently decreased at all treated females compared to the control females. Statistically significant decrease of total bilirubin (T-Bil) concentration was recorded only in females at the dose level 1000 mg/kg/day (but in range of historical control). Value of bile acids (BA) was decreased in females at the middle and at the highest dose levels in comparison with the control females but without statistical significance. Values of other biochemical parameters of treated females were quite balanced to the control group. The values of biochemical parameters were in range of historical control (except triglycerides at the dose level 500 mg/kg/day). The concentration of triglycerides (TG) was slightly higher than the upper limit of historical control.Satellite femalesThe following statistically significant differences were found out in treated satellite females: increased values of creatinine (CREA) and potassium (K) and decreased value of albumin (ALB) value. Value of bile acids (BA) was increased in comparison with satellite control females but without statistical significance. Values of other biochemical parameters of treated satellite females were quite balanced to the satellite control group. All values of biochemical parameters were in range of historical control.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males Statistically significant differences of pH and volume were not found in treated males. The volume was slightly increased in treated males compared to the control males. Colour of urine was changed in treated males. Presence of proteins and blood 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. Occurrence of leucocytes was recorded only in urine of two males at the dose level 500 mg/kg/day. Satellite malesStatistically significant differences of pH and volume were not found in satellite treated males. Presence of leucocytes was 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. The values of all other parameters of treated males were similar to control.

Behaviour (functional findings):

no effects observed

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 was quite well-balanced. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated males.Satellite males No significant differences were detected in examined parameters.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 except slight decrease at the lowest dose level. 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, non-treatment-related

Description (incidence and severity):

- Absolute Organ WeightsMales Statistically significant differences were not recorded. Slight decrease of absolute weight of thymus was recorded in all treated males compared to control group of males (without dose dependence). Weight of other organs was similar in treated and control males. Satellite malesStatistically significant differences were not detected in satellite treated males. Weight of organs of satellite treated males was similar or only insignificantly increased compared to satellite control males. Females Absolute weight of pituitary gland was significantly decreased in females at the dose levels 500 and 1000 mg/kg/day. Weight of other organs was similar in treated and control females. Satellite femalesStatistically significant differences were not recorded in satellite treated females. Absolute weights of organs were similar in satellite treated and control females. - Relative Organ WeightsMales Statistically significant differences were not recorded. Relative weights of thymus, adrenal gland and testes were statistically insignificantly decreased in all treated males compared to the control males. Relative weights of other organs of treated males were similar to control males. Satellite malesRelative weight of thymus was decreased insignificantly in satellite treated males. Relative weights of adrenal glands, spleen and testes were insignificantly increased in treated satellite males compared to the satellite control males. Relative weights of other organs were similar in satellite treated and satellite control males.Females Relative weight of liver was statistically significantly decreased at the dose level 500 mg/kg/day. Relative weight of pituitary gland was significantly decreased in females at the dose level 1000 mg/kg/day. Relative weights of thymus and kidneys were insignificantly increased in females at the dose level 1000 mg/kg. Weights of other organs were quite well-balanced in treated and control animals. Satellite femalesRelative weights of organs were similar in satellite treated and control females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males Control: no macroscopical findings were recorded in 6 males. 250 mg/kg/day: no macroscopical findings were recorded in 6 males. 500 mg/kg/day: finding related to the test substance administration – orange coloring of kidneys was recorded in 1 male. 1000 mg/kg/day: mostly findings related to the test substance administration – orange coloring of kidneys was recorded in 2 males. In one male flabby consistency and reducement of left testis and reduced size of epididymis were noted.Satellite malesControl satellite: no macroscopical findings were recorded in 6 males. 1000 mg/kg/day satellite: no macroscopical findings were recorded in 5 males.One male died on the 43rd day of study (intubation error). The test substance in the lungs was found in this male and the initial autolysis of the organs was observed.Females Control: no macroscopical findings were recorded in 6 females. 250 mg/kg/day: no macroscopical findings were recorded in 6 females.500 mg/kg/day: no macroscopical findings were recorded in 6 females.1000 mg/kg/day: cyst on the left uterine horn was detected in one female.Satellite femalesControl satellite: dilatation of uterus was observed in 1 female. 1000 mg/kg/day satellite: slight dilatation of uterus was observed in 2 females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

MalesThe incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-500-1000 mg/kg/day and 0S-1000S in satellite groups further in the text. In 1-1-2 males no histological findings were diagnosed.In reproductive organs these findings were observed: unilateral or bilateral atrophy of tubules (1-/-1) and mild unilateral spermatogranuloma (0-/-1) in the testes, slight focal chronic inflammation of prostate gland in 3-/-0 males.In the kidneys were revealed mild hydronephrosis (3-0-3) and solitary hyaline casts (1-0-0).Then only sporadic findings which were not connected with test substance treatment were found out.These all histopathological findings did not relate to the test substance treatment. Satellite males In 3-4 satellite males no histological findings were diagnosed.In reproductive organs slight foci of chronic inflammation in the prostate gland was observed in 3-0 male. In other organs these sporadic findings were detected: hydronephrosis in kidneys (0-1) and focal chronic inflammation in epididymides (1-0).These all histopathological findings did not relate to the test substance treatment.Females The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-1000 mg/kg/day and 0S-1000S in satellite groups further in the text. In 0-0 female no histological findings were diagnosed.In reproductive organs findings related with previous pregnancy and delivery of pups were observed: accumulation of lipophages and siderophages in mesometrium of uterus (5-6) and hemosiderin in mucosa of uterus (6-6), lobular hyperplasia of mammary gland (6- 6). In other organs these sporadically findings were detected: hydronephrosis in the kidneys (0-1), cyst in the thymus (2-0) and placental site trophoblastic tumor in uterus (0-1).Satellite females In 1-1 satellite female no histological findings were diagnosed.In reproductive organs hydrometra (2-4) (related to oestrous cycle), musocal (1-0) and squamous cell cyst (0-1) in uterus were observed.In other organs these sporadic findings were detected: hyaline casts (0-1) and chronic pyelitis (1-1) in the kidneys, cyst (3-0) and focal hemorrhage (0-1) in the thymus.

Histopathological findings: neoplastic:

not specified

Details on results:

Repeated oral administration of the test substance, Reactive Orange 13, to rats by gavage at the dose levels of 250, 500 and 1000 mg/kg/day did not cause any mortality. The effect of the test substance treatment on growth of animals was not adverse. The test substance did not affect utilization of nutrients of food: body weight increments, food consumption and food conversion were similar between control and treated animals. Individual variability of body weight increments was adequate to species, sex and age of animals used in the study and to period of gravidity or lactation in females. Water consumption in satellite treated males and females was slightly lower compared to satellite control groups during the whole study (exc. the 9th week in the recovery period).Urinalysis did not reveal any statistically significant changes of pH and volume in treated males. Only colour of urine (light orange) was changed in treated males at all dose levels. During biometry of organs in males, significant changes of absolute and relative weights of organs related to administration of the test substance were not detected. Most of observed significant changes of absolute and relative weights of organs in females (pituitary gland, liver) were considered unrelated to the test substance treatment due to absence of macroscopic or microscopic changes of structure of these organs.Haematological examination of males and females showed sporadic findings only.In males statistically significantly increased value of reticulocytes at the dose level 1000 mg/kg/day was recorded.In females the values of activated partial thromboplastin time in all treated females were decreased with dose dependency compared to the control females. The value of activated partial thromboplastin time was statistically significantly changed in females at dose levels 500 and 1000 mg/kg/day.All haematological parameters were in range of historical control.All findings observed in both sexes (but in absence of a treatment-related clinical signs of toxicity) were considered to be of no toxicological significance.Biochemical examination of treated males and females revealed minimal changes. In males and females total bilirubin concentration was statistically significantly decreased at the dose level 1000 mg/kg/day. The values of total bilirubin were dose dependently decreased at all treated females compared to the control group.In satellite treated females delayed changes only were recorded (statistically significantly decreased value of albumin and statistically significantly increased value of creatinine and potassium). All biochemical parameters were in range of historical control (except triglycerides in females at the dose level 500 mg/kg/day). The concentration of triglycerides was slightly higher than the upper limit of historical control.Sporadic macroscopic findings related to the colour of the test substance treatment were recorded during the pathological examination of treated males at the dose level 500 and 1000 mg/kg/day (orange coloring of kidneys).Microscopic evaluation showed that the test substance orally administered at the dose of 1000 mg/kg (the highest dose level) did not cause any pathological changes in the male and female organs. The test substance treatment did not produce changes detectable in functional observation of animals. Changes related to the colour of the test substance - coloured faeces were found out during health condition control, clinical observations and detailed clinical examination in males and females of the dose levels 500 and 1000 mg/kg/day.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect related to the test substance observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

see Attached background material

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES and FEMALES was established as 1000 mg/kg body weight/day. All findings observed in both sexes were considered to be of no toxicological significance.

Executive summary:

The test substance, Reactive Orange 13, 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 29th July 2016.

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

males – after mating period – totally for 49 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating,

non-mated females – for 25 days after the end of mating period.

After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.   

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.

Repeated oral administration of the test substance, Reactive Orange 13, to rats by gavage at the dose levels of 250, 500 and 1000 mg/kg/day did not cause any mortality.

The effect of the test substance treatment on growth of animals was not adverse. The test substance did not affect utilization of nutrients of food: body weight increments, food consumption and food conversion were similar between control and treated animals. Individual variability of body weight increments was adequate to species, sex and age of animals used in the study and to period of gravidity or lactation in females.

Water consumption in satellite treated males and females was slightly lower compared to satellite control groups during the whole study (exc. the 9th week in the recovery period).

Urinalysis did not reveal any statistically significant changes of pH and volume in treated males. Only colour of urine (light orange) was changed in treated males at all dose levels.

During biometry of organs in males, significant changes of absolute and relative weights of organs related to administration of the test substance were not detected.

Most of observed significant changes of absolute and relative weights of organs in females (pituitary gland, liver) were considered unrelated to the test substance treatment due to absence of macroscopic or microscopic changes of structure of these organs.

Haematological examination of males and females showed sporadic findings only.

In males statistically significantly increased value of reticulocytes at the dose level 1000 mg/kg/day was recorded.

In females the values ofactivated partial thromboplastin timein all treated females were decreased with dose dependency compared to the control females. The value ofactivated partial thromboplastin timewas statistically significantly changed in females at dose levels 500 and 1000 mg/kg/day.

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

Biochemical examination of treated males and females revealed minimal changes. In males and females total bilirubin concentration was statistically significantly decreased at the dose level 1000 mg/kg/day. The values of total bilirubin were dose dependently decreased at all treated females compared to the control group.

In satellite treated females delayed changes only were recorded (statistically significantly decreased value of albumin and statistically significantly increased value of creatinine and potassium).

All biochemical parameters were in range of historical control (except triglycerides in females at the dose level 500 mg/kg/day). The concentration of triglycerides was slightly higher than the upper limit of historical control.

Sporadic macroscopic findings related to the colour of the test substance treatment were recorded during the pathological examination of treated males at the dose level 500 and 1000 mg/kg/day (orange coloring of kidneys).

Microscopic evaluation showed that the test substance orally administered at the dose of 1000 mg/kg (the highest dose level) did not cause any pathological changes in the male and female organs.

The test substance treatment did not produce changes detectable in functional observation of animals. Changes related to the colour of the test substance - coloured faeces were found out during health condition control, clinical observations and detailed clinical examination in males and females of the dose levels 500 and 1000 mg/kg/day. 

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES and FEMALES was established as 1000 mg/kg body weight/day. All findings observed in both sexes were considered to be of no toxicological significance.