Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

EC Number:

250-792-9

EC Name:

Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

Cas Number:

31765-95-4

Molecular formula:

C44H24Cu2N8O17S4.4Na

IUPAC Name:

tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

Test material form:

solid: particulate/powder

Details on test material:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Laboratory rat has been chosen because our 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- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: males, females: sexually adult, 7 - 9 weeks on arrival; dose-range finding experiment: 9 weeks on arrival- Weight at study initiation: average cca 379 d- 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): pelleted diet for rats and mice - Altromin for rats/mice- Water (e.g. ad libitum): drinking water ad libitum- Acclimation period: at least 5 days (dose-range test – 5 days, main study – 6 days) ENVIRONMENTAL CONDITIONS- Temperature (°C): 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 APPLICATION FORM AND ADMINISTRATION 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 30 minutes together with occasional mixing with glass rod. The solution was then stirred by magnetic stirrer (600 rpm) for 15 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.PREPARATION OF DOSING SOLUTIONS: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. The test substance was dissolved in ultrasonic bath for 10 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 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The test substance was dissolved in ultrasonic bath for 30 min together with occasional mixing with glass rod. The solution was stirred by magnetic stirrer (600 rpm) for 15 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 10 minutes of ultrasonication and 10 minutes of mixing by magnetic stirrer at 500 rpm. Conc. level 1000 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 30 minutes of ultrasonication together with occasional mixing with glass rod and 15 minutes of mixing by magnetic stirrer at 600 rpm.HOMOGENEITY OF THE APPLICATION FORM Conc. level 10 mg/ 10 mL: The samples were taken after 10 minutes in ultrasonic bath and 10 minutes of mixing by magnetic stirrer (500 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 30 minutes in ultrasonic bath together with occasional mixing with glass rod and 15 minutes of mixing by magnetic stirrer (600 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 ANALYSISIt 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, Direct Black 112, 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:

Once a day, 7 days per week at the same time (7.00 – 10.00 am).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 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 ANIMALSDuring 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 PROCEDUREAnimals 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.METHODS OF INVESTIGATIONBody 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 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.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.Total protein, total bilirubin, urea, creatinine, glucose, transaminases (AST, ALT), cholesterol, albumin, alkaline phosphatase (ALP), phosphorus and calcium, triglycerides, cholinesterase, bile acids, sodium, potassium and chloride ions.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.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 (Repeated Dose Toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (Reproduction part of study – all animals). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.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 - In all males and females: pituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland + seminal vesicles, testes, all gross lesions, thyroid gland;- In males and females of Repeated Dose Toxicity part of study additionally: adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord – thoracic, spleen, stomach, thymus, thyroid gland, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mesenteric, paraaortal, oesophagus, eye;The mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used. In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes and kidneys, intestines, forestomach, stomach, mesenteric lymph nodes and rectum were examined at the lowest and middle dose level groups. In Reproductive Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Organs with macroscopical changes were examined also 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). DATA PROCESSING All the primary data (body weight, food consumption, water consumption, health condition control, general clinical observation, detailed clinical observation, functional observation, haematological examination, biochemistry, urinalysis, examination of vaginal smears, sperm examination, biometry of organs, necropsy findings, histopathological examination) were recorded in special protocols. These primary data were used for calculations and processed to tables.For the evaluation of Repeated Dose Toxicity part of study the first-six males and the first-six birth giving mothers of each basic group and satellite males and females were used. For the evaluation of Reproduction Toxicity part of study all males and females of basic groups were used.STATISTICAL ANALYSISFor statistical evaluation the software Statgraphic Centurion (version XVII, USA) was used. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.The results statistically significant on probability level 0.05 were indicated in the summary tables.As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used. For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level 0.05).

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: 1st - 7th weekBODY WEIGHT: Yes- Time schedule for examinations: 1st - 7th weekFOOD CONSUMPTION AND COMPOUND INTAKE: YesWATER CONSUMPTION AND COMPOUND INTAKE: Yes- Time schedule for examinations: 1st - 7th week in the satellite group onlyHAEMATOLOGY: Yes- Time schedule for collection of blood: parental males – 64th day of study, satellite males – 78th day of study, parental females – 13th day of lactation period, pups – 2 pups per litter – 4th day of lactation, 2 pups per litter – 13th day of lactation, satellite females – 78th day of study- Anaesthetic used for blood collection: Yes (ether)- Animals fasted: Not specified- How many animals: 6 males + 6 females of each group and of satellite groupCLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: parental males – 64th day of study, satellite males – 78th day of study, parental females – 13th day of lactation period, pups – 2 pups per litter – 4th day of lactation, 2 pups per litter – 13th day of lactation, satellite females – 78th day of study- Animals fasted: cca 18 h before blood collection- How many animals: 6 males + 6 females of each group and of satellite groupURINALYSIS: Yes- Time schedule for collection of urine:- Metabolism cages used for collection of urine: Yes- Animals fasted: Not specifiedNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: at the end of administration period and recovery period- Dose groups that were examined: 6 males + 6 females of each group and of satellite group- Battery of functions tested: the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex, motor activity assessment, individual observations of grip strength using grip strength meter (pectoral legs, pelvis legs)

Sacrifice and pathology:

GROSS PATHOLOGY: YesHISTOPATHOLOGY: Yes

Statistics:

For statistical evaluation the software Statgraphic Centurion (version XVII) 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 of males and females - mean pup weight - litter weight - anogenital distance of pups - selected haematology parameters - blood biochemistry parameters - data from urinalysis (pH, volume) - data from biometry of organs As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used. For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).The non-parametric tests were used for statistical evaluation of - selected reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of corpora lutea, number of implantations) - selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count) The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level 0.05).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Males Soft consistency of faeces was observed in males of the dose levels 500 and 1000 mg/kg/day from the 1st day of application of the test substance. Changes related to the colour of the test substance – coloured faeces and urine were recorded in males of the dose levels 125 (from the 7th day of application of the test substance), 500 and 1000 mg/kg/day (from the 1st day of application).Satellite malesSoft consistency of faeces was observed in satellite treated males from the 1st day of application of the test substance. Changes related to the colour of the test substance – coloured faeces and urine were recorded in satellite treated males from the 1st day of application.Females Soft consistency of faeces was observed in females of the dose levels 125 (from the 2nd day of application of the test substance), 500 and 1000 mg/kg/day (from the 1st day of application of the test substance). Changes related to the colour of the test substance – coloured faeces and urine were recorded in females of the dose levels 125 (from the 2nd day of application of the test substance), 500 and 1000 mg/kg/day (from the 1st day of application).Satellite femalesSoft consistency of faeces was observed in satellite treated females from the 1st day of application of the test substance. Changes related to the colour of the test substance – coloured faeces and urine were recorded in satellite treated females from the 1st day of application.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males Statistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Body weight of treated males at all dose levels was insignificantly decreased throughout the whole administration period. The difference was dependent on dose level (exc. the 2nd week).Weight increments in treated males were mostly slightly decreased in comparison with control.Satellite malesStatistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Body weight of satellite treated males was insignificantly decreased throughout the whole administration and recovery period.Weight increments of satellite treated males were similar or slightly decreased against satellite control males in administration period. Viceversa increased weight increments were recorded in satellite treated males in recovery period.FemalesStatistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Body weights of treated females and control females were quite balanced during pre-mating period. In pregnancy period body weight of treated females was slightly decreased or similar compared to control without dependence on dose level. In lactation period body weight was slightly decreased in treated females. On the day 12 and 13 the difference was dependent on dose level.Weight increments in treated females were decreased compared to control females in the 1st week of administration. In the 2nd week of administration low or negative body weight increment was recorded in control as well as in treated animals. The evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study.Satellite femalesStatistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Body weight of satellite treated females was comparable with satellite control animals for the nearly whole time of application and recovery period.No marked differences in weight increments were recorded between satellite treated females and satellite control females. 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 quite similar to consumption of control males practically during the whole study. Decrease of consumption was noted only at the 1st week of study - in males of the dose level 1000 mg/kg/day). Satellite malesThe food consumption of satellite treated males was quite similar to consumption of satellite control males during the whole administration and recovery period (exc. the 1st week).FemalesIn the 1st week of pre-mating period the food consumption of treated females was decreased and in the 2nd week it was quite balanced compared to control females.During pregnancy and lactation period the food consumptions of treated females was similar to control females. Satellite femalesThe food consumption of satellite treated females was quite similar to consumption of satellite control females during the whole administration and recovery period (exc. the 1st week).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

MalesThe food conversion of treated males compared to control animals was variable (without treatment relation) during the pre-mating period. In the 6th and 7th week conversion was decreased in males of the dose levels 125, 500 and 1000 mg/kg/day compared to control males.Satellite malesThe food conversion of satellite treated males was decreased only in the 4th and 5th week of the study. The food conversion of treated females was not affected in the pre-mating period. In pregnancy period the food conversion of treated females was similar to control or slightly decreased against control group, dose dependence was not recorded. In lactation period the food conversion was decreased in females of the dose level 500 mg/kg/day (day 1 – 4) and females of the dose level 1000 mg/kg/day (day 1 – 4 and day 4 – 12).Satellite femalesThe food conversion of satellite treated females was variable during administration and recovery period without obvious influence of the test substance.

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

effects observed, non-treatment-related

Description (incidence and severity):

Satellite malesThe water consumption of satellite treated males was visibly higher compared to satellite control group during the application period. In recovery period the water consumption of satellite treated males was similar to satellite control.Satellite femalesThe water consumption of satellite treated females was slightly higher compared to satellite control group during the application period. In recovery period the water consumption of satellite treated females was decreased against satellite control.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males Only two statistically significant changes of haematological parameters were detected: increase of total leucocyte count in males of the dose level 500 mg/kg/day and decrease of total erythrocyte count in males of the dose level 1000 mg/kg/day (without dose dependence). Total leucocyte count was insignificantly increased also at the dose level 1000 mg/kg/day. The values of WBC at the dose levels 500 and 1000 mg/kg/day were higher than historical control values. The value of total erythrocyte count at the dose level 1000 mg/kg/day was in range of historical control. Relative count of reticulocytes was insignificantly and dose-independently increased in treated groups, the value at the dose level 1000 mg/kg/day was slightly higher than historical control values. Other haematological parameters of treated males were not influenced by administration of the test substance. 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 Only the concentration of fibrinogen was statistically significantly increased: at the dose level 125 and 1000 mg/kg/day (without dose dependence). The values of other haemocoagulation parameters were quite balanced in treated and control females except insignificant dose dependent change of differential leucocyte count. The percentage portion of granulocytes was slightly increased in treated males viceversa the portion of lymphocytes was decreased. All values of 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, treatment-related

Description (incidence and severity):

MalesThe following statistically significant and dose dependent changes of biochemical parameters were detected: decrease of total protein (T-Pro) and albumin (ALB) and decrease of ALP in males of the dose levels 500 and 1000 mg/kg/day. The values of total protein at the dose level 1000 mg/kg/day and albumin at the dose level 500 and 1000 mg/kg/day were lower than historical control values. Creatinine (Crea) concentration was statistically significantly decreased in males of the dose levels 125 and 1000 mg/kg/day. The value of urea (BUN) was significantly decreased only at the dose level 1000 mg/kg/day. The value of Bile acids was dose-independently and insignificantly increased in males of the dose levels 125 and 1000 mg/kg/day. Values of other biochemical parameters of treated males were quite well-balanced compared to control.The values of biochemical parameters (exc. above mentioned total protein and albumin) were in range of historical control.Satellite malesStatistically significant decrease of creatinine concentration and increase of cholinesterase were 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 and dose dependent decrease of total protein (T-Pro) and albumin (ALB) values was recorded in males of the dose levels 500 and 1000 mg/kg/day. The values of total protein at the dose level 1000 mg/kg/day and albumin at the dose level 500 and 1000 mg/kg/day were lower than historical control values. Cholinesterase (CHE) concentration was insignificantly decreased at the dose level 1000 mg/kg/day. Values of other biochemical parameters of treated females were quite balanced to the control group. The values of biochemical parameters (exc. total protein and albumin) were in range of historical control.Satellite femalesThe following statistically significant differences were found out in treated satellite females: increased values of ALP and anorganic phosphorus (IP) and decreased cholinesterase (CHE) 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 females were quite balanced to the control group. All values of biochemical parameters were in range of historical control.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males Statistically significant differences were recorded for volume of urine in males at the dose level 1000 mg/kg/day. The volume was decreased dose-dependently.Colour of urine was changed in treated males and intensity of colouring was depended on dose level. Increased presence of proteins was detected at the dose level 1000 mg/kg/day. The values of pH and specific gravity were slightly increased in males of the dose level 1000 mg/kg. Occurrence of leucocytes was recorded only in urine of males of the dose level 1000 mg/kg. Satellite malesStatistically significant differences of pH and volume were not found in satellite treated males. 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 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, non-treatment-related

Description (incidence and severity):

Males Absolute weight of prostate gland and seminal vesicles was statistically significantly decreased at the dose levels 500 and 1000 mg/kg/day (without dose dependence). Absolute weight of thymus was insignificantly increased at the dose levels 500 mg/kg/day. Weight of other organs was similar or only insignificantly decreased in treated males compared to control. Satellite malesStatistically significant differences were not detected in satellite treated males. Weight of organs of satellite treated males was similar or only insignificantly decreased compared to satellite control males. Females Statistically significant differences were not recorded in treated females. Absolute weight of thymus was insignificantly and dose dependently decreased in treated females. Weight of other organs was similar in treated and control females. Satellite femalesAbsolute weight of spleen was significantly increased in satellite treated females. Differences in uterine weight related to oestrous cycle. Absolute weights of other organs were insignificantly changed in satellite treated females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males Control: no macroscopical findings were recorded in 6 males. 125 mg/kg/day: no macroscopical findings were recorded in 6 males. 500 mg/kg/day: mostly findings related to the test substance administration – coloured content of stomach and/or intestines was recorded in 6 males. Mesenteric lymph nodes were coloured by the test substance in 6 males. In 2 males paraaortal lymph nodes were coloured by the test substance. Submandibular lymph nodes and urine were coloured by the test substance in 1 male. Whitish colour of surface of spleen was observed in 2 males.1000 mg/kg/day: mostly findings related to the test substance administration – coloured content of stomach and/or intestines was recorded in 6 males. Mesenteric lymph nodes were coloured by the test substance in 6 males. In 1 male paraaortal lymph nodes were coloured by the test substance. Submandibular lymph nodes and urine were coloured by the test substance in 2 males. Swelling of kidneys was observed in two males. Change of urine colour occurred in 2 males.Satellite malesControl satellite: in 1 male flabby consistency a reducement of testis was noted. 1000 mg/kg/day satellite: mostly findings related to the test substance administration –mesenteric lymph nodes were coloured by the test substance in 6 males. Submandibular lymph nodes were coloured by the test substance in 3 males. In one male enlargement of one testis was observed. Females Control: no macroscopical findings were recorded in 6 females. 125 mg/kg/day: only findings related to the test substance administration – coloured content of stomach and intestines was recorded in 2 females. 500 mg/kg/day: mostly findings related to the test substance administration – coloured content of stomach and/or intestines was recorded in 5 females. Mesenteric lymph nodes were coloured by the test substance in 2 females. Submandibular lymph nodes were coloured by the test substance in 1 female. 1000 mg/kg/day: mostly findings related to the test substance administration – coloured content of stomach and/or intestines was recorded in 6 females. Mesenteric lymph nodes were coloured by the test substance in 6 females. In 2 females paraaortal lymph nodes were coloured by the test substance. Submandibular lymph nodes were coloured by the test substance in 1 female. Cyst was detected in oviduct of one female.Satellite femalesControl satellite: dilatation of uterus was observed in 1 female. 1000 mg/kg/day satellite: mostly findings related to the test substance administration –mesenteric lymph nodes was coloured by the test substance in 6 females. Submandibular lymph nodes were coloured by the test substance in 1 female. Dilatation of uterus was observed in 3 females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MalesIn some organs of digestive system histological changes related to the test substance treatment were recorded. Presence of brown pigment (of various intensity) was detected in mucosa of small intestine (0-0-2-2 males), large intestine (0-0-1-5 males), caecum (0-0-0-1 males) and rectum (0-0-2-1 males). Also the following changes was probably caused by the test substance administration: vacuolation of forestomach epithelium and polynuclear or miscellaneous infiltration of forestomach mucosa in 0-0-0-2 males, oedema of stomach submucosa in 0-0-0-3 males, polynuclear infiltration of small intestine mucosa in 0-0-0-3 males, polynuclear infiltration of large intestine mucosa or submucosa in 0-0-0-2 males, polynuclear infiltration of mucosa or submucosa of caecum in 0-0-1-3 males and polynuclear, mononuclear or miscellaneous infiltration of rectal mucosa or submucosa in 0-0-2-4 males. Macrophages containing brown or brownish pigment (of various intensity) were found out in mesenteric lymph nodes of 0-4-2-6 males and in paraaortal lymph nodes of 0-/-1-3 males. Presence of brown pigment (of various intensity) was noted also in tubular epithelium of kidneys: in 0-1-3-3 males. Then sporadic findings without connection to the test substance treatment were found out. Tubular degeneration or/and atrophy was diagnosed in testes of 1-/-/-3 males. Chronic inflammation was observed in prostate gland of 1-/-/-3 males. In liver of 2-/-/-2 males focal infiltration (polynuclear, mononuclear or miscellaneous) was registered. Focal necrosis of myocardium occurred in heart of 1-/-/-1males. Chronic progressive nephropathy (very early stage) and hydronephrosis was diagnosed in 1-0-0-1 males. In thymus of 1-/-/-1 males cysts were observed. The following probably treatment related changes were recorded in satellite treated males: brown pigment in forestomach mucosa of 0-1 males, in small intestine mucosa of 0-3 males, in large intestine mucosa of 0-6 males, in mucosa of caecum in 0-3 males, in rectal mucosa of 0-4 males; macrophages containing brown or brownish pigment in mesenteric lymph nodes of 0-5 males and in paraaortal lymph nodes of 0-1 male. Other findings did not relate to the test substance treatment. Tubular degeneration or tubular atrophy was observed only in testes of 3-5 satellite males. In prostate gland the following microscopical changes were detected: sporadic acinar atrophy in 2-1 satellite males, chronic inflammation in 1-3 satellite males. In kidneys sporadic basophilic tubules occurred in 2-1 satellite males.Females In some organs of digestive system histological changes related to the test substance treatment were recorded. Presence of brown pigment (of various intensity) was detected in mucosa of small intestine (0-0-2-4 females), large intestine (0-0-0-6 females), caecum (0-0-1-2 females) and rectum (0-0-2-4 females). Also the following changes was probably caused by the test substance administration: polynuclear infiltration of large intestine mucosa or submucosa in 0-0-2-3 females, polynuclear infiltration of mucosa or submucosa of caecum in 0-0-0-4 females and polynuclear, mononuclear, polynuclear or miscellaneous infiltration of rectal mucosa or submucosa in 0-0-1-4 females and focal erosion of rectal mucosa in 0-0-1-1 females. Macrophages containing brown or brownish pigment (of various intensity) were found out in mesenteric lymph nodes of 0-4-4-6 females and in paraaortal lymph nodes of 0-/-/-1 females. Presence of brown pigment (of various intensity) was noted also in tubular epithelium of kidneys: in 0-0-6-6 females. Accumulation of siderophages in endometrium or/and mesometrium of uterus (in 5-/-/6 females) related to previous pregnancy of females. Then the following sporadic findings without connection to the test substance treatment were found out: focal infiltration (polynuclear or miscellaneous) in liver of 1-/-/-1 females, oedema of stomach submucosa in 1-0-1-2 females, cysts in thymus of 1-/-/-2 females and in thyroid gland of 2-/-/-0 females. Satellite females The following probably treatment related changes were recorded in satellite treated females: presence of brown pigment (of various intensity) in small intestine mucosa of 0-5 females, in large intestine mucosa of 0-5 females, in mucosa eventually in lymphatic tissue of caecum in 0-5 females, in rectal mucosa of 0-6 females; macrophages containing brown or brownish pigment (of various intensity) in mesenteric lymph nodes of 0-4 females and in paraaortal lymph nodes of 0-3 females and brown pigment in tubular epithelium of kidneys (of various intensity) in 0-6 females. Other findings did not relate to the test substance treatment: focal mononuclear of miscellaneous infiltration in liver of 2-2 females, mild hemosiderosis in spleen of 4-4 females, cysts in thymus of 3-1 females, endometrial cyst in uterus of 1-1 females, focal glial infiltration in brain of 2-0 females. Hydrometra of uterus (non-pathological finding) related to the oestrous cycle – in 2-2 females.

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Details on results:

The test substance caused only insignificant decrease of body weight in treated males and females of all dose levels. Most of observed significant changes of absolute and/or relative weights of organs (prostate gland with seminal vesicles and thymus in males, spleen in females) were considered unrelated to the test substance treatment due to absence of treatment related macroscopic or microscopic changes of structure of these organs. Vice versa increase of relative weight of kidneys especially in males is probably related to the test substance administration. At the middle and the highest dose level the kidney weight increase was accompanied by histological findings. The reversible influence of the test substance treatment on urinary system and urine properties was confirmed during urinalysis and examination of water consumption. Urine volume was evidently decreased in treated males of the highest dose levels, colour of urine was changed at all dose levels and increased presence of protein and leucocytes in urine was recorded at the highest dose level. Water consumption was increased in both sexes (in males the difference against control was marked). Histopathological examination of kidneys revealed presence of brown pigment in tubular epithelium in males as well as in females (middle and the highest dose level). In females brown pigment occurred there even at the end of recovery period. Histolopathological examination of intestines, rectum and paraaortal lymph nodes of males and females at the middle and the highest dose level demonstrated presence of brown pigment too. In mesenteric lymph nodes macrophages containing the pigment were noted also at the lowest dose level. Accumulation of pigment (residues of the test substance) in above mentioned organs persisted also after ending of the test substance treatment, it was observed in satellite animals. Presence of pigment in organs of digestive system was accompanied by microscopical inflammatory changes – infiltrations (mostly polynuclear) and/or oedema of mucosa eventually of submucosa. Clinical examinations of treated parental males and females and pups detected no serious clinical symptoms of toxicity of the test substance. Soft consistence of faeces, faeces and urine coloured by the test substance were found out in treated males and females of all dose levels. The behaviour, health condition, clinical status and activity of treated animals were similar compared to control and no serious changes were found out. Changes related to the colouration of the test substance were observed also during pathological examination of males and females of the middle and the highest dose level (and sporadically in females of the lowest dose level): coloured content of stomach and/or intestines, change of colour of mesenteric lymph nodes and eventually paraaortal and submandibular nodes. Discoloration of mesenteric lymph nodes and eventually submandibular lymph nodes persisted in satellite males and females. Haematological examination showed toxicologically significant increase of total leucocyte count in males of the middle and the highest dose levels. The values were out of historical control range and this leucocytosis could be probably related to microscopical inflammatory changes in digestive system organs. Other changes of haematological parameters (decrease of total erythrocyte count in males of the highest dose level, increase of fibrinogen concentration in females of the lowest and the highest dose level) were regarded as adaptive changes due to their reversible pattern and/or lack of clear dose-response relationship and because the values fit well into the limits of historical control.Clinical biochemistry showed treatment related changes. Total protein and albumin were reversibly decreased in both sexes of the middle and the highest dose level. The concentrations were changed in a dose dependent manner and they were lower than historical control values. Decreased blood concentrations of these parameters probably were probably caused by increased renal excretion of proteins which was confirmed during urinalysis. Other significant changes of biochemical parameters (decrease of ALP activity, urea and creatinine concentration in males) were considered to be of no toxicological significance, the values of these parameters were in historical control range.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

The tables are enclosed in the attached document.

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES and FEMALES was established as 125 mg/kg body weight/day. The value of NOAEL was proposed based on the following findings detected at the dose levels 500 and 1000 mg/kg/day: microscopical changes in organs of digestive system, lymph nodes and kidneys, results of clinical biochemistry – decrease of albumin and total protein blood concentration, results of haematology – increase of total leucocyte count and results of urinalysis – increased presence of leucocytes and proteins in urine. Very sporadic or sporadic microscopical changes detected in mesenteric lymph nodes at the dose level 125 mg/kg/day were not accompanied by other toxicologically significant results.

Executive summary:

The test substance, Direct Black 112, 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 125, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 12th day of lactation,

males – after mating period – totally for 49 days,

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

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

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

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 to rats by gavage did not cause any death at any dose levels.

The test substance did not significantly influence the growth of treated animals. Body weights and body weight increments were insignificantly decreased in treated males and females and this change was reversible only in females.  

The test substance treatment did not produce changes detected in functional observation of animals. Changes related to the test substance (faeces and urine coloured by the test substance, soft consistency of faeces) were found out during health condition control, clinical observations and detailed clinical examination in males and females of the dose levels 125, 500 and 1000 mg/kg/day. 

Water consumption was reversibly increased at the dose level 1000 mg/kg/day in both sexes especially in males. Urinalysis revealed the influence of the test substance treatment on urine volume in males of the dose level 1000 mg/kg/day (significant difference). The difference was reversible. Colour of urine was changed in treated males of all dose levels, markedly at the dose levels 500 and 1000 mg/kg/day. In urine of males at the dose level 1000 mg/kg increased occurrence of proteins and leucocytes was confirmed. The above mentioned changes of water consumption and urine properties are considered as treatment related.

Haematological examination of males revealed the following statistically significant and reversible changes of haematological parameters: increase of total leucocyte count in males of the dose level 500 mg/kg/day and decrease of total erythrocyte count in males of the dose level 1000 mg/kg/day (without dose dependence). Total leucocyte count was insignificantly increased also at the dose level 1000 mg/kg/day and the values of WBC at the dose levels 500 and 1000 mg/kg/day were higher than historical control values. The value of total erythrocyte count at the dose level 1000 mg/kg/day was in range of historical control. The change of WBC was probably connected with the test substance treatment. Haematological examination of females demonstrated only statistically significant increase of fibrinogen concentration at the dose levels 125 and 1000 mg/kg/day. Fibrinogen was changed dose-independently and the values were in range of historical control.

During biochemical examination of males statistically significant changes of some biochemical parameters were detected: dose dependent decrease of total protein and albumin concentration and decrease of alkaline phosphatase activity at the dose levels 500 and 1000 mg/kg/day, dose independent decrease of creatinine concentration at the dose levels 125 and 1000 mg/kg/day and decrease of urea concentration at the dose level 1000 mg/kg/day. All above mentioned parameters exc. creatinine were changed reversibly. The values of total protein at the dose level 1000 mg/kg/day and albumin at the dose level 500 and 1000 mg/kg/day were lower than historical control values. Statistically significant, reversible and dose dependent decrease of albumin and total protein concentration was recorded also in females and the values were lower than historical control values. The differences of albumin and total protein concentration in both sexes were considered as the test substance treatment related.

Reversible and dose-independent decrease of absolute weight of prostate gland with seminal vesicles was found out in males of the dose levels 500 and 1000 mg/kg/day. Relative weight of prostate gland with seminal vesicles was only insignificantly decreased at the dose level 500 mg/kg/day. Significant changes of relative weights were recorded in the following organs of males: thymus – increase at the dose level 500 mg/kg/day and kidneys – increase at the dose levels 125 and 1000 mg/kg/day. Relative weight of kidneys was insignificantly increased also at the dose level 500 mg/kg/day. This change of kidneys weight was probably treatment related even if the dose dependence was not confirmed. In females only statistically significant increase of absolute and relative weight of spleen was noted at the end of recovery period. Weights of kidneys in treated females were insignificantly and dose dependently increased compared to control.

During pathological examination changes related to the test substance administration were observed in males of the dose levels 500 and 1000 mg/kg/day and in females of all dose levels (sporadically at the dose level 125 mg/kg/day): coloured content of stomach and/or intestines, change of colour of mesenteric lymph nodes and eventually paraaortal and submandibular nodes. Changed colour of urine occurred only in males. Discoloration of mesenteric lymph nodes and eventually submandibular lymph nodes persisted in satellite males and females.

Histological changes related to the test substance treatment were recorded in organs of males and females. Irreversible presence of brown pigment (of various intensity) was detected in mucosa of intestines, rectum, in mesenteric and paraaortal lymph nodes, in tubular epithelium of kidneys, reversible infiltrations (mostly polynuclear) eventually oedema in forestomach, stomach, intestines and rectum of males and females at the dose levels 500 and 1000 mg/kg/day. In animals of the dose level 125 mg/kg/day brown pigment occurred only in mesenteric lymph nodes.