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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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:

yes

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 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.

Details on mating procedure:

- M/F ratio per cage: 1 male + 1 female per cage- Length of cohabitation: max. 14 days- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy- Further matings after two unsuccessful attempts: no- After successful mating each pregnant female was caged: individually

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).

Details on study schedule:

- Parental males (totally 49 days of administration):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 43rd day – 63rd day (administration period) → 64th day (necropsy) - Satellite males (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy) - Parental females:1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration)→ gestation → lactation → day 12 post partum - Satellite females (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy) - Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25 days after the end of mating period - Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25th day after confirmed mating

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

Parental animals: Observations and examinations:

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 only

Oestrous cyclicity (parental animals):

Oestrous cycles were monitored before the beginning of treatment to select females with regular cyclicity for the study. Vaginal smears of all females were monitored daily for two weeks.

Sperm parameters (parental animals):

Parameters examined in male parental generations: epididymis weight, prostate gland with seminal vesicles weight, testes weight, pituitary gland weight, thyroid gland weight, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS- Performed on day 4 postpartum: no PARAMETERS EXAMINEDThe following parameters were examined in offspring: number and sex of pups, body weight, stillbirths, live births, postnatal mortality, anogenital distance (AGD), presence of nipples in male pups, presence of gross anomalies, physical or behavioural abnormalities, T4 hormon concentration, thyroid gland weight GROSS EXAMINATION OF DEAD PUPS:yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE - Male animals: All surviving animals (49th day of administration, satellite males 63rd day of administration period) - Maternal animals: All surviving animals (12th day post partum) GROSS NECROPSY - Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. HISTOPATHOLOGY / ORGAN WEIGHTS The tissues indicated in tables were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE - The offspring were sacrificed at 4th or 13th day of lactation. - These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: GROSS NECROPSY - Gross necropsy consisted of macroscopic examination. HISTOPATHOLOGY / ORGAN WEIGTHS Thyroid glands were weighed.

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).

Reproductive indices:

See Fertility parameters table in attached tables.

Offspring viability indices:

See Reproduction data table in attached tables.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

MalesIn control males and treated males of all dose levels no signs of diseases were recorded during the application period. Only changes related to the colour of the test substance – coloured faeces were recorded at the dose level 1000 mg/kg/day from the 7th day of application to the end of administration period.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).FemalesIn control females and treated females of all dose levels no signs of diseases were recorded during the application period. Only changes related to the colour of the test substance – coloured faeces were recorded at the dose level 1000 mg/kg/day: from the 7th day of application to the end of administration period.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).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the whole study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

MalesDifferent body weight on the 1st day of administration was caused by sequential including of animal groups to the study. Body weight of treated males at all dose levels was insignificantly decreased throughout the whole administration period. The difference was dependent on dose level from the 4th week of the study to the end of study. Body weight increments were slightly decreased in treated males or similar in control and treated males.FemalesStatistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Pre-mating period: Different body weight on the 1st day of administration was caused by sequential including of animal groups to the study. Body weights of treated females were slightly decreased in pre-mating period compared to control (without dose dependence). Weight increments were quite balanced in control and treated females.Pregnancy: Mean body weight and body weight increments on the day 7, 14 and 20 of pregnancy was slightly decreased in females of the dose level 500 mg/kg/day in comparison with control. Body weight and weight increments of females of the dose levels 125 and 1000 mg/kg/day were not markedly changed compared to control.Lactation: Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. Mean body weights and weight increments of mothers at the dose level 1000 mg/kg/day were slightly decreased at the end of lactation period (on the 12th and 13th day). Body weight and weight increments of mothers at the dose levels 125 and 500 mg/kg/day were comparable to control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MaleIn the 1st week of the study food consumption of males of the dose level 1000 mg/kg/day was evidently decreased against control and then no marked differences were observed between treated and control males.FemalePre-mating period: The mean food consumption of treated females was decreased in the 1st week of pre-mating period. Pregnancy: Non-pregnant females were not included in the evaluation of food consumption during pregnancy. The mean food consumption of pregnant females treated by the test substance was similar to control group. Lactation: Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period. The mean food consumptions of treated mothers were slightly higher at the dose levels 500 mg/kg/day (from the 1st to the 4th day) and 1000 mg/kg/day (throughout the lactation period) in comparison with control.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Males onlyBlood samples from all adult parental males were assessed for serum levels for thyroid hormone thyroxine (T4 total). Mean concentrations of T4 hormone at all dose levels were similar to the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

MalesFull histopathology of the preserved organs and tissues was performed for high dose and control animals. Because of probably treatment related changes at the highest dose level and some macroscopical findings at the lowest and the middle dose level the histopathological examination of macroscopically changed organs (all males) and then of kidneys, stomach, forestomach, intestines, rectum and mesenteric lymph nodes (only males of Repeated Dose Toxicity part of study) was performed at the middle and the lowest dose level. The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-125-500-1000 mg/kg/day further in the text. Microscopical examination of reproductive organs, thyroid and pituitary gland did not reveal presence of treatment related changes, only the spontaneous changes were found in both control and treated animals. Tubular degeneration or/and atrophy was diagnosed in testes of 4-/-/-5 males. Retention of sporadic spermatids (maximally in 1 % of tubules) was detected in testes of 0-/-/-2 males. Germ cells in tubules and spermatic granuloma occurred in epididymis of 1-/-/-1 males. In prostate gland of 4-/-/-4 males chronic inflammation was observed. Microscopical findings detected in other organs are described in Repeated dose toxicity part of study. Insignificant and very sporadic changes are mentioned only in individual tables FemalesFull histopathology of the preserved organs and tissues was performed for high dose and control animals. Because of probably treatment related changes at the highest dose level and some macroscopical findings at the lowest and the middle dose level the histopathological examination of macroscopically changed organs (all females) and then of kidneys, intestines, stomach, forestomach, mesenteric lymph nodes and rectum (only in females of Repeated Dose Toxicity part of study) was performed at the middle and the lowest dose level. The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-125-500-1000 mg/kg/day further in the text. Microscopic examination of reproductive organs, thyroid gland and pituitary gland did not reveal presence of treatment related changes. The changes related to previous pregnancy were found in both control and treated females: accumulation of siderophages in mesometrium and/or endometrium of uterus in 7-/-/-10 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 2-3-1-0 females. Follicular cysts were described in ovaries of 3-/-/-0 females. Microscopical findings detected in other organs are described in Repeated Dose Toxicity part of study.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

Oestrous cycles were monitored before the beginning of treatment to select females with regular cyclicity for the study. Vaginal smears of all females were monitored daily for two weeks.Four females were placed into the satellite group for irregular oestrus cycle.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Motility of sperms was not significantly changed in treated males compared to control.Slightly increased percentage of affected sperms was recorded during sperm morphology examination of treated males (without dose dependence). Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.

Reproductive performance:

no effects observed

Description (incidence and severity):

Evidence of copulation was found out in all females of the dose levels 125 and 1000 mg/kg/day. In one control female and one female of the dose level 500 mg/kg/day copulation was not confirmed. No treatment related changes of mean duration of mating was observed in treated groups. Number of females achieving pregnancy was not influenced by the test substance treatment. The highest number of pregnant females was recorded at the dose level 1000 mg/kg/day. All pregnant females gave birth live pups. No abortion was recorded. Number of females bearing live pups was not influenced by the test substance treatment. The highest number of pregnant females was recorded at the dose level 1000 mg/kg/day. Mean duration of pregnancy was similar in treated and control groups. Mean number of corpora lutea, number of implantations and number of live born pups were not significantly changed in treated females. Slight decrease of number of corpora lutea and implantations was noted at the dose level 500 mg/kg/day. Sex ratio was well-balanced in the control as well as in treated groups. Number of stillborn pups was not increased in mothers of treated groups. Mean weights of litter at birth, at postnatal day 4 and 13 within were insignificantly changed in treated groups compared to control. The differences were not dependent on dose level. Mean pup body weights of treated and control groups were quite balanced during the whole lactation period. Measurement of an anogenital distance in pups showed unaffected difference between males and females and was similar in all groups. No difference in pup weight at the time of anogenital distance measurement was recorded among the groups. No treatment-related findings were observed in pups at macroscopical examination.The values of mating indexes showed that mating was not negatively affected by the test substance treatment. Fertility indexes were higher in animals of the dose level 1000 mg/kg/day. Gestation index was not changed in treated groups compared to control. No treatment related changes of pre-implantation, post-implantation and postnatal losses were recorded in treated groups.

Effect levels (P0)

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 observed at the highest dose

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean weight of litter within all weighing intervals was insignificantly changed in treated groups compared to control. The difference was not dependent on dose level. Mean pup body weights of treated and control groups were quite balanced during the whole lactation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Blood samples from the 13 day old pups were assessed for serum levels of thyroid hormone (T4). Pup blood was pooled by litter. No statistically significant differences of T4 value were recorded in pups from treated groups. Hormone concentrations in pups from treated groups were similar to control.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Negligible differences were recorded in thyroid gland weight of pups between control and treated groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination was performed in all pups (except cannibalism). Macroscopical findings were observed sporadically and did not related to the test substance administration.Control: 92 pups were examined. Five pups were not examined due to cannibalism. Flatulence of stomach and stomach without milk was observed in 2 female pups and 3 male pups. Flatulence of stomach was detected also in one stillborn pup.125 mg/kg/day: 106 pups were examined. Two pups were not examined due to cannibalism. No macroscopical findings were recorded.500 mg/kg/day: 93 pups were examined. No macroscopical findings were recorded.1000 mg/kg: 129 pups were examined. Two pups were not examined due to cannibalism. In one died male pup only autolysis of organs was found out. No macroscopical findings were recorded in other pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

NUMBER AND SEX RATIO OF PUPSThe total numbers of live pups at first check of litter after parturition, on the 4th day and 13th day of lactation were not influenced by the test substance treatment. Total number of live pups in mothers of the dose level 1000 mg/kg/day was markedly higher than in the control group (due to higher number of mothers at this dose level). Negligible differences were recorded in mean numbers of pups between control and treated groups.Sex ratio was not changed: number of female pups was similar to number of male pups in litters of control group and treated groups.DEVELOPMENT OF PUPSThe presence and number of nipples in male pups were counted on day 13 of lactation - the presence of nipples in male pups was not recorded.The anogenital distance (AGD) of each pup was measured on 4th of lactation. No statistically significant differences between control and treated groups were recorded in values of corrected anogenital distance. The values of pups from treated groups were similar to control.No differences in postnatal development of pups were observed at the control and treated groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

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 observed at the highest dose

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Tables are listed in attached document.

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg body weight/day. All changes observed in parental males and females and in their offspring at all dose levels were considered to be of no toxicological significance.

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 (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.

The test substance treatment did not significantly influence physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period). Body weights and body weight increments were insignificantly decreased in treated males and females.  

Significant changes of reproductive organs weights were found out only in males: absolute weight of prostate gland with seminal vesicles was significantly decreased in males of the dose levels 125, 500 and 1000 mg/kg/day (without dose dependence). Relative weight of prostate gland with seminal vesicles was decreased only insignificantly and dose independently. Absolute weight of thyroid gland was significantly decreased in males at the dose level 500 mg/kg/day.

Examination of sperm motility and morphology in parental males did not reveal adverse effect of the test substance treatment on sperm quality.

Numbers of corpora lutea, implantations and pups were not influenced by the test substance administration at any dose level too. No adverse effect of the test substance treatment was observed during examination of thyroxine hormone blood concentration in parental males not even during pathological and histopathological examination of reproductive organs in treated parental animals. Also evaluation of body weight of pups, development of pups, blood concentration of thyroxine hormone in pups and macroscopic examination of pups did not manifest any adverse effect of the test substance treatment at any dose level.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg body weight/day. All changes observed in parental males and females and in their offspring at all dose levels were considered to be of no toxicological significance.