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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted by the Council on the 29th July 2016

Deviations:

yes

Remarks:

see Any other information on materials and methods

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, - Age at study initiation: 9 - 11 weeks- Weight at study initiation: cca 400 g (males), cca 252 g (females)- Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage- Diet (e.g. ad libitum): maintenance pelleted diet for rats and mice (Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany)- Water (e.g. ad libitum): drinking water ad libitum- Acclimation period: 5 days (dose-range finding experiment), 13 days (main study)ENVIRONMENTAL CONDITIONS- Temperature: 22 ± 3°C- Humidity: 30 - 70 %- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on exposure:

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

Details on mating procedure:

Animals were mated from the 29th day of study. Mating 1:1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

The animals were treated 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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF EXPERIMENTAL ANIMALS:During the acclimatisation period, the health condition of all animals was controlled daily. Normal course of the oestrus cycle of all females was controlled during 14 days before start of application. Females with abnormal oestrus cycle were removed from mating. Then the animals were randomly divided into the control and test groups and they were marked individually. MATING PROCEDURE:Animals were mated from the 29th day of study. Mating 1:1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.EXPERIMENTAL DATA COLLECTIONHealth condition control: daily - during the acclimatization and the experimental part Body weight: males and satellite animals - the first day of administration and then weekly, females - the first day of administration and then weekly, during pregnancy: 0., 7th, 14th, 20th day during lactation: 1st, 4th, 12th and 13th day pups (litters) - 1st, 4th, 12th and 13th day pups – individually – 4th day of lactationFood consumption: weekly and on the same days as body weight (except the mating period) satellite males and females – weeklyWater consumption: satellite males and females – twice a weekClinical observations: males and females - daily during the administration period pups - as soon as possible after delivery and then dailyMortality control: twice dailyDetailed clinical observation: before the first application and then weekly (except the mating period) Functional observation: at the end of administration/observation period Laboratory examinations: - vaginal smears: daily – 1st – 14th day of study; daily in mating period (max. 14 days); on necropsy day- urinalysis: the last day of administration/observation period – only males- haematology: males – after the end of application period parental females – on the 13th day of lactation satellite animals – after the end of observation period- biochemistry: males and nonpregnant females – after the end of application period 2 pups per litter - on the 4th day of lactation parental females and pups – on the 13th day of lactation satellite animals – after the end of observation period- anogenital distance measurement: pups – 4th day of lactation- pathological examination: males and nonpregnant females – after the end of application period 2 pups per litter - on the 4th day of lactation parental females and pups – on the 13th day of lactation satellite animals – after the end of observation period- weight of organs: during necropsy- sperm observation: parental males – during and after necropsy (not performed in satellite males) - histopathological examination: after necropsyMETHODS OF INVESTIGATION - Body WeightThe body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too. Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period. - Food ConsumptionIn a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period. - Food Conversion Food conversion in % (weight increment/food consumption x 100) was calculated for animals of Repeated Dose Toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females. - Water Consumption The drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study. - Mortality ControlAll rats during the treatment periods were examined for vitality or mortality twice daily. - Health Condition Control All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before application, during application and immediately after application. - Clinical Observations of Males and FemalesAll rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.m.). Animals were observed in natural conditions in their cages. - Clinical Observation of PupsAll pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded. - Detailed Clinical Observation This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, breathing, tonic or clonic movements, stereotypes or bizarre behaviour. The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina. - Functional Observation This observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover, the individual observations of grip strength were performed using grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons. - Laboratory examinations Examination of Vaginal SmearsVaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of oestrous cycle of females. Only females with regular cyclicity were put into the study. Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa. Vaginal smears have been made also on necropsy day to determine the stage of oestrous cycle. UrinalysisThis examination was performed only in 6 males of each group and in satellite males. In females, this examination was not performed (dams should not be removed from the pups for long time). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered by 2 mL of drinking water for 100 g of body weight by gavage to the stomach. Haematological ExaminationThis examination was performed only in 6 males and 6 females of each group and in satellite males and females. The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation system. Biochemical ExaminationThis examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.Blood samples from the day 13 pups and the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total). Treatment related changes of T4 total blood serum levels, of thyroid gland weight and microscopic structure were not observed in the day 13 pups therefore assessment of blood serum level of T4 total was not performed in day 4 pups. Anogenital Distance (AGD) MeasurementThe AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalised to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight. Nipples ExaminationThe presence and number of nipples in male pups were counted on day 13 of lactation. Pathological ExaminationDuring the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative. Observation of SpermIn all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm MotilitySperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm. Sperm MorphologySperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck ¬– were recorded. Biometry of OrgansAt the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation. Histopathological Examination Samples of the following tissues and organs were collected at necropsy and fixed:pituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland + seminal vesicles, testes, all gross lesions, thyroid gland, adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord – thoracic, spleen, stomach, thymus, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lLymph nodes – mesenteric, paraaortal, oesophagus, eyeThe mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used. In 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 macroscopic 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).

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: weekly BODY WEIGHT: Yes - Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

Oestrous cycles were monitored before the beginning of treatment to select females with regular cyclicity for the study.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS- Performed on day 4 postpartum: no PARAMETERS EXAMINEDThe following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, pup weight, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples in male pups GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE - Male animals: After the end of administration period the male animals of main groups were sacrificed, satellite animals were observed for the next 14 days without treatment. - Maternal animals: Parental females: day 12 post partum Satellite females: 78th day Non-pregnant females (without evidence of copulation): 25 days after the end of mating period Non-pregnant females (with evidence of copulation): 25th day after confirmed mating GROSS NECROPSY - Revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. HISTOPATHOLOGY / ORGAN WEIGHTSPituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland and seminal vesicles, testes, thyroid gland and all gross lesions were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE2 pups per litter – 4th day of lactation; other pups - 13th day of lactation GROSS NECROPSYMicroscopical evaluation of thyroid gland of pups, evaluation of prenatal and early postnatal development and growth of pups.ORGAN WEIGTHSThyroid gland weight.

Statistics:

For 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.In general: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 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.

Clinical signs:

no effects observed

Description (incidence and severity):

MalesNo clinical changes were recorded in control and treated males during the application period. Only changes related to the colour of the test substance – coloured faeces were recorded at the dose level 500 mg/kg/day from the 29th day of study and at the dose level 1000 mg/kg/day from the 2nd day of application to the end of administration period.FemalesAt all control and treated females, no clinical changes were recorded during the whole study. Only changes related to the colour of the test substance – coloured faeces were recorded at the dose level 500 mg/kg/day from the 29th day of study and at the dose level 1000 mg/kg/day from the 2nd day of application to the end of administration period.

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 was relatively well-balanced in comparison with control males. Statistical analysis was performed for necropsy body weight. Statistically significant differences were not found. Body weight increment of all treated males was not adversely affected by the test substance treatment. FemalesPre-mating period: Mean body weight of treated females was similar compared to the control group. Body weight increments were variable within the 1st and 2nd week of application. Negative body weight increment during the 1st week of application was recorded in control females. Pregnancy: Females without parturition (non-pregnant females) were not included in the evaluation of mean body weight increments during pregnancy. Mean body weight of all treated groups was comparable to the control group during whole pregnancy. The mean body weight increments of all treated pregnant females were similar to the control females. Lactation: Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The body weight of females was similar with control females during the rest of lactation period. The mean body weight increments of treated mothers were comparable to control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MalesFood consumption of treated males was relatively well-balanced in comparison with control males.FemalesPre-mating period: The mean food consumption of treated females was similar to control in 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 during the lactation period were quite well balanced compared to control mothers.

Food efficiency:

not specified

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 concentration of T4 hormone at the dose level 1000 mg/kg/day was insignificantly decreased in comparison with control group. Concentration of other dosed groups 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 mainly for high dose and control animals. Because of macroscopic findings in males at the lowest and the middle dose levels the histopathological examination of macroscopically changed organs was performed in one male (kidneys) at the lowest dose level and in two males (kidneys and testes) at the middle dose level.The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-250-500-1000 mg/kg/day groups further in the text. In 5-0-1-7 males no histological findings were diagnosed.In reproductive organs these findings were observed: unilateral or bilateral atrophy of tubules (1-/-1-1) and mild unilateral spermatogranuloma (0-/-0-1) in the testes, slight focal chronic inflammation of prostate gland in 5-/-/-1 males.In the kidneys were revealed mild hydronephrosis (3-0-0-3), solitary hyaline casts (1-0-0-0) and glomerulonephropathy (0-1-0-0).Then only sporadic findings which were not connected with test substance treatment were found out.These all histopathological findings did not relate to the test substance treatment. Microscopic 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. FemalesFull histopathology of the preserved organs and tissues was performed for high dose and control animals. The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-1000 mg/kg/day groups further in the text. In 1-0 female no histological findings were diagnosed.In reproductive organs findings related with previous pregnancy and delivery of pups were observed: accumulation of lipophages and siderophages in mesometrium of uterus (10-11), hemosiderin in mucosa of uterus (11-11) and lobular hyperplasia of mammary gland (6- 6). In uterus was detected placental site trophoblastic tumor (0-1).In other organs these sporadically findings were detected: hydronephrosis in the kidneys (0-1) and cyst in the thymus (2-0). Microscopic examination of reproductive organs, thyroid gland and pituitary gland did not reveal presence of treatment related changes.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

no effects observed

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.Increased percentage of affected sperms was not recorded during sperm morphology examination at any dose level. 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):

The values of mating indexes showed that mating was not negatively affected by the test substance treatment. Fertility indexes were higher in animals at the dose level 500 mg/kg/day; in other treated groups was similar or lower compared to the control group.Gestation index was not changed in treated groups compared to control. Post-implantation losses were increased at the dose levels 250 and 500 mg/kg/day compared to the control. Post-natal losses were slightly higher at the dose level 500 and 1000 mg/kg/day compared to the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect related to the test substance observed

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Only two cases of cannibalism were recorded.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean weight of litter within all weighing intervals was not statistically significantly changed in treated groups compared to control. An increase in mean litter weight was recorded at the dose level 1000 mg/kg/day during all weighing intervals.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 were recorded in pups from treated groups against control pups. All values were within the range of historical control.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid gland weight:No significant differences were recorded in pups from treated groups in comparison with the control pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination was performed in all pups (except cannibalism). Macroscopic findings were observed sporadically and did not related to the test substance administration.Control: 150 pups were examined. No macroscopical findings were recorded. 250 mg/kg/day: 131 pups were examined. In 2 stillborn pups from 2 litters non-aerial lungs were found out. No macroscopical findings were recorded in others pups.500 mg/kg/day: 152 pups were examined. No macroscopical findings were recorded. One pup was not examined due to cannibalism. 1000 mg/kg/day: 160 pups were examined. No macroscopical findings were recorded. One pup was not examined due to cannibalism.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Number and sex ratio of pups were not significantly affected by the test substance administration. No differences in postnatal developmental were observed in pups at the treated groups – presence of nipples in male pups was not recorded and anogenital distance in treated male and female pups in comparison with the control pups was similar. Concentration of thyroxine hormone T4 in day 13 pups from the treated groups was similar to the control group.Microscopical evaluation of thyroid gland of pups did not show any findings related to the test substance treatment. Also evaluation of prenatal and early postnatal development and growth of pups did not show any adverse effect of the test substance treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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 related to the test substance observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Tables are listed in attached document.

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, Reactive Orange 13, was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on the 29th July 2016. Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment (see the Annex 2) and approved by Sponsor.

The treated groups were administered daily for the following periods:

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

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

- males – after mating period – totally for 49 days,

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

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

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

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

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

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

The number of females achieving pregnancy, sperm parameters and microscopical structure of reproductive organs in both parental males and females seem to be not affected by the test substance administration.

Motility of sperms was not significantly changed in treated males compared to control. Increased percentage of affected sperms was not recorded during sperm morphology examinationat any dose level. Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.

All parental males were assessed for serum levels of thyroid hormone thyroxine (T4). Statistically insignificant decrease of the concentration of hormone was recorded in males at the dose level 1000 mg/kg/day.

Statistically significant changes of absolute and relative weights of genital organs in males and females were not recorded.

Number and sex ratio of pups were not significantly affected by the test substance administration. No differences in postnatal developmental were observed in pups at the treated groups – presence of nipples in male pups was not recorded and anogenital distance in treated male and female pups in comparison with the control pups was similar. Concentration of thyroxine hormone T4 in day 13 pups from the treated groups was similar to the control group.

Microscopical evaluation of thyroid gland of pups did not show any findings related to the test substance treatment. Also evaluation of prenatal and early postnatal development and growth of pups did not show any adverse effect of the test substance treatment.

Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. The ability of parental animals to successfully mate, to achieve pregnancy and to give birth live pups were not influenced by the test substance administration.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on evaluation of the results of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the test substance, Reactive Orange 13, did not cause any adverse effects that could be considered as a symptom of reproduction or developmental toxicity. NOAEL was established at 1000 mg/kg/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. According to these findings, the test substance need not be classify for reproductive or developmental toxicity according to Regulation (EC) No. 1272/2008.