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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1.3. 2016-1.3. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

219-708-8

EC Name:

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

Cas Number:

2503-73-3

Molecular formula:

C42H29N7O13S4.4Na

IUPAC Name:

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

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

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

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

TEST ANIMALS-Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500- Age at study initiation: sexually adult (9 weeks on arrival)- Fasting period before study: no- Housing: All the study proceeded in SPF (Specified Pathogen Free) animal house of CETA in SPF conditions (according to internal SOP No. 12).- Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satelliteanimals - 2 rats of the same sex in one cage- Diet: ad libitum, maintenance pelleted diet for rats and mice - Altromin for rats/mice, Manufacturer:Altromin Spezialfutter GmbH & Co. KG, Germany- Water: ad libitum- Acclimation period: 6 days, During the acclimatisation period the health condition of all animals was controlled daily.Then the animals were randomly divided into the control and test groups and they weremarked individually.- Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight- Identification of animals: the animals have been identified by the colour marks on their fur, each cage was marked with the number of animals, sex, number of cage, name and dose level of the test substanceENVIRONMENTAL CONDITIONSTemperature (°C): 22±3°C- Humidity (%): 30-70%- Air changes (per hr): 15 air- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Sex:

male/female

Details on test animals or test system and environmental conditions:

STUDY TIME SCHEDULETest substance delivery: 09. 12. 2015Determination of stability and homogeneity: 01. 03. – 04. 03. 2016Dose-range finding experiment: 13. 04. – 10. 05. 2016Main Study:Animal arrival: 17. 08. 2016Acclimatisation: 6 daysPre-exposure period: 23. 08. – 05. 09. 2016Administration: from 06. 09. 2016AdministrationParental 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) → 64thday (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 partumSatellite 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 periodNon-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 matingUrine collection: only males – 63rd and 77th day of studyBlood collection for haematology and biochemistry:parental males – 64th day of studysatellite males – 78th day of studyparental females – 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation2 pups per litter – 13th day of lactationsatellite females – 78th day of studyNecropsy: parental males – 64th day of studysatellite males – 78th day of studyparental females – 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation, other pups - 13th day of lactationsatellite females – 78th day of studynon-pregnant females – 26th day after the end of mating period or confirmed matingHistopathological examination: 02. 01. – 01. 03. 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The application form for analysis was prepared in the same manner as for application to animals –i.e. solution in water for injection.Concentration Level 10 mg/10mLCa 0.1 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer (500 rpm) for 10 minutes.Concentration Level 1000 mg/10 mLCa 10 g of the test substance was weighed with wider end of glass Pasteur pipette into a 150 mLglass beaker calibrated to 100 mL, mixed using glass rod during of vehicle adding and the beakerwas replenished by the vehicle. The solution was stirred by magnetic stirrer (600 rpm) for 15 minutes,let to stand for min. 17 hours without mixing and then stirred by magnetic stirrer (900 rpm) for 45 minutes again.VEHICLE-Aqua pro iniectione (water for injection) Producer: Ardeapharma Ševětín a.s., Czech Republic- Batch No. and expiration: 1508310535, exp. 08/2017 (DRFE)1511090667, exp. 11/2017 (DRFE)1603110144, exp. 03/20181605100264, exp. 05/20181606130339, exp. 06/2018

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:

The stability and the homogeneity of application form were determined in CETA analytical laboratories (Analytical Group I).Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a high-performance liquid chromatography based on a method developed at the test facility.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days per week

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.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

The Dose-Range Finding Experiment was performed as it is described in Annex 2 of this final report. According to results of Dose-Range Finding Experiment the following dose levels – 250, 500 and 1000 mg/kg/day were chosen for the main test. The dose levels were approved by Sponsor.- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:

MORTALITY CONTROL- Time schedule: twice dailyHEALTH CONDITION CONTROL- Time schedule: daily - during the acclimatization and the experimental partCAGE SIDE OBSERVATIONS: Yes- Time schedule: males and females - daily during the administration periodThis observation was made in order to record possible clinical effects after application and all changesin behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cagesDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: males and females before the first application and then weekly- At the first part of observation the behaviour of animals in the cage was monitored: piloerection,posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.- The second part was the observation during the removal from cage: reaction to handling, elasticity ofskin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.BODY WEIGHT: Yes-males and satellite animals - the first day of administration and then weekly,females - the first day of administration and then weekly, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 4th day, 12th day and 13th daypups (litters) - 1st , 4th day, 12th day and 13th daypups – individually – 4th day of lactationFOOD CONSUMPTION- Time schedule: parental males - weekly (except the mating period)parental females - weekly during premating periodduring pregnancy and lactation – on the same days as body weightsatellite males and females – weeklyIn a specified day the remainder of pellets was weighed in each cage, the new food was weighed outand the food consumption for the previous week was computed.In males mean values were calculated for each week of the study (except of mating period in parentalmales). Food consumption for animal/day was calculated from mean values of each group.The same way of calculation of mean food consumption was used for females in premating period and for satellite females. In pregnancy and lactation period mean individual values (grams/animal/day)were calculated for each week of the study. Mean food consumption of parental females for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption of pregnant females.FOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: YesWATER CONSUMPTION: Yes- Time schedule for examinations: satellite males and females – twice or three times a week (the 1stand 2nd week – twice a week and from 3rd week to the end of study a week)- The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.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 periodExamination 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. Vaginal smears were prepared and examined according to internal SOP No. M/74.

Oestrous cyclicity (parental animals):

daily – 1st – 14th day of study daily in mating period (max. 14 days)on necropsy dayVaginal 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. Vaginal smears were prepared and examined according to internal SOP No. M/74.

Sperm parameters (parental animals):

In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/74 and M/82.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 mo rphology 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.

Litter observations:

CLINICAL OBSERVATIONAll 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 in the 1st day post-partum and in the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.BODY WEIGHTTime schedule: 1st, 3rd and 4th day

Postmortem examinations (parental animals):

SACRIFICE- Male animals:42th day of study- Maternal animals: day 3 post partumGROSS NECROPSY- During 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.HISTOPATHOLOGY / ORGAN WEIGHTSThe tissues indicated in Table [No.: 5] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

Macroscopic examination-all pups (except cannibalism)

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used.Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.

Reproductive indices:

Male mating index= (number of males with confirmed mating/number of males cohabited) x 100Female mating index=(number of sperm-positive females/number of females cohabited) x 100Male fertility index= (number of males impregnating a females/number of males cohabited) x 100Female fertility index= (number of pregnant females/number of sperm-positive females) x 100Gestation index=(number of females with live born pups/number of pregnant females) x 100Survival index=(number of live pups on day 4 post partum/number of pups born alive) x 100

Offspring viability indices:

Pre-implantation loss: Number of corpora lutea – number of implantationsPost-implantation loss: Number of implantations – number of live birthsPost-natal loss: Number of live births – number of alive at postnatal day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

MALESHealth Condition Control Males were not distributed into the groups during the check-in, acclimatisation and the pre-exposure period (monitoring of oestrous cycle in females). No signs of diseases were recorded during that time. In control males and treated males of all dose levels no signs of diseases were recorded during the whole study. Only changes related to the colour of the test substance – coloured faeces were recorded in treated males: from the 9th day of study at the dose level 500 mg/kg/day and from the 4th day of study at the dose level 500 mg/kg/day.Clinical ObservationNo clinical changes were recorded in control and treated males during the application period. Only changes related to the colour of the test substance – coloured faeces were recorded (at the dose level 500 mg/kg/day: from the 8th day of study, at the dose level 1000 mg/kg/day: from the 3rd day of study).FEMALESHealth Condition ControlFemales were not distributed into the groups during the check-in, acclimatisation and the pre-exposure period with monitoring of oestrous cycle. No signs of diseases were recorded during that time. In 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 500 mg/kg/day: from the 9th day of study, at the dose level 1000 mg/kg/day: from the 4th day of study).Clinical ObservationAt all control and treated females, no clinical changes were recorded during the whole study. Only changes related to the colour of the test substance – coloured faeces were recorded (at the dose level 500 mg/kg/day: from the 8th day of study, at the dose level 1000 mg/kg/day: from the 3rd day of study).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

MALES: There were no unscheduledd deaths during the whole study.FEMALES: One female from the highest dose level (No. 161) died on the 14th day of pregnancy. It was considered as probably accidental death without treatment relationship.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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 of the dose levels 250 and 1000 mg/kg/day at the end of the study was quite analogous to control group. Body weight of males of the dose level 500 mg/kg/day was slightly decreased compared to control males. Body weight increments were similar in control and treated males for the whole study.Statistically significant differences in necropsy body weight were not found in treated males. FEMALESPre-mating periodDifferent 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. Weight increments were quite balanced in control and treated females.PregnancyMean body weight of all treated groups was slightly decreased in comparison with control (without dose dependence). Weight increments of treated females and control females were quite balanced during pregnancy. LactationOnly mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. Mean body weight of all treated groups was slightly decreased in comparison with control (without dose dependence). 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):

MALESNo marked differences were observed between treated and control males.FEMALESPre-mating periodThe mean food consumption of treated females was similar to control in pre-mating period. PregnancyFemales without parturition (non-pregnant or aborted 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. LactationOnly 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 lower at the dose levels 250 and 1000 mg/kg/day in comparison with control.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological examination showed some significant changes of haemocoagulation parameters (shortened prothrombin time, irregular changes of fibrinogen concentration in males, prolonged prothrombin time and decrease of fibrinogen concentration in females) and red blood component (decrease of haemoglobin concentration in males and increase of mean corpuscular volume in females). The changes were regarded as adaptive changes due to their reversible pattern and/or lack of clear dose-response relationship and because the values fit well into the limits of historical control.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

MALESBlood samples from the all adult parental males were assessed for serum levels for thyroid hormone thyroxine (T4 total). Mean concentrations of T4 hormone at the dose levels 250 and 500 mg/kg/day were insignificantly decreased in comparison with control. Concentration of dose level 1000 mg/kg/day was similar to the control group. Statistically significant differences were not detected in treated males.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, 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 (kidneys) 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 (males No. 21 – 26 and 41 – 46) was performed at the middle and the lowest dose level.The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-250-500-1000 mg/kg/day further in the text. 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. Tubular degeneration or/and atrophy was diagnosed in testes of 4-/-/-4 males. In three control males degeneration or atrophy was observed only in sporadic tubules (to 3 %), only in one control male 100 % of tubules of one testis was affected. In males of the highest dose level degeneration or atrophy was detected maximum in 4 % of tubules. Germ cells in epididymal tubules occurred in 2-/-/-3 males and in prostate gland the following findings were observed: sporadic acinar atrophy in 4-/-/-4 males and focal chronic inflammation 2-/-/-2 males. Microscopical findings detected in other organs are described in Repeated dose toxicity part of study.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 (in kidneys) 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 (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 0-250-500-1000 mg/kg/day further in the text. In female spontaneously died on the 14th day of pregnancy beginning autolysis of reproductive organs was observed. 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 9-/-/-10 females. Dilatation of oviduct was described in 1-1-/-0 females. Microscopical findings detected in other organs are described in Repeated dose toxicity part of study.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Observation of SpermMotility 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):

Fertility ParametersThe values of mating indexes showed that mating was not negatively affected by the test substance treatment.Fertility indexes were lower in control and in animals of the dose level 250 mg/kg/day. Gestation index was slightly decreased only at the dose level 1000 mg/kg/day due to one female died during pregnancy. Evidence of copulation was found out in all females (till the 5th day of mating period). Slightly decreased number of females achieving pregnancy was recorded in control group and the lowest dose level. Nearly all pregnant females (exc. one female of the highest dose level -died on the 14th day of pregnancy) gave birth live pups. No abortion was recorded. Number of females bearing live pups was slightly decreased at control, the lowest and the highest dose level. At control and at the dose level 250 mg/kg/day it corresponded to the number of pregnant females. 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. Sex ratio was well-balanced at the lowest and the middle dose level, increased number of female pups was found out at control and at the highest dose level. Weight of litters at birth, at PND 4 (postnatal day) and PND 13 was slightly decreased at treated groups but the differences were not dependent on dose level. 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

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 biologically or statistically significiant changes.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant differences were not recorded.Mean weight of litter within all weighing intervals was insignificantly decreased 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

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical Examination – Hormone T4Blood 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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Weight of thyroid gland was statistically significantly increased in male and female pups of treated mothers (exc. male pups at the dose level 500 mg/kg/day). The difference was not dependent on dose level.

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: 173 pups were examined. One female pup was not examined due to cannibalism. No macroscopical findings were recorded in pups.250 mg/kg/day: 159 pups were examined – in one litter one pup without testis was found out. In one died pup marked flatulence of stomach and intestines was observed. No macroscopical findings were recorded in other pups.500 mg/kg/day: 174 pups were examined – in one male pup spleen was placed on the contrary side; stomach without milk was noted in one male pup and one female pup of one litter. Four pups of one litter died during early postnatal period, the following findings were diagnosed: oedema of lungs and anaemia of organs in one male pup; only partial cannibalism in two female pups; pale and autolysed organs and increased right eyeball in one female pup. No macroscopical findings were recorded in other pups. 1000 mg/kg: 167 pups were examined. One female pup was not examined due to cannibalism. In two female pups (of two various litters) stomach without milk was found out. No macroscopical findings were recorded in other pups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Evidence of copulation was found out in all females (till the 5th day of mating period). Slightly decreased number of females achieving pregnancy was recorded in control group and the lowest dose level. Nearly all pregnant females (exc. one female of the highest dose level -died on the 14th day of pregnancy) gave birth live pups. No abortion was recorded. Number of females bearing live pups was slightly decreased at control, the lowest and the highest dose level. At control and at the dose level 250 mg/kg/day it corresponded to the number of pregnant females. 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. Sex ratio was well-balanced at the lowest and the middle dose level, increased number of female pups was found out at control and at the highest dose level. Weight of litters at birth, at PND 4 (postnatal day) and PND 13 was slightly decreased at treated groups but the differences were not dependent on dose level. 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

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 biologically or statistically significant changes.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

yes

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:

Introduction

The test substance, Direct Blue 78, was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on the 29^thJuly 2016.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day).The dose levels for study were determined on the basis of results of adose-range finding experiment (see the Annex 2) and approved by Sponsor.

 

The treated groups were administered daily for the following periods:

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

pregnant females – during pregnancy and till the 12^thday of lactation,

males – after mating period – totally for 49 days,

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

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

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

 

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

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

Reproduction part of study:

The test substance treatment did not affect 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). 

 

Some significant changes of reproductive organs, pituitary and thyroid gland weights without relation to the test substance treatment were found out in males: decrease of absolute weight of prostate gland with seminal vesicles at the dose levels 250 and 500 mg/kg/day, decrease of absolute weight of pituitary gland at the dose levels 500 and 1000 mg/kg/day, decrease of relative weight of pituitary gland at the dose level 500 mg/kg/day, decrease of absolute weight of thyroid gland at all dose levels and decrease of relative weight of thyroid gland at the dose level 250 and 1000 mg/kg/day. Most of these changes were not dependent on dose level. In thyroid gland only absolute weight was changed in a dose dependent manner. In females only

thyroid gland weights were changed significantly (but without dose dependence): increase of absolute and relative weight at the dose level 250 mg/kg/day and relative weight at the dose level 500 mg/kg/day and decrease of absolute weight at the dose level 1000 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 treatment 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, concentration of thyroxine hormone in pups and macroscopical examination of pups did not reveal any influence 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.