Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulphonic acid, 3-[(4-amino-2-methoxyphenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.07.2016 – 19.10.2017

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 29th July 2016

Deviations:

yes

Remarks:

see Any other information ...

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 15-[(Z)-2-{4-[(E)-2-{4-[(E)-2-{2,5-dimethyl-4-[(E)-2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]-2-sulfophenyl}ethenyl]-3-sulfophenyl}diazen-1-yl]-10,12-dioxa-2,3-diaza-11-cupratricyclo[11.4.0.0^{4,9}]heptadeca-1(17),2,4,6,8,13,15-heptaene-6-sulfonate

EC Number:

943-210-6

Molecular formula:

Not applicable. UVCB Substance

IUPAC Name:

tetrasodium 15-[(Z)-2-{4-[(E)-2-{4-[(E)-2-{2,5-dimethyl-4-[(E)-2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]-2-sulfophenyl}ethenyl]-3-sulfophenyl}diazen-1-yl]-10,12-dioxa-2,3-diaza-11-cupratricyclo[11.4.0.0^{4,9}]heptadeca-1(17),2,4,6,8,13,15-heptaene-6-sulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: by Sponsor, Batch No. 8004/2008- Expiration date of the lot/batch: 02/2018- Storage condition of test material: The test substance was stored in dry and dark place at room temperature- Stability under test conditions:testing laboratory analysis of homogeneity and stability: _____________________________(.............the both application forms (1000 mg/10 mL and 10 mg /10 mL) of the test substance, Direct Blue 85, 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. .............)TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: see below Details on oral exposureFORM AS APPLIED IN THE TEST (if different from that of starting material)solution in water for injection

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL (SPF quality - guaranteed)

Details on species / strain selection:

- according to guideline- random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex did not exceed ± 20% of the mean weight

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, RČH CZ 11760500- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: sexually adult, 7-9 weeks on arrival- Weight at study initiation: males 401 - 428 g, females 252 - 264 g- Fasting period before study: no- Housing: SPF conditions according to internal SOP No.12; sterilized soft wood fibers Lignocel;Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – 1 M + 1 F 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): complete pelleted diet for rats and mice in SPF breeding - Altromin for Rats/Mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany- Water (e.g. ad libitum): ad libitum; quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law- Acclimation period: 12 days (DRFE 5 days)ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3- Humidity (%): 30-70- Air changes (per hr): approximately 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 /12STUDY TIME SCHEDULEAdministration (from 04.10.2016)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 ) → 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) → 64th 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)→ 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) → 64th 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) → 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) → 25th day after confirmed matingObservationsUrinalysis: only males – 63rd and 77th day of studyBlood collection for haematology and biochemistry: parental males – 64th day of study satellite males – 78th day of studyparental females - 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation; 2 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 matingEnd of histopathological examination: 19.10.2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

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 vehicle in ultrasonic bath for 30 min; the solution was stirred by magnetic stirrer (800 rpm) for 30 minutes. 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 two hours after preparation of application form.VEHICLEwater for injection; batch no.: 1606130339; exp. 06/2018- Concentration in vehicle:The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

Details on mating procedure:

Animals were mated from the 29th day of study.- M/F ratio per cage: 1:1- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

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.

Frequency of treatment:

7 days per week at the same time

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)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

(vehicle only)

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:

- Dose selection rationale: The dose levels for study were determined on the basis of results of a dose-range finding experiment.- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:

HEALTH CONDITION CONTROL: Yes- Time schedule: daily - during the acclimatization and the experimental partCLINICAL OBSERVATIONS: Yesmales and females - daily during the administration period in natural conditions in cagespups - as soon as possible after delivery and then dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: before the first application and then weekly (except the mating period)BODY WEIGHT: Yes- Time schedule for examinations:males - the first day of administration and then weekly,females - the first day of administration and then weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 4th, 12th and 13th day,satellite males and females - the first day of administration and then weekly.FOOD CONSUMPTION:- Food consumption determined and group mean daily diet consumption calculated as g food/animal/day: Yes- Time schedule: weekly and on the same days as body weight (except the mating period); satellite males and females – weeklyFOOD EFFICIENCY:- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weightedaverages from the consumption and body weight gain data: YesTime schedule: males - weekly (except the mating period), females - weekly during premating period,during pregnancy and lactation – on the same days as body weight, satellite males and females – weekly

Oestrous cyclicity (parental animals):

yes, before beginning of treatment; vaginal smears of all females were monitored daily for two weeks

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:- sperm motility, sperm morphology- in all males (except the satellite group)

Litter observations:

CLINICAL OBSERVATION OF PUPS- Time schedule:All 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.Anogenital distance measurement: 4th day of lactationNipples examination (the presence and number of nipples in male pups): counted on day 13 of lactation. BODY WEIGHTpups (litters) - 1st , 4th day, 12th day and 13th daypups – individually – 4th day of lactation

Postmortem examinations (parental animals):

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, stomach, small intestine, large intestine and caecum were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.In Reproduction Toxicity part of study the histopathology of the reproductive organs, pituitary gland and thyroid gland was performed for all high dose and control animals and organs with macroscopical changes were examined at the lowest and middle dose level groups. Detailed histological examination was performed on testes of all high dose and control animals from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). SACRIFICEAfter the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment. GROSS NECROPSYDuring 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 [5] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: The macroscopic examination was performed in all pups. No pathological findings were recorded at the dose levels 250 and 500 mg/kg/day and in control pups. Stomach – empty was recorded at the dose level 1000 mg/kg/day in the 16 pups from one female. These pups died after parturition.No histopathological findings were recorded during examination of thyroid gland in pups.GROSS NECROPSYDuring 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 [5] were prepared for microscopic examination and weighed, respectively.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Changes related to the colour of the test substance – coloured excrements were recorded in males at the dose levels 500 and 1000 mg/kg/day.In one female at the dose level 1000 mg/kg/day hunched posture and piloerection were recorded during the study.

Mortality:

no mortality observed

Description (incidence):

Female No.162 (at the dose level 1000 mg/kg/day) died on the 14th day of application due to intubation error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

M: Body weight was significantly decreased in males at the dose level 1000 mg/kg/day during the whole study. At the dose level 500 mg/kg/day the body weight was significantly decreased at the 2nd and 3rd week of study, from the 4th to the end of study this was slightly decreased without statistical significance. Body weight increments in treated males were variable in comparison with control males during the application period. The negative body weight increment was recorded at the dose level 1000 mg/kg/day at the 3rd week of study.F: Pre-mating periodMean body weight was decreased in females at the dose level 1000 mg/kg/day during the whole pre-mating period and at the dose level 500 mg/kg/day at the 2nd week of application period.The mean body weight increments of treated females were decreased (except 1st week of application in females at the dose level 500 mg/kg/day when this was increased)PregnancyFemales without parturition (non-pregnant females) were not included in the evaluation of mean body weight increments during pregnancy. Significant decrease of mean body weight was recorded in females at the dose level 1000 mg/kg/day during the whole pregnancy period and in females at the dose level 500 mg/kg/day at the 14th and 20th day of pregnancy period. The mean body weight increments of pregnant females at the dose levels 250 and 500 mg/kg/day were similar to the control females. At the dose level 1000 mg/kg/day the mean body weight increments was decreased. LactationOnly mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. During lactation significantly decreased mean body weight was detected in females at the dose level 1000 mg/kg/day during the whole lactation period and in females at the dose level 500 mg/kg/day on 1st and 4th day of lactation period. The mean body weight increments of treated pregnant females were similar to the control females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

M: Food consumption of all treated males and control males was similar (except the 2nd and 5th week of application at the dose level 1000 mg/kg/day when this was decreased).F: Pre-mating periodThe mean food consumption in females at the dose levels 500 and 1000 mg/kg/day and control females was similar. The mean food consumption in females at the dose level 1000 mg/kg/day was decreased. PregnancyFemales without parturition (non-pregnant females) were not included in the evaluation of food consumption during pregnancy. Decrease of mean food consumption in females at the dose level 1000 mg/kg/day was recorded during the whole pregnancy period. In females at the dose level 250 mg/kg/day the mean of food consumption was decreased in the time interval 14-20 day of pregnancy.LactationOnly mothers (females with live pups born) were included in evaluation of food consumption during lactation period.Decrease of mean food consumption in females of all treated groups was recorded during the whole lactation period.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological examination of males was without changes and females showed sporadic findings only; all findings were without dose dependence. Only differential percentage counts of lymphocytes and granulocytes were significantly changed without dose dependency at the dose level 500 mg/kg/day. The concentration of fibrinogen (FIB) was statistically significantly increased in the satellite treated females. All findings observed in both sexes (but in absence of a treatment-related clinical signs of toxicity) were considered to be of no toxicological significance.All haematological parameters were in range of historical control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical examination showed increased value of protein total and albumin in both sexes (irreversible in females). Reversibly increased relative weight of liver and kidneys were detected in males and females. This probably related to the adaptation process of organism to the test substance administration. Increased activity of hepatic enzymes could be also related with metabolism of test substance in organism. For complete elimination of these symptoms (increased value of protein total and albumin) be probably needed to longer time of recovery period. The concentration of thyroid hormone thyroxine (T4) in males was not significantly changed.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

During the functional observation markedly decreased number of upstanding in males at the middle and highest dose levels and in females at the highest dose level was recorded. Biochemical examination showed decreased value of creatinine in males in all treated groups and in females at the highest dose level. Also increased activity of AST and ALT in males and females was detected. These values (values of creatinine, values of activity of AST and ALT) were out of historical control limits. Decrease of these parameters related to serious damage of muscular matter which was confirmed during the functional observation and by the histopathological examination.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

M: During the examination of microscopical structure of reproductive organs toxicologically significant findings in males at the highest dose level were found out (in testes: tubular dystrophy, tubular atrophy, brown pigment in interstitium; in epididymis: germ cells and/or cell debris in lumen of tubules, mononuclear infiltration of epithelium and/or interstitium, brown pigment in interstitium, oligospermia or azoospermia). F: The changes related to previous pregnancy or oestrus cycle were found in controls and treated animals: in uterus – siderophages and/or lipophages in endometrium and/or mesometrium in 12-/-/-4 females, hydrometra in 0-2-1-1 females. In rectum, brown pigment in mucosa in 0-0-2-4 females, infiltration of mucosa and/or submucosa in 0-0-0-1 female and focal erosion in 0-0-0-1 females were found out. Cyst in thymus in 2-/-/-3 females was found out. Atrophy of muscle fibres in skeletal muscle in 0-0-0-4 females was detected. Thrombus in rectal vein or inflammation of vessels in 0-/-/-2 females was found out. Other microscopical changes observed in organs occurred only sporadically or they did not relate to test substance treatment.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

Oestrous cycles were monitored before treatment started to select for the study females with regular cyclicity daily for two weeks. Irregular cycle was demonstrated in one female only – this female was not included in groups for reproduction part of study.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Higher number of males with decrease of sperm motility was recorded at the dose level 1000 mg/kg/day. Increased percentage of affected sperms (mainly flattened head) was recorded at sperm morphology examination in males in all treated groups. Decreased absolute weight of prostate gland+seminal vesicles and epididymis was recorded in males at the highest dose levels. Also the higher number of males with decreased sperm motility was recorded there.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Number of females achieving pregnancy was seriously affected by the test substance treatment - in females at the highest dose level was the lowest. Number of implantations was decreased at the middle and highest dose level. One female at the highest dose level with abort was recorded.Male and female fertility index and gestation index at the highest dose level were decreased markedly. Post-implantation loses and post-natal loses were increased at the highest dose level.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: determined on the basis results of histopathological examinations and examination of reproduction parameters

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The mean of number of pups and sex ration (M/F) was lower at the highest dose level compared to control group.No presence of nipples in male pups was recorded and anogenital distance in treated male and female pups was not significantly affected.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In one female at the highest dose level the number of pups was not determined by the reason of cannibalism of pups immediately after parturition. Also in one female all pups died during the lactation period. During the macroscopic examination of these pups empty stomach (without milk) in five pups and haematoma in head in two pups were recorded.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In pups at the highest dose level the mean body weight and mean weight of litter were markedly decreased.

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):

Concentration of thyroxine hormone T4 in pups from treated groups was similar with the control group. During the microscopical evaluation of thyroid gland in pups no findings were found out.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In one female all pups died during the lactation period. During the macroscopic examination of these pups empty stomach (without milk) in five pups and haematoma in head in two pups were recorded.

Histopathological findings:

no effects observed

Description (incidence and severity):

During the microscopical evaluation of thyroid gland in pups no findings were found out. Concentration of thyroxine hormone T4 in pups from treated groups was similar with the control group.

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:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathological examinations, examination reproduction parameters

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Deviations:

During the study, short increase of temperature (31.03.2017 - for ten hours, temperature from 25.1 to 25.9°C and 01.04. 2017 for thirteen hours, temperature from 25.4 to 26.7°C) in animal room was recorded. This microclimatic deviation did not affect the welfare of animals and had no impact on the results of the study.

One deviation from the Study Plan occurred. 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. The differences between these two versions are not substantial for performance of study (some corrections in number of pups for T4 examination, changes in list organs to weight determination, calculation of fertility parameters etc.). In this study, these changes were respected.

No other deviations from study plan or test guidelines were observed during the study performance.

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 500 mg/kg body weight/day. The value of NOAEL was determined on the basis results of histopathological examinations and examination of reproduction parameters. 

Executive summary:

Introduction

The test substance, Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt, 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 28th July 2015.

 Further in the text the simplified name is used for the test substance, i.e. “Direct Brown 115, Direct Brown 116”, instead of “Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt.”

 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 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. 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 parental animals and pups were removed for weighing and histopathological examination.

 

Results

Reproduction part of study:

Parental males and females

The dose level 250 mg/kg/day – No significant changes in growth on treated animals were recorded. Increased percentage of affected sperm mainly flattened head in males was recorded.

No significant changes of weight of organs, macroscopic examination and microscopical structure of reproductive organs were recorded in both sexes. 

The dose level 500 mg/kg/day – decreased the body weight was recorded in both sexes during the study. Coloured excrements and/or urine in males and females were observed during the clinical examination. In males increased percentage of affected sperm mainly flattened head was recorded. No significant changes of weight of organs and macroscopic examination were recorded in both sexes. 

During the examination of microscopical structure of reproductive organs only sporadic finding in males was found out. 

The dose level 1000 mg/kg/day – decreased the body weight was recorded in both sexes during the study. In females also decreased food consumption was recorded.

Biometry of organs showed significantly decreased absolute weight of prostate gland+seminal vesicles and epididymis and significantly increased relative weight of pituitary gland and thyroid gland in males.

In males increased percentage of affected sperm mainly flattened head and higher number of males with decreased of sperm motility in males were recorded.

No significant changes of weight of organs and macroscopic examination were recorded in both sexes. 

During the examination of microscopical structure of reproductive organs toxicologically significantly findings in males were found out (in testes: tubular dystrophy, tubular atrophy, brown pigment in interstitium; in epididymis: germ cells and/or cell debris in lumen of tubules, mononuclear infiltration of epithelium and/or interstitium, brown pigment in interstitium, oligospermia or azoospermia). In females findings in uterus which related to females gravidity or oestrus cycle were found out.  

Pups

Mean number of pups per litter and mean number of male and female pups was decreased at the dose level 1000 mg/kg/day. Weight of litters was decreased in all treated groups and weight of pups was decreased at the dose levels 500 and 1000 mg/kg/day. At the dose level 1000 mg/kg/day these finding was changed markedly.

The anogenital distance in pups both sexes at the dose level 1000 mg/kg/day and in female pups at the dose level 250 mg/kg/day was significantly increased. Presence of nipples in male pups was not recorded. Body weight of male pups at the dose level 1000 mg/kg/day was significantly decreased.

Concentration of thyroxine hormone T4 in pups was similar with the control group.

The macroscopic findings were recorded in one pup at the dose level 500 mg/kg/day and 5 pups at the dose level 1000 mg/kg/day.

During the microscopical evaluation of thyroid gland in pups no findings were found out.

Reproduction parameters

One female with abortion was recorded at the dose level 1000 mg/kg/day. Number of females achieving pregnancy was decreased at the dose levels 250 and 1000 mg/kg/day. Number of implantations was decreased at the dose levels 500 and 1000 mg/kg/day. The number of females with pups was reduced at the dose levels 250 and 1000 mg/kg/day (at the dose level 1000 mg/kg/day only five female with pups was recorded). In one female at the dose level 1000 mg/kg/day all pups died during the study - cannibalism of whole litter.

Male and female fertility index in all treated group was decreased (at the dose level 1000 mg/kg/day this was markedly). Gestation index was decreased at the dose level 1000 mg/kg/day.

 

Conclusion

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 500 mg/kg body weight/day. The value of NOAEL was determined on the basis results of histopathological examinations and examination of reproduction parameters.