3-methyl-1,1-diphenylurea

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.01.-02.09.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 decrease of relative humidity (to 22.18 %) in SPF animal room was observed during study. The deviation from the limits was short-term and did not influence the wellness of animals and the study results.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,1-diphenylurea

EC Number:

236-039-7

EC Name:

3-methyl-1,1-diphenylurea

Cas Number:

13114-72-2

Molecular formula:

C14H14N2O

IUPAC Name:

3-methyl-1,1-diphenylurea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: 3-Methyl-1,1-diphenylurea- Physical state: white solid powder- Composition of test material, percentage of components: main component: 3-Methyl-1,1-diphenylurea CAS:13114-72-2 >98% (w/w) impurities: unlisted additives: unlisted-Molecular formula: C14H14N2O-Molecular weight: 226,27-Batch No.: 14015- Expiration date of the lot/batch: 11/2019- Stability under test conditions: stable- Storage condition of test material: in closed container, in dry room at room temperature (at laboratory conditions)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 21760118- 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, satellite animals - 2 rats of the same sex in one cage- Diet: ad libitum, Complete pelleted diet for rats in SPF breeding - ST 1, manufacturer: VELAS a.s., Hrabanov 535, 289 22 Lysá nad Labem CZ 801080-01, Diet was sterilised before using.- Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex. Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.- Water: ad libitum- Acclimation period: 5 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 were marked individually. ENVIRONMENTAL CONDITIONS- Temperature (°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 STUDY TIME SCHEDULETest substance delivery: 16. 12. 2014 Determination of stability and homogeneity:19.01. – 29. 01. 2015Dose-range finding experiment: 11. 02. – 10. 03. 2015Main Study:Animal arrival:11. 03. 2015Acclimatisation: 5 daysAdministration started: 17. 03. 2015 Administration and observation: Parental males:1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study Satellite males:1st day → 42nd day (administration) → 56th day (recovery period)Parental females:1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 3 post partum Satellite females:1st day → 42nd day (administration) → 56th day (recovery period)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The application form was prepared by mixing with olive oil. Two concentrations of application form were prepared (10 mg/10 ml and 1000 mg/10 ml). Concentration 10 mg/10 mlThe 50 mg of the test substance was mixed and dissolved in 50 mL of the vehicle in ultrasonic bath for 15 minutes. Then the application form (yellow solution without visible solid particles) was shaked for 45 minutes (400 Mot).Concentration 1000 mg/10 mlThe 5 g of the test substance was mixed and dissolved in 50 mL of the vehicle in ultrasonic bath for 15 minutes. Then the application form (milky suspension) was shaked for 45 minutes (400 Mot).VEHICLEOlive oilBatch No.: 5523502 Producer: Dr. Kulich Pharma, Hradec Králové, Czech Republic

Details on mating procedure:

- M/F ratio per cage: 1:1- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy- After successful mating each pregnant female was caged individually

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:

Parental males:1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study Satellite males:1st day → 42nd day (administration) → 56th day (recovery period)Parental females:1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 3 post partum Satellite females:1st day → 42nd day (administration) → 56th day (recovery period)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)

Frequency of treatment:

7 days per week

Details on study schedule:

Administration and observation: Parental males:1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study Satellite males:1st day → 42nd day (administration) → 56th day (recovery period)Parental females:1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 3 post partum Satellite females:1st day → 42nd day (administration) → 56th day (recovery period)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Basic groupsvehicle 12F+12M140 mg/kg/bw 12F+12M280 mg/kg/bw 12F+12M560 mg/kg/bw 12F+12MSatellite groupsvehicle 6F+6M560 mg/kg/bw 6F+6M

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-range finding experiment with 21-day application period was performed with 4 groups of treated animals without control group. On the basis of the results given above the following dose levels – 140, 280 and 560 mg/kg/day were chosen for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

Examinations

Parental animals: Observations and examinations:

MORTALITY CONTROL- Time schedule: dailyHEALTH CONDITION CONTROL- Time schedule: daily - during the acclimatization and the experimental part CAGE 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 changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.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 of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina. BODY WEIGHT: Yes- Time schedule for examinations: males - weekly females - weekly in premating and mating periodduring pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 3rd and 4th day;satellite males and females - weeklyFOOD CONSUMPTION- Time schedule: parental males - weekly (except the mating period) parental females - weekly during premating period during pregnancy and lactation – on the same days as body weight satellite males and females – weeklyIn 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 of mating period in parental males). 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 1st and 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.

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/45.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.

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 partum GROSS 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.

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:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Increased percentage of affected sperm and decreased sperm motility in treated males at the lowest and highest dose levels was recorded. At the lowest dose level damage of sperm was only mild. The test substance had effect on sperm quality.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of parental males showed only irrelevant changes of micriscopical structure of reproductive organs. Tubular degeneration and/or tubular atrophy of germ epithelium in testes occurred only in sporadic tubules and germ cell detritus and decreased sperm content in epididymis was also diagnosed sporadically. Above mentioned microscopic changes were not considered as toxicologically relevant. Increased weight of prostate gland and decreased weight of pituitary gland was detected during biometry of organs. Increased percentage of affected sperm and decreased sperm motility in treated males at the lowest and highest dose levels was recorded. At the lowest dose level damage of sperm was only mild. The test substance had effect on sperm quality. Examination of reproductive system of parental females showed changes of uterus: focal accumulation of siderophages in endometrium or mesometrium, focal accumulation of siderophages and lipophages and haematoma in mesometrium. These changes related with previous gravidity. Other microscopical changes were found out only sporadically. During examination of biometry of organs significantly decreased absolute weight of pituitary gland, uterus and ovaries in females at the highest dose level was recorded. These weight reductions of reproductive organs were not evaluated as toxicologically significant by the reason of an insufficient number of mothers at that dose level.

Details on results (P0)

OBSERVATION OF PARENTAL MALESBODY WEIGHT AND BODY WEIGHT INCREMENTThe different mean body weight of groups before application was caused by gradual start-up of application. The loss of body weight was recorded at the dose level 560 mg/kg/day in the 1st week of application period. Statistically significantly decreased the body weight was detected at the dose levels 280 and 560 mg/kg/day during application period (except the 3rd, 4th and 6th week of application period at the dose level 280 mg/kg/day). Dose-dependent decreased body weight increment was recorded in all treated groups at the 1st week of application period (except at the dose level 560 mg/kg/day where fall of body weight was detected). Decreased body weight increment at the dose level 560 mg/kg/day was recorded in the 2nd week of application period. FOOD CONSUMPTIONDecreased mean of food consumption was recorded at the dose level 560 mg/kg/day in the 1st and 2nd week of application period. HEALTH CONDITION CONTROL AND GENERAL CLINICAL OBSERVATIONMales In control males and treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In treated males at the dose level 140 mg/kg/day no clinical changes of health condition before, during and after application of the test substance were observed. At the dose level 280 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding and disquiet/irritation. No clinical changes in males at the dose level 280 mg/kg/day were detected during afternoon clinical observation.At the dose level 560 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding, disquiet/irritation, salivation, piloerection. Bizarre behaviour - automutilation (injured tail or ears, sore in skin on the neck) and symptoms of stress (mainly - focal loss of fur, salivation and disquiet/irritation) at the dose level 560 mg/kg/day was observed irregularly during the study. Satellite malesIn satellite control males and satellite treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In satellite treated males the following changes were observed immediately after application: poking the nose to the bedding, salivation and piloerection. During clinical observation the bizarre behaviour - automutilation (injured tail) and symptoms of stress (salivation, disquiet/irritation) were observed haphazardly. Swelling of mandibular in one treated male was observed during the study – this finding did not relate with the test substance treatment. HISTOPATHOLOGICAL EXAMINATIONonly irrelevant changes of micriscopical structure of reproductive organs. Tubular degeneration and/or tubular atrophy of germ epithelium in testes occurred only in sporadic tubules and germ cell detritus and decreased sperm content in epididymis was also diagnosed sporadically. Above mentioned microscopic changes were not considered as toxicologically relevant. BIOMETRY OF REPRODUCTIVE ORGANSAbsolute weight: decreased absolute weight of testes at the dose level 140 mg/kg/day was detected (without statistical significance). Insignificantly decreased weight of epididymis at the dose level 560 mg/kg/day was found out. Absolute weight of prostate gland was significantly increased at the dose level 560 mg/kg/day and on the contrary weight of pituitary gland was significantly decreased. Relative weight: Relative weight of testes was increased at the dose levels 280 and 560 mg/kg/day (at the dose level 280 mg/kg/day the difference statistically significant). Increased weight of prostate gland at the dose levels 280 and 560 mg/kg/day was detected (at the dose level 560 mg/kg/day the difference statistically significant). At the dose level 560 mg/kg/day weight of pituitary gland was insignificantly decreased. OBSERVATION OF SPERMHigher number of males with decreased sperm motility was recorded at the dose levels 140 and 560 mg/kg/day. Increased number of males with affected sperms at the dose levels 140 and 560 mg/kg/day was detected. OBSERVATION OF PARENTAL FEMALESBODY WEIGHT AND BODY ICREMENTBefore mating periodSignificant loss of body weight in females at the dose level 560 mg/kg/day before mating period was recorded. PregnancyFemales without parturition (non-pregnant females) were not included in the evaluation of body weight increments during pregnancy. Decreased body weight was detected at the dose level 560 mg/kg/day in the 14th and 20th day of pregnancy and at the dose level 280 mg/kg/day in the 20th day of pregnancy. The body weight increment in females at the dose level 560 mg/kg/day was decreased marked at the 14th and 20th day of pregnancy. LactationOnly mothers (females with live born pups) were included in the evaluation of body weight increment during lactation period. During lactation decreased body weight at the dose levels 280 and 560 mg/kg/day was detected. On the 3rd day of lactation period loss of body weight of mothers was recorded at the dose level 560 mg/kg/day. Disbalance of body weight increment was recorded during lactation period. FOOD CONSUMPTIONPre-mating periodDecreased mean food consumption at the dose level 560 mg/kg/day was recorded. PregnancyFemales without parturition (non-pregnant females) were not included in the evaluation of food consumption during pregnancy. On the 20th day of pregnancy mean food consumption in mothers at the dose level 560 mg/kg/day was lower than in control females. LactationOnly mothers (females with live born pups) were included in evaluation of food consumption during lactation period.Mean food consumption in females at the dose level 560 mg/kg/day was lower than in control females. HEALTH CONDITION CONTROL AND CLINICAL OBSERVATIONFemales In control females and treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In treated females at the dose level 140 mg/kg/day no changes of health condition before, during and after application of the test substance were observed.At the dose level 280 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding, salivation, hunched posture and disquiet/irritation.Clinical findings (piloerection and hunched posture) in females at the dose level 280 mg/kg/day were detected during afternoon clinical observationAt the dose level 560 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding, hunched posture, salivation, piloerection. Bizarre behaviour - automutilation (injured tail, sore in skin on the neck), symptoms of stress (mainly - focal loss of fur, salivation, disquiet/irritation) and hunched posture at the dose level 560 mg/kg/day was observed irregularly during the study. Satellite femalesIn satellite control females and satellite treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In satellite treated females the following changes were observed immediately after application: poking the nose to the bedding, piloerection, secretion around nostrils and disquiet/irritated. BIOMETRY OF REPRODUCTIVE ORGANSNon-pregnant females and females with were not used for calculation of means and evaluation of organs weight. Absolute weight of ovaries, uterus and pituitary gland was decreased at the dose level 560 mg/kg/day with statistical significance. Relative weight of ovaries, uterus and pituitary gland was decreased at the dose level 560 mg/kg/day without statistical significance. HISTOPATHOLOGICAL EXAMINATIONshowed changes of uterus: focal accumulation of siderophages in endometrium or mesometrium, focal accumulation of siderophages and lipophages and haematoma in mesometrium. These changes related with previous gravidity. Other microscopical changes were found out only sporadically. REPRODUCTION PARAMETRSAll treated and control females were mated. Presence of sperm was not found in four females at the dose level 560 mg/kg/day. Abortion in females was not recorded. The number of females achieving pregnancy at the dose level 560 mg/kg/day was markedly lower than in the control group. Duration of mating and pregnancy of treated groups were relatively well-balanced to the control group. The number of females bearing live pups and females with live pups at day 4 after parturition was decreased at the dose levels 280 and 560 mg/kg/day. The number of corpora lutea and number of implantations were relative well-balanced to the control group. Number of pups per litter was markedly lower at the dose level 560 mg/kg. Mean weight of litters and weight of pups was markedly decreased at the dose level 560 mg/kg/day. Abnormal pups were found out in all treated group, but at the dose levels 280 and 560 mg/kg/day macroscopic findings were with toxicological importance. Mating index of treated males and treated females at the dose level 560 mg/kg/day was decreased. Fertility index of treated males and treated females at the dose levels 280 and 560 mg/kg/day was lower than at the control group (at the dose level 560 mg/kg/day the difference was marked). Gestation index was decreased at the dose level 560 mg/kg/day. Markedly decreased survival index at the dose level 560 mg/kg/day was detected. Pre-implantation losses of treated mothers were relatively similar with the control group. Post-implantation losses and post-natal losses at the dose level 560 mg/kg/day were higher than control group.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

other: decrease sperm motility, affected sperms

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Stillborn pups in all treated groups was recorded: one pup at the dose level 140 mg/kg/day, 10 pups at the dose level 280 mg/kg/day and 12 pups at the dose level 560 mg/kg/day. Four pups in the control group, one pup at the dose level 140 mg/kg/day, one pup at the dose level 280 mg/kg/day and eight pups at the dose level 560 mg/kg/day died during lactation period. Cannibalism in mothers at all groups was recorded: 1 pup in the control group, 6 pups at the dose level 140 mg/kg/day, 12 pups at the dose level 280 mg/kg/day and 7 pups at the dose level 560 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical evaluation was performed for mean body weight of pups and weight of litters. Dose-dependent decreased mean weight of the pups and mean weight of litters in all treated groups was detected with statistical significance (except mean weight of litters at the dose level 140 mg/kg/day in the 1st day after parturition when it was not statistically significant).

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination was performed in all euthanized or died pups. Foci with addicted skin in top of head in 4 pups at the dose level 280 mg/kg/day and 5 pups at the dose level 560 mg/kg/day was detected. In skin of head haematoma in 9 pups at the dose level 560 mg/kg/day was recorded. Pale colour of liver in 7 pups and pale colour of kidneys in 5 pups at the dose level 140 mg/kg/day was found out. Anaemic organs in abdominal cavity in 2 pups at the dose level 140 mg/kg/day was observed.

Histopathological findings:

not examined

Other effects:

not examined

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:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other:

Organ:

other: decrease of number of pups, weight of pups and weight of litters,

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The value of NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and for DEVELOPMENT OF PUPS was established as 140 mg/kg body weight/day.

Executive summary:

At the dose level 140 mg/kg/day increased number of males with affected sperm and with decreased sperm motility were recorded. Decreased weight of pups and weight of litters was also recorded. 

The course of mating, pregnancy and lactation of parental animals, weights of reprodutive organs and pituitary gland, number of pre-implantation, post-implantation and post-natal losses of mothers and number, sex ratio of pups were not adversely affected by the test substance treatment.    

 

At the dose level 280 mg/kg/day decreased body weight of males and pregnant females was recorded. In males weight of testes and prostate gland was increased. Decreased number of pups and decreased mean weight of pups and litters was found out. Increased number of stillborn pups and pups which were lost during lactation period were recorded. During macroscopical examination of pups the pups with findings (foci with addicted skin in top of head) was recorded. The fertility index was also decreased at the middle dose level. Others reproduction parameters (mating index, gestation index, pre-imlantation and post-implantation losses) were not affected.

 

At the dose level 560 mg/kg/day loss of weight and/or decreased body weight of males and pregnant females was recorded. In males following changes related to reproduction were recorded: increased number of males with affected sperm, decreased sperm motility, increased weight of prostate gland, decreased weight of pituitary gland. The microscopical structure of reproductive organs was not significantly affected – only sporadic findings were found out.

Markedly decreased number of mothers was recorded. Decreased weight of ovaries, uterus and pituitary gland in femaleswas not evaluatedas significant by the reason ofan insufficientnumber of mothers. In the reproductive system of mothers microscopical findings were recorded only sporadically and these mainly related with previous gravidity.

 

 

The following changes showed examination of pups: markedly decreased total number of pups, decreased mean weight of pups and weight of litters, increased number of still born pups and pups which were lost during the lactation period. In all pups macroscopical findings (foci with addicted skin in top of head and/or haematoma on skin of head) were found out.

All calculated reproduction parameters (indexes of mating, fertility, gestation and survival) were decreased. Post-implantation and post-natal losses were higher than others groups. But for evaluation these parameters at the dose level 560 mg/kg/day low number of mother was achieved.