Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Study period:
From October 15,2012 to April 30,2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline performed on a similar substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: SPF breeding, VELAZ- Age at study initiation: 10 weeks on arrival- Weight at study initiation: body weight - group mean ± standard deviation (g)MALEGroup code 0 15 mg/kg bw 60 mg/kg bw 240 mg/kg bw248.58 ± 10.33 246.23 ± 13.77 255.17 ± 11.91 243.32 ± 6.97FEMALEGroup code 0 15 mg/kg bw 60 mg/kg bw 240 mg/kg bw201.92 ± 6.38 200.73 ± 9.63 205.87 ± 5.15 199.53 ± 8.19- Housing:2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage.- Bedding: sterilized soft wood fibers (Lignocel)- Diet: complete pelleted diet for rats and mice in SPF breeding- Water: drinking water ad libitum, quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry- Acclimation period:6 days- Health chech: 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):- Photoperiod: 12 hours cycle dark/light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua pro injectione
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Concentration Level 50 mg/10mLCa. 0.5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (800 rpm) for 60 minutes.Concentration Level 1000 mg/10 mLCa. 10 g of the test substance was weighted with wider end of glass Pasteur into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle and disslolved in ultrasonic bath for a 15 min. This solution was stirred by magnetic stirrer (800 rpm) for 30 minutes.
Details on mating procedure:
Animals were mated from the 15th 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 were determined by means of measuring of a peak area of the test substance by high-performance liquid chromatography based on the method developed at the test facility.The stability of the application form The stability of the application form was checked by determination of a concentration of the test substance within 120 min (at the time 0 min, 30 min, 60 min and 120 min). The samples were taken from the middle of the beaker content after required time intervals.The homogeneity of the application formThe homogeneity of the application form was checked by determination of a concentration of the test substance in three places of solution (at the bottom, in the middle and at the surface). Results of analysisFrom the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle in concentration level 50 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes starting with preparation of the application form.From the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle in concentration level 1000 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 60 minutes from starting with analysis of the application form.
Duration of treatment / exposure:
Parental males:1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of studySatellite males:1st day → 42nd day (administration) → 56th day (observation)Parental females:1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partumSatellite females:1st day → 42nd day (administration) → 56th day (observation)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of studyNon-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 at the same time (8.00 – 10.00 am). The vehicle control group was administered by aqua pro injectione in the same volume.
Details on study schedule:
Health condition control: daily - during the acclimatization and the experimental part Body weight: males - weekly females - weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st, 3rd and 4th day;pups (litters) – 0. or 1st, 3rd and 4th day;satellite males and females - weeklyFood consumption: 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 – weeklyWater consumption: satellite males and females – twice a weekClinical observations: males and females - daily during the administration periodpups - as soon as possible after delivery and then dailyMortality control: twice dailyDetailed clinical observation: before the first application and then weekly (except the mating period) Functional observation: at the end of administration/observation period Laboratory examinations: - vaginal smears: daily in mating period- urinalysis: the last day of administration/observation period – only males- haematology: at the end of administration/observation period- biochemistry: at the end of administration/observation period - pathological examination: males and nonpregnant females – at the end of administration periodparental females and pups - on the 4th day of lactationsatellite males and females - at the end of observation period- weight of organs: during necropsy- sperm observation: all males after necropsy (exc. satellite males)
Doses / concentrations
Remarks:
Doses / Concentrations:0, 15, 60 and 240 mg/kg/day Basis:actual ingested
No. of animals per sex per dose:
1. Control 0 mg/kg bw day 12 males + 12 females2. Low dose 15 mg/kg/day 12 males + 12 females3. Intermediate dose 60 mg/kg/day 12 males + 12 females4. High dose 240 mg/kg/day 12 males + 12 femalesSatellite groups:5. Control – vehicle – satellite: 0 mg/kg bw day 6 males + 6 females6. High dose – satellite: 240 mg/kg/day 6 males + 6 females
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yesmales and females - daily during the administration periodAll rats were observed daily during the administration period.This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day(11.00 – 13.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.DETAILED CLINICAL OBSERVATIONS: Yesbefore the first application and then weekly (except the mating period)Functional observation: at the end of administration/observation periodThis observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, 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.Functional ObservationThis observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period.During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.BODY WEIGHT: Yesmales - weeklyfemales - weekly in premating and mating period,during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st, 3rd and 4th day;satellite males and females - weeklyThe body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too.Weight increment was computed as an mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.FOOD CONSUMPTION:males - weekly (except the mating period)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 out and the food consumption for the previous week was computed.In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.Food conversion in % (weight increment/food consumption x 100) was calculated for animals of repeated dose toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females. (Numbers of females for repeated dose toxicity part of study were chosen at the end of study.)WATER CONSUMPTION: Yessatellite males and females – twice a weekThe drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study. OTHER:CLINICAL CHEMISTRY: Yesat the end of administration/observation periodThis examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.The following parameters were determined by automatic biochemical analysers SPOTCHEMTM EZ SP-4430 and SPOTCHEMTM EL SE-1520 (Arkray, Inc., Japan).GlucoseCholesterol, totalUreaBilirubin, totalAspartate aminotransferaseAlanine amonitransferaseAlkaline phosphataseCalciumPhosphorusProtein, totalProtein, albuminCreatinineSodiumPotassiumChlorideURINALYSIS: Yesthe last day of administration/observation period – only malesThis examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for long time). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.The following parameters were determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan).VolumeColourCloudOdourGlucoseProteinBilirubinUrobilinogenpHSpecific gravityBloodKetonesNitriteLeucocytes
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:
CAGE SIDE OBSERVATIONS: yesAll 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 0 or 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.BODY WEIGHT: Yespups (litters) – 0. or 1st, 3rd and 4th day;
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yesmales and nonpregnant females – at the end of administration periodparental females and pups - on the 4th day of lactationsatellite males and females - at the end of observation periodDuring 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: Yesafter necropsySamples of the following tissues and organs were collected at necropsy and fixed:Repeated dose toxicity part of study (6 males and 6 females from each main group + 6 males and 6 females from satellite groups)Adrenal glandsAortaBrain (incl. cerebellum and med. oblongata)CaecumCoagulating glandColonDuodenumPancreasRectumSalivary glandsSciatic nerveSeminal vesiclesSkeletal muscleSkinSpinal cord – thoracicSpleenStomachThymusThyroid gland incl. parathyroidTracheaUrinary bladderFemale mammary gland areaFemurHeartIleumJejunumKidneysLiverLungsLymph nodes – mesenteric, paraaortalOesophagusAll gross lesionsReproduction part of study (12 males and 12 females from each main group)Pituitary glandOvariesUterusCervix of uterusVaginaEpididymis/EpididymidesProstate glandSeminal vesicles and coagulating glandTestesAll gross lesionsThe mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.Detailed histological examination was performed on testes of males from Reproduction Toxicity part of study – for all high dose and control animals (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the following publications: 1. Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990); 2. Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997); 3. Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11; 4. Lonnie D. Russell et al.: Histological and histopathological evaluation of the testis, 1st ed., Published 1990 by Cache River Press in Clearwater.
Postmortem examinations (offspring):
SACRIFICE- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:GROSS NECROPSY- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]HISTOPATHOLOGY / ORGAN WEIGTHSparental females and pups - on the 4th day of lactation
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs and selected reproduction parameters – number of live born pups, number of corpora lutea, number of implantations, mean weight of pup on the 0./1st day and mean weight of pup on the 4th day. 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:
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
Description (incidence and severity):
highest dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
highest dose
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
highest dose
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)There were no unscheduled deaths during the whole study.MALEAt the lowest and the middle dose level no clinical changes after application of the test substance were recorded. The following clinical changes after application of the test substance were detected in all males of the highest dose level: test substance-coloured excrements and orange-brown colour of urine (changed colour of bedding: shavings) from the 1st week to the 6th week of application period.FEMALEThere were no unscheduled deaths during the whole study. One female of the middle dose level was euthanasied before the scheduled time of necropsy by the reason of bad health status (on the 24th day of pregnancy).At the lowest dose level no clinical changes after application of the test substance were recorded. In one female of the middle dose level piloerrection, disquietness and dischargefrom vulva was observed in the 6th week. The following clinical changes after application of the test substance were detected in all females of the highest dose level: test substance-coloured excrements and orange-brown colour of urine (changed colour of bedding: shavings) from the 1st week to the 6th week of application period.Health Condition Control MALEIn 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 of the lowest and the middle dose level no changes of health condition were found out before, during and immediately after application of the test substance. In males of the highest dose level the following changes were observed during the application period: orange-brown colour of urine (changed colour of bedding: shavings) and test substance-coloured excrements. FEMALEIn 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 of the lowest no changes of health condition were found out before, during and immediately after application of the test substance. In one female of the middle dose level piloerrection, disquietness and discharge from vulva was observed in the 6th week (for two days). In females of the highest dose level the following changes were observed during the application period: orange-brown colour of urine (changed colour of bedding: shavings) and test substance-coloured excrements. BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)MALEStatistically significantly decreased body weight was found in males of the highest dose level in the 2nd week of application. The body weight of males of the lowest dose level was similar compared to control. In males of the middle and the highest dose level decreased body weight was recorded in comparison with control. This difference was dependent on dose level. FEMALEPre-mating mating periodStatistically significantly decreased body weight was found in females of the highest dose level in the 2nd week of application. The mean body weight increments of the control females and females at the lowest and the middle dose level were quite balanced whilst weight increments of females of the highest dose level slightly decreased in the pre-mating period. Pregnancy Females without parturition (non pregnant or aborted females) were not included in the evaluation of mean body weight increments during pregnancy. The mean body weight increments of treated pregnant females at all dose levels were similar to control mothers in the 1st and in the 2nd week of pregnancy. In the 3rd week of pregnancy body weight increment of pregnant females of the highest dose level was decreased. Lactation Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The mean body weight increments of treated mothers at the lowest and the middle dose level and control mothers were similar. The mean body weight increment of mothers at the highest dose level was slightly increased against control but the body weight of mothers of the highest dose level remained decreased compared to control. FOOD CONSUMPTION:MALEThe mean food consumption of males of the lowest and the middle dose level was analogous to control males. The mean food consumption of males of the highest dose level was slightly decreased in the 1st week, from the 2nd to the 6th week it was similar to control. FEMALEPre-mating periodThe mean food consumption of females treated by the lowest and the middle dose levels was balanced with control females in pre-mating period. The food consumption of females of the highest dose level was decreased.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 lowest and the middle dose levels was similar to control. The mean food consumption of pregnant females of the highest dose level was decreased in the 3rd week of pregnancy.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 similar to control mothers. REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)Sperm motility was similar in control males and treated males. Presence of “non-motile sperms” was not recorded. Examination of sperm morphology did not reveal marked differences between control and treated males.REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)FEMALEAll females of control group and the lowest and the middle dose level were mated. Evidence of copulation was not found in one female of the highest dose level. The number of females achieving pregnancy was analogous in treated and control groups. Abortions occurred at the middle and the highest dose level. The duration of mating of females at the lowest and the middle dose level was similar to the control group. The duration of mating of females at the highest dose level was longer against control. The duration of pregnancy of females at all dose level was comparable to the control. The number of females bearing live pups was well-balanced in the control and the lowest dose level. In females of the middle and the highest dose level this number was apparently decreased compared to control. Mean number of corpora lutea was decreased at the highest dose level. Vice versa mean number of implantations was decreased at the middle dose level. Mean number of live pups at birth and at day 4 after parturition in treated mothers was similar to control. Mean weight of the litter at the highest dose level was markedly decreased against control. Mean weights of pup recorded at the 1st check of litter after parturition and on 4th day of lactation was slightly decreased at the highest dose level. Macroscopical findings were recorded in stomach of pups of treated mothers: sporadically at the lowest and the middle dose level and in more than 25% of pups at the highest dose level. No significant differences of mating indexes were observed. Fertility indexes were decreased at the middle and the highest dose level. At the middle and the highest dose level gestation index was markedly decreased. Survival indexes of treated groups were analogous to control.Pre-implantation losses were slightly increased at the middle dose level and post-implantation losses were increased at the middle and the highest dose level. No significant differences were detected in post-natal losses.ORGAN WEIGHTS (PARENTAL ANIMALS)MALEThe statistical analysis of the data revealed no significant intergroup differences in absolute and relative weight of reproductive organs and pituitary gland. Absolute and relative weight of prostate gland was slightly decreased in males of the highest dose level. Relative weight of testes was increased in treated males in a dose dependent manner. FEMALEThe statistical analysis of the data did not reveal significant intergroup differences in absolute and relative weights of ovaries, uterus and pituitary gland. Nonpregnant females and females with abortion were not used for calculation of means and evaluation of biometry results. Slight increase of absolute and relative weight of ovaries at the middle dose level and of relative weight at the highest dose level was described. Absolute and relative weights of examined organs were conformable to the control group.GROSS PATHOLOGY (PARENTAL ANIMALS)MALEThe incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-15-60-240 mg/kg/day further in the text.No macroscopical findings were recorded in 11-11-11-0 males. In 11-12-12-12 males no macroscopical changes were observed in testes, epididymides, seminal vesicles, coagulation glands and pituitary gland. Change of colour of organs and tissues (or contents of organs) were observed in males of the highest dose level: dark colour subcutis and skeletal muscle in 0-0-0-2 males, brown colour of stomach mucosa membrane and chyme in 0-0-0-11 males, brown colour of stomach chyme in 0-0-1-1 males. Other macroscopical changes were described sporadically. FEMALEThe incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels of 0-15-60-240 mg/kg/day further in the text. No macroscopical findings were recorded in 12-12-6-1 females. Change of colour of organs and tissues (or contents of organs) were observed in females of the middle and the highest dose level: dark or brown colour of stomach mucous membrane and chyme in 0-0-0-4 females, dark or brown colour of stomach chyme in 0-0-3-7 females, brown colour of mammary gland in 0-0-2-4 females, yellow or brown colour of fat tissue in 0-0-2-5 females, yellow or brown colour of subcutis in 0-0-2-11 females, yellow or brown colour of skeletal muscle in 0-0-0-11 females, brown colour of heart in 0-0-0-2 females, dark red or brown-red colour of spleen in 0-0-0-10 females, dark brown colour of liver and brown-red colour of kidneys in 0-0-0-3 females. Enlargement of spleen was observed in 0-0-0-4 females. Discharge from vagina was found out in 0-0-2-0 females and late resorptions or dead foetuses was detected in uterus of 0-0-3-1 females.HISTOPATHOLOGY (PARENTAL ANIMALS)MALEMicroscopic examination of testes, epididymis, seminal vesicles, coagulation glands and pituitary gland did not reveal presence of treatment related changes. FEMALEThe incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels 0-15-60-240 mg/kg/day further in the text. In 4-1-1-0 females no histological changes were detected. During microscopical examination of reproductive organs the following possible treatment related changes were registered. Presence of brown pigment was found out in ovaries of 0-0-0-12 females and in uterus of 0-1-3-11females. Inflammation was diagnosed in uterus of 0-0-4-1 females. In uterus the changes related to previous pregnancy were found: accumulation of siderophages in mesometrium or/and in endometrium in 8-9-4-5 females and haemorrhages or haematoma in mesometrium in 2-2-1-2 females.Hydrometra - the change associated with oestrous cycle was registered in uterus of 0-0-0-2 females.Other microscopical changes observed in reproductive organs occurred only sporadically or they did not relate to test substance treatment

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: FERTILITY

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)The statistical evaluation of the number of live born pups/per litter, number of corpora lutea and number of implantations was performed. No statistically significant intergroup differences were recorded. The total number of live pups at the middle and the highest dose levels was markedly decreased in comparison with the control. The total numbers of live pups per litter at the lowest dose level was slightly higher than control. Mean number of pups per litter in treated mothers was similar to control. In sex ratio no significant differences were recorded in treated groups. CLINICAL SIGNS (OFFSPRING)no dataBODY WEIGHT (OFFSPRING)The statistical evaluation of mean weight of pup on the 0./1st day and mean weight of pup on the 4th day was performed. No statistically significant intergroup differences were recorded. Mean body weights of litters at the lowest and the middle dose levels were slightly increased compared to control. Mean weight of the litter at the highest dose level was markedly decreased against control. Mean weights of pup recorded at the 1st check of litter after parturition at lowest and the middle dose levels and control were similar. Mean weights of pup recorded at the 1st check of litter after parturition at the highest dose level was slightly decreased. Mean body weight of pup on the 4th day of lactation was also slightly decreased at the highest dose level. GROSS PATHOLOGY (OFFSPRING)macroscopic examination was performed in all pups. Macroscopical findings were observed at pups of treated mothers: yellow colour of milk, empty stomach or flatulence of stomach. OTHER FINDINGS (OFFSPRING)Development of Pups Prenatal mortality was increased at the middle and the highest dose level. Number of stillborn pups was negligible at control group and at the lowest and the highest dose levels. Number of stillborn pups at the middle dose level was not exactly determined by the reason of total cannibalism of pups in one female. Mortality of pups in lactation period was sporadically detected only at the middle and the highest dose level. No differences in postnatal development of pups were observed at the control and treated groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: DEVELOPMENT

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Effects on reproduction at the dose level 240 mg/kg/day:

-         moderate increase of relative weight of testes in males (dose dependent)

       -    slight increase of relative weight of ovaries (without dose dependence)

-         decrease of body weight increments of females in pregnancy period

-         increase occurrence of abortions (increase of prenatal mortality)

-         decrease of number of females bearing live pups

-         decrease of mean number of corpora lutea

-         decrease of mean weight of litter

-         increase of post-implantation losses

-        increased occurrence of macroscopical findings in pups: change of colour of milk

-        increased occurrence of pathological findings in females: dead foetuses andinflammation in uterus[fd2] [RV3] – in aborted females

 

Effects on reproduction at the dose level 60 mg/kg/day:

-         euthanasia of one female before the scheduled time of necropsy by the reason of bad health status (abortion)

      -     slight increase of absolute and relative weight of ovaries (without dose dependence)

-         increase occurrence of abortions (with not clear dose/effect dependence: the effect diminishes at the highest dose)

-         decrease of number of females bearing live pups (consequence of decreasing number of mothers)

-        sporadical occurrence of macroscopical findings in pups: change of colour of milk

 

Effects on reproduction at the dose level 15 mg/kg/day:

- minimal occurence of macroscopiacal findings in pups: change of colours in milk

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was proposed as 15 mg/kg body weight/day. The value was set as a conservative value, determined on the basis of decreased number of females bearing live pups, increased prenatal mortality (increased number of females with abortions, but dose/response relationship is not clear) and decreased mean litter weight. Male ability to produce sperm that can fertilise eggs and female ability to be impregnated were not significantly changed. Therefore the value of 60 mg/Kg body weght/day has been used for risk assessment purposes
Executive summary:

The test substance, Direct Brown 44, 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 March 22nd 1996.

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 15, 60, 240 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 (240 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

 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 3rd day of lactation,

males – after mating period – totally for 42 days,

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

non-mated females – to the 54th day of study.

    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 control 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 during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups) 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 were removed for weighing and histopathological examination.

Results

    Repeated oral administration of Direct Brown 44 to rats by gavage at the dose levels 15, 60 and 240 mg/kg/day did not cause any mortality. One female of the dose level 60 mg/kg/day was euthanasied before the scheduled time of necropsy by the reason of bad health status (abortion on the 24th day of pregnancy)

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, absolute weight and relative weight of pituitary gland, spermiogenesis and sperm parameters, macroscopical structure of reproductive organs and pituitary gland of parental animals, number of post-natal losses of mothers, sex ratio and early postnatal development of pups were not adversely influenced by the test substance treatment.

    Evaluation of body weight of parental animals (decrease of body weight increments of females in pregnancy – at the dose level 240 mg/kg/day) and relative weights of reproductive organs (dose dependent increase of relative weight of testes – at the dose level 240 mg/kg/day), examination of number of live pups (decrease of the total number of live pups – at the dose levels 240 mg/kg/day), calculation of reproduction parameters (increased number of females with abortion and decreased number of females bearing live pups - at the dose levels 240 mg/kg/day), calculation of prenatal losses (increase of pre-implantation loss at the dose level 240 mg/kg/day), evaluation of pup weights (decrease of mean litter weigh - at the dose level 240 mg/kg/day), macroscopical examination of pups (change of colour of milk – at the dose level 240 mg/kg/day, sporadically at the dose levels 15 and 60 mg/kg/day), microscopical examination of reproductive organs of parental females (deposits of pigment in ovaries and uterus – at the dose level 240 mg/kg/day and inflammation in uterus in females with abortion – at the dose levels 60 and 240 mg/kg/day) revealed effects attributable to test substance.