Tetrasodium 4,4'-bis[[4-[(2-hydroxyethyl)amino]-6-(m-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From January 13th to December 10th, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-bis[[6-anilino-4-[(2-hydroxyethyl)methylamino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

237-600-9

EC Name:

Disodium 4,4'-bis[[6-anilino-4-[(2-hydroxyethyl)methylamino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

13863-31-5

Molecular formula:

C38H40N12Na2O8S2

IUPAC Name:

disodium 2,2'-ethene-1,2-diylbis[5-({4-anilino-6-[(2-hydroxyethyl)(methyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

CRL (SPF quality - guaranteed)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding
- Age at study initiation: sexually adult; males – 9 weeks on arrival, females – 9 weeks on arrival
- Fasting period: animals were without feed two hours before application and two hours after application of the test item
- Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage
- Diet: maintenance pelleted diet for rats and mice - Altromin for rats/mice
- Water: ad libitum
- Acclimation period: dose-range finding experiment: 5 days; main study: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua pro iniectione

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into glass beaker calibrated to 100 ml and dissolved in vehicle (aqua pro iniectione, ca 80 % of total volume) in ultrasonic bath together with occasional mixing by glass rod for 10 minutes. After this the glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer (400 rpm) for 10 min.
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 application forms were prepared daily just before administration and covered with aluminium foil to avoid potential degradation due to light. The administration of the test item to animals was performed during two hours after preparation of application form. The stirring of solutions continued during administration.
The test item was administered to the stomach by gavage. The animals were treated 7 days per week at the same time (7.00 – 10.00 am). The vehicle control group was administered by vehicle - aqua pro iniectione in the same volume.

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:

Stability and homogeneity were determined by means of measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility.

The application form for analysis was prepared in the same manner as for application to animals – i.e. solution in aqua pro iniectione.

Concentration level 10 mg/10 ml
Ca 0.10 g of the test item was weighed with wider end of glass Pasteur pipette into a 150 ml glass beaker calibrated to 100 ml and the beaker was slowly replenished by the vehicle. The test item was dissolved in ultrasonic bath for 8 min. The solution was stirred by magnetic stirrer at 350 rpm for 10 minutes.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability of test item.

Concentration level 1000 mg/10 ml
Ca 10.0 g of the test item was weighed with laboratory spoon into a 150 ml glass beaker calibrated to 100 ml. The beaker was slowly replenished by the vehicle together with mixing by glass rod. The test item was dissolved in ultrasonic bath together with occasional mixing by glass rod for 10 minutes. After this the application form (yellow fine slurry) was stirred by magnetic stirrer at 400 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability of test item.

Stability of the application form
The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.

Concentration level 10 mg/ 10 ml: time interval 0 min represents for this concentration the time after 8 minutes of ultrasonication and 10 minutes of mixing by magnetic stirrer at 350 rpm.
Concentration level 1000 mg/ 10 ml: time interval 0 min represents for this concentration the time after 10 minutes of ultrasonication together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer at 400 rpm.

Homogeneity of the application form
Concentration level 10 mg/ 10 ml: the samples were taken after 8 minutes in ultrasonic bath and 10 minutes of mixing by magnetic stirrer (350 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.
Concentration level 1000 mg/ 10 ml: the samples were taken after 10 minutes in ultrasonic bath together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer (400 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.

Results of analysis
Both the application forms of the test item (10 mg and 1000 mg /10 ml), at defined laboratory conditions are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

Duration of treatment / exposure:

DRFE
Administration: 19 days as total time of application in DRFE study; females became pregnant gradually, each female was applied from next day after confirmed mating to 19th day of pregnancy)

Main study
Administration
Parental males (totally 49 days of administration):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 43rd day – 63rd day (administration period) → 64th day (necropsy)
Satellite males (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)

Parental females:
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration)→ gestation → lactation → day 12 post partum
Satellite females (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)

Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25 days after the end of mating period

Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25th day after confirmed mating

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control - main study

Doses / concentrations 2

Dose / conc.:

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

Remarks:

main study

Doses / concentrations 3

Dose / conc.:

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

Remarks:

main study

Doses / concentrations 4

Dose / conc.:

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

Remarks:

main study

Doses / concentrations 5

Dose / conc.:

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

Remarks:

control - DRFE

Doses / concentrations 6

Dose / conc.:

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

Remarks:

DRFE

Doses / concentrations 7

Dose / conc.:

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

Remarks:

DRFE

Doses / concentrations 8

Dose / conc.:

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

Remarks:

DRFE

No. of animals per sex per dose:

DRFE
1. Control 0 6 males + 6 females
2. Low dose 100 mg/kg/day 6 males + 6 females
3. Intermediate dose 300 mg/kg/day 6 males + 6 females
4. High dose 1000 mg/kg/day 6 males + 6 females

Main study
The first six males and six females with delivered pups per group and satellite groups of animals (control and treated) are a part of the repeated dose toxicity part of study.
Basic groups:
1. Control 0 12 males + 12 females
2. Low dose 80 mg/kg/day 12 males + 12 females
3. Intermediate dose 250 mg/kg/day 12 males + 12 females
4. High dose 750 mg/kg/day 12 males + 12 females

Satellite groups:
5. Control – vehicle – satellite 0 6 males + 6 females
6. High dose – satellite 750 mg/kg /day 6 males + 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the result of the DRF study on the substance, the selected highest tested dose for the main study should have been 1000 mg/kg bw/day, since no significant toxicological effects have been observed at this dose and it could have been considered as limit dose. However, it should be noticed that this study has been performed not only to investigate the toxicological properties of the substance in itself, but also and mainly to compare the effects within the members of the category; therefore the highest tested dose has been selected as below reported, taking into account the existing following studies:

• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on kidney at 750 mg/kg bw/day
• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on testes at 750 mg/kg bw/day
• 3a-MSA – Subacute toxicity study – Effects to be confirmed on haematological parameters, liver and kidney starting from 200 mg/Kg bw/day
• 3a-MSA – Two generation toxicity study - Relevant toxicological effects mainly in terms of maternal toxicity at 1000 mg/kg bw/day and minor effects at 300 mg/kg bw/day

Indeed, in this respect all systemic studies concerning repeated dose, reproductive and developmental toxicity (OECD 422, 408 and 414) have been set on similar dose level in order to be able to compare all members of the category on the specific organ effects.
The lowest and the medium doses have been selected in order to have a coherent spacing of doses respect to the highest selected one, also taking into account all the available data on the repeated dose toxicity ( 3a-MSA, 1-DSA).

- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:

Body weight:
males and satellite animals - the first day of administration and then weekly,
females - the first day of administration and then weekly,
during pregnancy: 0, 7th, 14th, 20th day,
during lactation: 1st, 4th day, 12th day and 13th day
pups (litters) - 1st, 4th day, 12th day and 13th day
pups – individually – 4th day of lactation
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too.
Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

Food consumption: weekly and on the same days as body weight (except the mating period); satellite males and females – weekly
In 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 × 100) was calculated for animals of the repeated dose toxicity part of study. In the pre-mating period, the food consumption and conversion of females was calculated from values of all females.

Water consumption: satellite males and females – twice a week
The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

Mortality control
All rats during the treatment periods were examined for vitality or mortality twice daily.

Health condition control
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before application, during application and immediately after application.

Clinical observations of parental males and females
All rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.m.). Animals were observed in natural conditions in their cages.

Detailed clinical parental observation
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, breathing, tonic or clonic movements, stereotypes or bizarre behaviour. The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

Functional observation of parents
This observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. The individual observations of grip strength were performed using a grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.

Urinalysis
This 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 prolonged periods). 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 per 100 g of body weight by gavage to the stomach.
Urinalysis parameters:
Volume
Colour
Cloud
Odour
Glucose - GLU
Protein - PRO
Bilirubin - BIL
Urobilinogen - URO
pH
Specific gravity
Blood - BLD
Ketones - KET
Nitrite - NIT
Leucocytes - LEU

Haematological examination
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation system.
Haematological parameters:
Total erythrocyte count - RBC
Mean corpuscular volume - MCV
Haematocrit - HCT
Haemoglobin concentration - HGB
Total leucocyte count - WBC
Total platelets count - PLT
Partial thromboplastin time - APTT
Prothrombin time - PT
Fibrinogen - FIB
Lymphocytes - LYM
Monocytes - MON
Neutrophiles - NEU
Eosinophiles - EOS
Basophiles - BASO
Reticulocyte count was examined by the help of light microscope.

Biochemical examination
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals were fasted approximately for 18 hours before blood collection but they were supplied drinking water ad libitum.
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
Biochemical parameters:
Protein total - T-Pro
Alkaline phosphatase - ALP
Cholesterol total - T-Chol
Triglycerides - TG
Alanine aminotransferase - ALT
Aspartate aminotransferase - AST
Creatinine - Crea
Urea - BUN
Albumin - ALB
Bilirubin total - T-Bil
Glucose - GLU
Calcium - Ca
Phosphorus - IP
Cholinesterase - CHE
Bile acids - BA
Sodium - Na
Potassium - K
Chloride - Cl

Blood samples from parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) and rat thyroid stimulating hormone (TSH).

Oestrous cyclicity (parental animals):

Examination of vaginal smears
Vaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of oestrous cycle of females. Only females with regular cyclicity were put into the study. Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa. Vaginal smears have been made also on necropsy day to determine the stage of oestrous cycle.

Sperm parameters (parental animals):

Observation of sperm
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.

Sperm motility
Sperm 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 morphology
Sperm 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, and abnormal form of neck – were recorded.

Litter observations:

Clinical observation
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.

Biochemical examination
Blood samples from the day 13 pups (pooled sample, one male + one female pup) were assessed for serum levels of thyroid hormone thyroxine (T4 total) and rat thyroid stimulating hormone (TSH).

Anogenital distance (AGD) measurement
The AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalised to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.

Nipples examination
The presence and number of nipples in male pups were counted on day 13 of lactation. The presence of nipples in male pup on day 13 of lactation is undesirable (non-physiological).

Postmortem examinations (parental animals):

Pathological examination
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.

Biometry of organs
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymis/epididymides or uterus, prostate gland + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (reproduction part of study – all animals).
Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ × 100/ body weight. From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

Histopathological examination
Organs for histopathological examination
In all males and females: pituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland + seminal vesicles, testes, thyroid gland, all gross lesions.
In males and females of repeated dose toxicity part of study additionally: adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord – thoracic, spleen, stomach, thymus, thyroid gland, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mesenteric, paraaortal, oesophagus, eye

The mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

In 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. Because of treatment related changes were recorded at the highest dose level in the liver, the histopathological examination of liver was performed also in males and females at the middle and at the lowest dose level.
In reproductive toxicity part of study, the full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Detailed histological examination of testes was performed with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. Histopathological examination of macroscopically changed organs was performed in one female (uterus dilatation) at the dose level 80 mg/kg/day and in one female (ovarian cyst) at the dose level 250 mg/kg/day.

Postmortem examinations (offspring):

From one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

Statistics:

For statistical evaluation the software Statgraphic® Centurion (version XVII, USA) was used.
Males/females from control group were compared with males/females from 3 treated groups. Satellite males/females from control group were compared with satellite males/females from treated group. The results statistically significant on probability level (p ≤ 0.05) were indicated in the summary tables.

The parametric tests were used for statistical evaluation of
• body weight of males and females
• mean pup weight
• litter weight
• anogenital distance of pups
• selected haematology parameters
• blood biochemistry parameters
• data from urinalysis (pH, volume)
• data from biometry of organs

As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.
For normally distributed data have at first the variance check has been performed (Levene´s test)to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).

The non-parametric tests were used for statistical evaluation of
• selected reproduction parameters with non-continuous distribution (no. of live born pups, no. of pups, no. of implantations)
• selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)

The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤ 0.05).

Reproductive indices:

For each dose group the following fertility parameters were calculated:

Fertility parameters - group parameters
Male mating index = 100 × number of males with confirmed mating / number of males cohabited
Female mating index = 100 × number of sperm-positive females / number of females cohabited
Male fertility index = 100 × number of males impregnating a female / number of males cohabited
Female fertility index = 100 × number of pregnant females / number of sperm-positive females
Gestation index = 100 × number of females with live born pups / number of pregnant females

Offspring viability indices:

For each of parental females the following parameters were calculated:
viability index on PND4 = 100 × number of pups surviving on PND4 / number of pups born alive
post-implantation loss = number of implantations – number of live births
post-natal loss = number of live births – number of alive at PND4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of disease and clinical signs of intoxication were recorded during the application period in all treated animals. No clinical changes were recorded in control or treated males during the application period.
The behaviour, clinical status and activity of treated animals were similar during the study and not different from males of the control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males
There were no unscheduled deaths during the study.

Females
Two females died at the dose level 80 mg/kg/day. Female No.126 died on the 39th day of application due to the intubation error. Female No.127 died on the 41stday of application during complicated delivery.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males
Body weight was not affected by the test item treatment and was well-balanced between treated groups of males and control males. Body weight increment of all treated males was variable as well in the control group and not adversely affected by the test item.
Statistically significant differences in necropsy body weights were not found in treated males.

Females
Pre-mating period
Body weights of treated females were comparable with the control group of females. The negative body weight increment was recorded in females during the 1st week at the dose level 250 mg/kg/day. The mean body weight increments of all treated pregnant females were similar to the control females during the 2nd week

Pregnancy
Non-pregnant females and females with implantations but without live pups born were not included in the evaluation of mean body weight increments during pregnancy.
Mean body weight of all treated groups was comparable to the control group during whole pregnancy.
The mean body weight increments of all treated pregnant females were similar to the control females during whole pregnancy.

Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The body weight of females was similar with control females during the lactation period.
The mean body weight increments of treated mothers were comparable to control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males
The mean food consumption of treated males was comparable with the control males. No marked differences were recorded.

Females
The mean food consumption of treated females before mating was similar with the control females.

Pregnancy
Females without parturition (non-pregnant or females with implantations, but without pups) were not included in the evaluation of food consumption during pregnancy. The mean food consumption of pregnant females treated by the test item was comparable with the food consumption of control females.

Lactation
Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period. The mean food consumption of pregnant females treated by the test item was comparable with the food consumption of control females.

Description (incidence and severity):

Details are available in the repeated dose toxicity part of the study.

Description (incidence and severity):

Details are available in the repeated dose toxicity part of the study.

Ophthalmological findings:

not examined

Description (incidence and severity):

Details are available in the repeated dose toxicity part of the study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical examination - thyroid hormones
Blood (serum) samples from all adult parental males were assessed for thyroid hormones thyroxine (T4 total) and rat thyroid stimulating hormone (TSH). Mean concentrations of T4 hormone was significantly increased at the dose level 750 mg/kg/day, but in range of historical control. Mean concentrations of TSH hormone at all dosed groups were comparable with control group.

Description (incidence and severity):

Details are available in the repeated dose toxicity part of the study.

Description (incidence and severity):

Details are available in the repeated dose toxicity part of the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Males
Full histopathology of the preserved organs and tissues was performed for high dose and control animals (in the repeated dose toxicity part of the study).
As there were no treatment-related changes in reproductive organs at the highest dose level, histopathologic examination was not performed in animals from dose levels 80 and 250 mg/kg/day. The incidence of affected males is expressed in numeric form and ranges in sequence of the dose levels 0 - 750 mg/kg/day.

For examination of the reproduction toxicity of the test item, microscopic examination with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure was performed for all males of control (No 1-12) and high dose (No 61-72).
Microscopical examination of reproductive organs did not reveal presence of treatment-related changes (therefore examination of reproductive organs was performed only in control and high dosed males). The histological examination confirmed that the spermatogenesis was not affected by the test item treatment. In the testicles was detected the degeneration of the germinal epithelium and tubular atrophy in 1 – 3 males. This change was considered as non-specific change in animals. The focal atrophy of prostate was recorded in 3 – 1 males. This change occurs spontaneously in aging rats. The non-specific change as the focal lymphacytic infiltration of the interstitium was detected in prostate of 5 – 3 males. The cellular detritus in the alveolar lumen in coagulation gland was recorded in 0 – 2 males. Pituitary gland of both control and treated males was without any pathological lesions, only hemangiectasia was recorded in one male at the highest dose level. The incidence of other microscopical findings were very sporadic.

Organ/Diagnosis 0 80 250 750
Number of examined animals 12 0 0 12
Number of died animals/Mortality 0 0 0 0
Without findings 2 0 0 2
Pituitary gland: hemangiectasia 0 / / 1
Thyroid gland: basophilic precipitates of colloides 0 / / 1
Thyroid gland: focal hyperplasia 0 / / 1
Prostate gland: focal atrophy 3 / / 1
Prostate gland: focal chronic inflammation 0 / / 1
Prostate gland: edema and/or lymphocytic infiltration of the interstitium 5 / / 3
Prostate gland: cellular detritus in the alveolar lumen 0 / / 1
Coagulation gland: cellular detritus in the alveolar lumen 0 / / 2
Testicles: focal degeneration of the germinal epithelium/focal tubular degeneration 1 / / 3

Females
Full histopathology of the preserved organs and tissues was performed for high dose and control females (in the repeated dose toxicity part of the study). For examination of the reproduction toxicity of the test item, microscopic examination of the reproductive organs, pituitary and thyroid gland was performed for high dose females (Nos. 161-172) and control females (Nos. 101-112).
Because the treatment-related changes at the highest dose level in reproductive organs were not found, only histopathological examination of macroscopically changed organs was performed. Macroscopical changes on the uterus (dilatation) was recorded only in female No. 129 at the dose level 80 mg/kg/day. Histologically was detected hydrometra of uterus. Macroscopical change in ovary (cyst) was detected in female No.149 at the dose level 250 mg/kg/day. Histologically was detected luteal cyst in ovary.
The incidence of affected females is expressed in numeric form and ranges in sequence for the dose levels 0 – 80 – 250 – 750 mg/kg/day.
Microscopic examination of reproductive organs, thyroid gland and the pituitary gland did not reveal the presence of treatment-related changes. The changes related to pregnancy were found in control and treated females: accumulation of siderophages in mucosa of the uterus was recorded in 2 – / – / – 1 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 4 – 1 – / – 1 females. Pituitary gland of both control and treated rats was without any pathological lesions, only hemangiectasia was recorded in 1 – / – / – 1 females. The non-specific change the dilatation of retinal ovary was detected in 2 – / – 0 – 1 females. The incidence of other microscopical findings was very sporadic and unrelated to treatment. These findings are mentioned in individual tables.

Organ/Diagnosis 0 80 250 750
Number of examined animals 12 1* 1* 12
Number of died animals/Mortality 0 2 0 0
Without findings 1 0 0 4
Pituitary glandHypophysis: hemangiectasia 1 / / 1
Thyroid gland: basophilic precipitates in colloides of follicle 0 / / 1
Ovary: luteal cysts 1 / 1 0
Ovary: dilatation of the retinal ovary 2 / 0 1
Ovary: proliferation of the interstitial glands 0 / 0 1
Uterus: siderophages in the mucosa 2 0 / 1
Uterus: hydrometra 4 1 / 1

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycles were monitored before treatment started to select females for the study with regular cyclicity. Vaginal smears of all females were monitored daily for two weeks. It was found that all the females had regular oestrus cycle.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology. Male rats ability to produce sperm that can fertilise eggs was not affected by the test item administration

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproduction parameters
Evidence of copulation was found out in all females.
A decreased number of females achieving pregnancy was recorded at the control dose level as well as at the dose level 750 mg/kg/day. No abortions were recorded in the treated groups. The mean duration of pregnancy was similar in treated and control groups.
The mean number of implantations was comparable at all groups of treated females with the control females, except the highest dose level.
The statistically significantly increased number of implantation was recorded at the dose level 750 mg/kg/day in comparison with control.

The mean number of live pups at the first litter check after parturition was comparable in treated groups with the control group of females.
The stillborn were not found at any group of treated females as well as in females at the control group.
The mean weights of litters of treated groups at birth, at PND 4 (postnatal day) and PND 13 were comparable with the litter weights of control group.

Fertility parameters
The values of mating indexes showed that mating was not negatively affected by the test item treatment.
Fertility indexes were higher in the treated groups than in the control.
The gestation index was similar or higher in the treated groups in comparison with the control group; the viability index on PND 4 was comparable in treated and control group.
Post-implantation losses were slightly higher in treated groups in comparison with the control group. Post-natal losses were comparable in all groups.

Details on results (P0)

Repeated oral administration of the test item to rats by gavage did not cause the death of animals. Two females died at the dose level 80 mg/kg/day (female No. 126 died due to intubation error and female No. 127 died during the complicated delivery). These deaths were accidental and not treatment-related.

The effect of the test item treatment on growth of animals was not adverse. The food consumption was similar between control and treated animals. Individual variability of body weight increments was adequate to species, sex and age of animals used in the study and to period of gravidity or lactation in females.

Test item treatment did not produce clinical changes in health status of animals.
All twelve males per group (Nos. 1-12, 21-32, 41-52, 61-72) and females (101-112, 121-132, 141-152, 161-172) were examined from the main groups.

Test item treatment did not affect male ability to produce sperm that can fertilise eggs and ability of females to become pregnant.

Mean concentrations of T4 hormone was statistically significantly increased at the dose level 750 mg/kg/day, but in range of historical control. Mean concentrations of TSH hormone at all dosed groups were comparable with control group at all dose levels.

The body weight of parental males and females was not affected by the test item application.
Evaluation of the absolute and relative weight of reproductive organs of male and female, as well as the weight of pituitary and thyroid gland, did not reveal any statistically significant differences in organ weight.
Microscopic examination of reproductive organs, thyroid gland and pituitary gland in males revealed tubular degeneration in testicles in one control male and in three males at the dose level 750 mg/kg/day. Because the relative weight of testicles and spermiogenesis of the males were not affected with the test item treatment, this change was considered as non-specific, unrelated to the effect of the test item treatment. The incidence of other microscopical findings were in most cases spontaneous or agonal changes, or sporadic, not associated with the test item treatment.
Microscopic examination of reproductive organs, thyroid gland and pituitary gland in females did not reveal presence of treatment related changes. Sporadic findings were of spontaneous origin.

Examination of sperm motility and morphology in treated parental males did not show any differences in comparison with the control males.
Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. The ability of parental animals to successfully mate, to achieve pregnancy and to give birth live pups was not influenced by the test item administration. The number of implantations was statistically significantly increased at the dose level 750 mg/kg/day, but without toxicological significance. The post-implantation losses were very slightly increased in treated groups in comparison with the control.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Description (incidence and severity):

The total number of live pups at the first litter check after parturition, on the 4th day and 13th day of lactation, was higher in all treated groups than in control group. No stillborn pups were recorded in any group of females.
The mean number of live pups at the first litter check after parturition was in all treated groups comparable with the control group of females.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of litters and pups was comparable or slightly higher than in the control group. The statistically significantly increased weights of pup were detected at the dose levels 80 and 250 mg/kg/day

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical examination – thyroid hormones
Blood samples from the 13-day old pups (pooled sample; one male and one female pup per litter) were assessed for serum levels of thyroid hormone thyroxine (T4) and rat thyroid stimulating hormone (TSH). No statistically significant differences were recorded in the mean concentration of hormone T4 and TSH in pups from treated groups against control pups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Corrected anogenital distance did not show significant changes among the treated groups and control group. No differences in the postnatal development of pups were observed in the control and treated groups.


.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The presence of nipples in male pups was checked on day 13 of lactation and none were present (nipple presence in male pups on day 13 of lactation is undesirable).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The weight of the thyroid glands in male and female pups was comparable with the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The clinical observation of pups was made daily during the study. The macroscopic examination was performed on all surviving pups and/or pups found dead. The examination could not be performed on pups that disappeared due to cannibalism and/or in dead pups with autolysed organs.

Control: 99 of 100 born pups were examined.
Female No. 106: one pup of the nineteen pups disappeared because of cannibalism.
No macroscopical findings were recorded in other pups/litters.

80 mg/kg/day: 111 of 113 born pups were examined.
Female No. 121: one pup of thirteen disappeared because of cannibalism.
Female No. 131: one pup of twenty-one disappeared because of cannibalism.
No macroscopical findings were recorded in other pups/litters.

250 mg/kg/day: 154 of 157 born pups were examined.
Female No. 141: two pups of sixteen disappeared because of cannibalism.
Female No. 142: one pup of fourteen disappeared because of cannibalism.
No macroscopical findings were recorded in pups/litters.

750 mg/kg/day: 136 of 136 born pups were examined.
No macroscopical findings were recorded in other pups/litters.

Histopathological findings:

no effects observed

Description (incidence and severity):

Histopathological examination of thyroid glands of pups of females from the high dose group did not reveal any pathological finding.

Details on results (F1)

The test item did not affect the number of pups and their development. The total number of pups and their mean body weights at first litter check after parturition and during the next intervals were not negative affected by the test item treatment.
Macroscopical examination of pups did not show negative effect of the test item administration on development of pups. No differences in postnatal developmental were observed in pups at the treated groups – presence of nipples in male pups was not recorded. Corrected anogenital distance in treated pups was comparable to the control pups.
No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups. Microscopical evaluation of thyroid gland of pups did not show any findings related to the test item treatment.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: AGD, nipples retention

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Dose range finding experiment

DRF 422	dose (mg/kg)				notes
	0	100	300	1000
For dams (per dose)
Mortality	0	0	0	0
Body Weight F [group mean ± standard deviation (g)]	442.12 ± 21.36	447.87 ± 37.48	450.16 ± 31.02	433.70 ± 33.40	at 20th day of pregnancy
Clinical signs: description, severity, time of onset and duration	No effects	No effects	No effects	No effects
Haematological findings	No effects	No effects	No effects	No effects
Number of pregnant dams	6/6	6/6	6/6	6/6
Implantations [mean ± SD]	15.53 ± 1.86	15.17 ± 3.66	17.17 ± 1.94	16.17 ± 2.79
Resorptions [mean ± SD]	1.00 ± 1.26	0.33 ± 0.52	0.67 ± 0.82	2.17 ± 4.83
Corpora lutea [mean ± SD]	16.33 ± 1.03	16.50 ± 1.38	17.67 ± 2.25	17.33 ± 2.66
Pathological examination	No effects	No effects	No effects	No effects
For foetuses/offspring (per dose)
Total number of live foetuses	86	89	99	84
Total number of dead foetuses	0	1	0	0
External, soft tissue and skeletal malformations and other relevant alterations	No effects	No effects	No effects	No effects

Main study

	dose (mg/kg)						notes
	0	80	250	750	0S	750S
For dams (per dose)
Number of pregnant dams	8/12	10/12	12/12	9/12	Not examined	Not examined
Females with abortion	1	0	0	0	Not examined	Not examined
Females with live pups born	7/12	8/12	12/12	9/12	Not examined	Not examined
Mortality and day of death	0	2/12 one died on the 39th day due to anintubation error, one died on the 41st day during complicated delivery	0	0	0	0
Clinical signs: description, severity, time of onset and duration	No effects	No effects	No effects	No effects	No effects	No effects
Haematological findings	No effects	No effects	No effects	No effects	No effects	Statistically significantly decreased value of reticulocytes [2.18 % vs 2.65 %]
Clinical biochemistry findings	No effects	No effects	No effects	No effects	No effects	No effects	Statistically significantly increased value of T-Chol was reported in females at the dose level 250 mg/kg/day (in the historical control range). Statistically significant difference was found out in treated females in value of Ca at the dose levels 250 and 750 mg/kg/day, but still in the historical control range. The value of TG was statistically insignificantly increased at the dose level 250 mg/kg/day in comparison with control (over the historical control range). The values of AST and ALT were increased at the dose level 750 mg/kg/day, without statistical significance but out of the range of historical control. The value of ALP was insignificantly increased at the dose level 750 mg/kg/day. Statistically significantly decreased value of CHE and increased value of T-Chol were noted in satellite treated females in comparison with the satellite control females. The value of ALT and AST were statistically insignificantly decreased in treated satellite females (in range of the historical control).
Mean number of implantations	13.63	16.75	14.50	18.56	Not examined	Not examined	The number of implantations was statistically significantly increased at the dose level 750 mg/kg/day, but without toxicological significance.
Number of live born pups - at birth (mean)	14.29	14.13	13.08	15.11	Not examined	Not examined
Mean number of resorptions (early and late)	n.a.	n.a.	n.a.	n.a.	Not examined	Not examined
Number of stillborn pups (total number/mean)	0/0	0/0	0/0	0/0	Not examined	Not examined
Mean Post-natal losses (live births minus alive at day 4)	0.14	0.25	0.25	0.00	Not examined	Not examined
Mean Post-implantation losses (implants minus live births)	0.86	2.63	1.42	3.44	Not examined	Not examined	The post-implantation losses were very slightly increased in treated groups in comparison with the control.
Number of corpora lutea	Not examined	Not examined	Not examined	Not examined	Not examined	Not examined
Mean duration of pregnancy (days)	21.57	22.38	22.00	21.78	Not examined	Not examined
Body weight	No effects	No effects	No effects	No effects	No effects	No effects
Body weight changes	No effects	No effects	No effects	No effects	No effects	No effects
Reproductive organ weight changes	No effects	No effects	No effects	No effects	No effects	No effects	absolute and relative weights of ovaries, uterus, pituitary gland and thyroid gland in treated females were comparable with the control group
Histopathological findings: nature and severity	No significant treatment-related changes	No significant treatment-related changes	No significant treatment-related changes	No significant treatment-related changes	Not examined	Not examined
Necropsy findings	No significant treatment-related changes	No significant treatment-related changes	No significant treatment-related changes	No significant treatment-related changes	Not examined	Not examined	Since treatment-related changes at the highest dose level in reproductive organs were not found, only histopathological examination of macroscopically changed organs was performed.
Pituitary gland: hemangiectasia	1/6	Not examined	Not examined	1/6	Not examined	Not examined
Thyroid gland: basophilic precipitates in colloides of follicle	0/6	Not examined	Not examined	1/6	Not examined	Not examined
Ovary: luteal cysts	1/6	Not examined	1/6	0/6	Not examined	Not examined
Ovary: dilatation of the retinal ovary	2/6	Not examined	0/6	1/6	Not examined	Not examined
Ovary: proliferation of the interstitial glands	0/6	Not examined	0/6	1/6	Not examined	Not examined
Uterus: siderophages in the mucosa	2/6	0/6	Not examined	1/6	Not examined	Not examined
Uterus: hydrometra	4/6	1/6	Not examined	1/6	Not examined	Not examined
Absolute uterine weight [g±SD]	0.6236 ± 0.2108	0.6015 ± 0.1575	0.5120 ± 0.1005	0.5941 ± 0.0642	Not examined	Not examined
Results of the thyroid hormone measurements	Not examined	Not examined	Not examined	Not examined	Not examined	Not examined	Only parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) and rat thyroid stimulating hormone (TSH) by ELISA kit. Data was reported in the repeated-dose part.
For males (per dose)
Absolute weight of reproductive organs	No effects	No effects	No effects	No effects	Not examined	Not examined
Observation of Sperm	No effects	No effects	No effects	No effects	Not examined	Not examined	A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology.
Histopathological findings	No treatment-related changes	Not examined	Not examined	No treatment-related changes	Not examined	Not examined	As there were no treatment-related changes in reproductive organs at the highest dose level, histopathologic examination was not performed in animals from dose levels 80 and 250 mg/kg/day. The histological examination confirmed that the spermatogenesis was not affected by the test item treatment. In the testicles was detected the degeneration of the germinal epithelium and tubular atrophy in 1 – 3 males. This change was considered as non-specific change in animals. The focal atrophy of prostate was recorded in 3 – 1 males. This change occurs spontaneously in aging rats. The non-specific change as the focal lymphacytic infiltration of the interstitium was detected in prostate of 5 – 3 males.
For foetuses/offspring (per dose)
Total number of live born pups	100	113	157	136	Not examined	Not examined
Sex ratio	61M/39F	55M/58F	81M/76F	70M/66F	Not examined	Not examined
Mean pup weight at birth (g)	6.58	7.22	7.19	6.79	Not examined	Not examined
Mean pup weight at day 4 after parturition (g)	9.79	11.00	11.04	10.39	Not examined	Not examined
Mean pup weight at day 13 after parturition (g)	24.72	28.19	27.97	25.73	Not examined	Not examined
External, soft tissue and skeletal malformations and other relevant alterations	No effects	No effects	No effects	No effects	Not examined	Not examined
Number and percent of foetuses and litters with malformations and/or variations	0	0	0	0	Not examined	Not examined

Applicant's summary and conclusion

Conclusions:

NOAEL (No Observed Adverse Effect Level) for reproduction and development was established as 750 mg/kg body weight/day in both sexes. No biologically significant changes were observed.

Executive summary:

Study design

Before the start of study with laboratory animals, the stability and homogeneity of application form were determined at the test facility. A dose-range finding experiment (DRFE) was performed at doses of 100, 300 an 1000 m/k/day to determine the dose levels for the main study.

The doses of 80, 250, 750 mg/kg/day were determined on the basis of results of DRFE and approved by Sponsor.

Methods

Wistar rats (SPF quality) were used for testing. The test item was administered in the form of a solution in aqua pro iniectione. Oral application by stomach tube was performed daily. The animals were without feed two hours before application and two hours after application of the test item. The main study includes 3 treated groups (doses 80, 250, 750 mg/kg/day) and one control group (vehicle only) and two satellite groups of animals - one control group (vehicle only) and one treated group (750 mg/kg/day).

Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

The first 6 males and 6 mothers who delivered pups per group (as per internal SOP) and a satellite groups of animals (control and treated) are part of the repeated dose toxicity study and examined with respect to toxicity of the test item. Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects up to 14 days post treatment. All 12 males and females per group are a part of the reproduction study and examined with respect to reproduction parameters.

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 up to the 12th day of lactation; males – after mating period – 49 days in total; non-pregnant females (mated females without parturition) – for 25 days after confirmed mating; non-mated females – for 25 days after the end of the mating period. After the end of the administration period, the animals in the main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.    

During the study, clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or at the specified time intervals. Detailed clinical observation was carried out weekly. Functional observations were performed at the end of the application and observation periods. Vaginal smears were prepared daily, 2 weeks before start of the administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy. Reproduction parameters relevant to pups (number of pups, weight of litters and weight of pups, sex and vitality of pups, measurement of anogenital distance, nipple retention, serum levels of thyroid hormones (T4 and TSH in pups) were also recorded. The study was completed with urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of the main groups, the sperm parameters, sperm motility and sperm morphology were examined. Selected organs from adult animals and pups were removed for weighing and histopathological examination.

Results - reproduction part of the study

Repeated oral administration of the test item did not cause any mortality (except two females from the dose level 80 mg/kg/day – female No. 126 died due to intubation error and female No. 127 during the complicated delivery). These deaths were accidental and not treatment-related.

All 12 males per group (Nos. 1-12, 21-32, 41-52, 61-72) and females (101-112, 121-132, 141-152, 161-172) were examined from the main groups.

Test item treatment did not affect male ability to produce sperm that can fertilise eggs and ability of females to become pregnant.  

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in males. Mean concentrations of T4 hormone was significantly increased at the dose level 750 mg/kg/day, but in range of historical control. Mean concentrations of TSH hormone at all dosed groups were comparable with control group at all dose levels.

The body weight of parental males and females was not affected by the test item administration.

No macroscopical findings related to the test item treatment were recorded during the pathological examination of treated males and females reproductive organs.

No significant changes of absolute and relative weights of genital organs, pituitary and thyroid gland in males and females were recorded.

Microscopic examination of reproductive organs, thyroid gland and pituitary gland in males revealed tubular degeneration in testicles in 1 control male and in 3 males at the dose level 750 mg/kg/day. Because the spermiogenesis of the males was not affected with the test item treatment, this change was considered as non-specific, unrelated to the effect of the test item treatment. The incidence of other microscopical findings were in most cases spontaneous or agonal changes, or sporadic, not associated with the test item treatment.

Microscopic examination of reproductive organs, thyroid gland and pituitary gland in females did not reveal presence of treatment related changes. Sporadic findings were of spontaneous origin.

Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. The ability of parental animals to successfully mate, to achieve pregnancy and to give birth live pups was not influenced by the test item administration. The number of implantations was statistically significantly increased at the dose level 750 mg/kg/day, but without toxicological significance. The post-implantation losses were very slightly increased in treated groups in comparison with the control.

The test item did not affect the number of pups and their development. The total number of pups and their mean body weights at first litter check after parturition and during the next intervals were not adversely affected by the test item treatment. Macroscopical examination of pups did not show negative effect of the test item administration on development of pups. Corrected anogenital distance in treated pups was comparable to the control pups.  

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups. Microscopical evaluation of thyroid gland of pups did not show any findings related to the test item treatment.

Conclusions

Accordingly, the NOAEL for reproduction and development was established as 750 mg/kg body weight/day in both sexes. No biologically significant changes were observed.