Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The test substance did not cause any adverse effects up to the highest dose tested in an OECD422 study, 100 mg/kg bw/day. Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD and used as the highest dose in the OECD422 study. There was no evidence of treatment related effect in the OECD422 on fertility at the top dose of 100 mg/kg bodyweight per day. 

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
29 October 2013 to 13 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with OECD Guideline with GLP certificate.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
no deviations considered to have an impact on the outcome of the study and interpretation of the results.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: Young adult rats, approximately 11 weeks old at starting and 13 weeks at mating. The age range within the study was kept to the minimum.
- Weight at study initiation: Males: 351 g – 411 g, Females: 208 g - 242 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment.
- Fasting period before study: Not specified
- Housing: Rodents were group-housed, up to 3 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting). Cage type: Type II and/or III polypropylene/polycarbonate. Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 24.0 °C
- Humidity (%): 32 – 62 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 October 2013 (start of treatment) To: 13 December 2013 (last necropsy)

Animal identification:
Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth,
Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was formulated in PEG 400, as a visibly stable homogenous formulation at 5, 15 and 50 mg/mL concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared up to 7-day intervals, based on the stability test performed at the Analytical Laboratory of CiToxLAB Hungary Ltd. Analysis of PREPOLYMER D formulation samples of 3 – 150 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 24 hours at room temperature and 7 days in refrigerator. Homogeneity and stability of the test item in the vehicle (PEG 400) was verified by a GC/FID method supplied by the Sponsor before the first dosing.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected by the Sponsor in consultation with the Study Director based on available data, formulation and analytical trials and information from previous experimental work, including the results of a preliminary dose range finding study in the rat.
- Concentration in vehicle: 5, 15 and 50 mg/mL concentrations according to the dose level and volume selected
- Amount of vehicle: A constant volume of 2 mL was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.
- Lot/batch no. (if required): BCBK9981V
- Purity: Not specified
Details on mating procedure:
- M/F ratio per cage: 1/1 for the mating period
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 4 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Sperm positive females were caged individually.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity were performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment. One set was taken to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution for concentration measurements.

Analytical method:
To each of the 1 mL formulation sample, 5 mL of Hexane and 1 mL of saturated Sodium-chloride solution were added. The samples were stirred vigorously for 45 minutes.
5 and 15 mg/mL formulations: the Hexane phase was subjected to GC analysis without further dilution.
50 mg/mL formulation: the hexane phase was diluted by 5 fold prior to GC analysis.

Results:
Test item content of the dosing formulations was determined on 3 occasions during the study. The measured concentrations of PREPOLYMER D evaluated for each test item-dose group varied between 90 and 103%. No test item was detected in the control samples. These results were within acceptable ranges (90 % - 110%).
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 4 days mating period, through gestation and up to the day before the necropsy (at least 4 days post-partum dosing). Females showing no-evidence of copulation were sacrificed, 25-27 days after the end of the mating period.
Frequency of treatment:
Once daily
Details on study schedule:
Dosing of both sexes began after an acclimation period (A) of 5 days after the animal arrival; the animals were dosed for 2 weeks before mating, during the mating/post-mating, and were continued up to the day before the necropsy.

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.

Females were dosed for 14 days pre-mating, for up to 4 days mating period, through gestation and up to the day before the necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed, 25-27 days after the end of the mating period.

All F1 offspring were terminated on Day 4 post-partum; in order to allow for overnight fasting of dams prior to urine collection on PPD5, offspring were euthanized on PND/PPD 4, and the dams on PPD/PND 5.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day (control)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 male + 12 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data, formulation and analytical trials and information from previous experimental work, including the results of a preliminary dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 13/227-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. During Phase 2 (14 days’ Repeat Dose Phase) in the DRF, treatment-related mortality (4 out of 6) was observed at 200 mg/kg bw/day in females. There was no mortality in the lower dose groups (100 and 50 mg/kg bw/day). From this information, a dose of 200 mg/kg bw/day caused lethal effects and was hence well in excess of the MTD, so although no toxicity was observed at 100 mg/kg bw/day, this dose level was considered to be the MTD.The oral route was selected as it is a possible route of exposure to the test item in humans.
- Randomization: All parental (P) animals were sorted according to body weight by computer and divided to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC+4.0 software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Positive control:
No positive control
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment. All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical.

The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

5 males and 5 females/group, “subgroup A”: Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 24 and 27 am; females on PPD 4 am). In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes. Selected animal were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rats were dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.

Fore/hind grip strength measurements were conducted using a grip strength meter, an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.

Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of at least 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings are made to produce the appropriate parameters. At the first instance the data from the high dose and control groups was evaluated for distance travelled in 5 minute segments. Changes were seen in the high dose females, therefore the evaluation was extended to the lower groups. The results are tabulated for information with individual data for each parameter (distance travelled, average speed, % of time spent immobile, number of rearings and average duration of rearing).

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), PPD4 and PPD5 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
Observation of the delivery process and nursing instinct
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

CLINICAL PATHOLOGY
All animals selected for blood sampling was fasted (overnight period of food deprivation, after the litter has been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

For terminal blood sampling all selected animals (subgroup B, 5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times
The following parameters were evaluated in animals selected:
RBC Red Blood Cell (erythrocyte) count, (10 to power 12/L) M/mL (Method: Automatic laser cell count)
WBC White Blood Cell (leukocyte) count, (10 to power 9/L) K/mL (Method: Automatic laser cell count)
Hgb Haemoglobin concentration, (g/dL) (Method: Determination of cyan-methemoglobin absorbance)
Hct Haematocrit (relative volume of erythrocytes) (%) (Method: Computed by equipment)
MCV Mean Corpuscular (erythrocyte) Volume (fL) (Method: Laser cell volume determination)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg) (Method: Computed by equipment)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL) (Method: Computed by equipment)
RDW Red Cell (erythrocyte) volume (%) (Method: Distribution Width Laser detection)
Plt Platelet (thrombocyte) count, (109/L) K/mL (Method: Automatic laser cell count)
MPV Mean Platelet Thrombocyte volume (fL) (Method: Cell volume determination by laser)
RETIC % Reticulocyte count (%) (Method: Comparative value based on laser light detection)
NE % Neutrophil (%) (Method: Cell differentiation based on myeloperoxidase activity)
LY % Lymphocyte (%) (Method: Cell differentiation based on myeloperoxidase activity)
MO % Monocyte (%) (Method: Cell differentiation based on myeloperoxidase activity)
BA % Basophil (%) (Method: Cell differentiation based on myeloperoxidase activity)
EO % Eosinophil (%) (Method: Cell differentiation based on myeloperoxidase activity)
LUC % Large Unstained Cells (%) (Method: Cell differentiation based on myeloperoxidase activity)
Coagulation:
APTT Activated Partial Thromboplastin Time (sec) (Method: Micronized silica method)
PT Prothrombin Time (sec) (Quick method (Biggs, R., andR.G. MacFarlane(1962) Human Blood Coag.and its Disorders, Oxford)

Blood smears were prepared for all subgroup B animals. Evaluation of blood smears was not performed.

Clinical chemistry
The following parameters were evaluated in animals selected:
Glucose Blood sugar concentration (mmol/L) (Method: Colorimetric test (540 nm))
T-BIL Total Bilirubin concentration (μmol/L) (Method: End-point colorimetric (dual-wavelength) test (400 & 460 nm))
Urea Urea concentration (mmol/L) (Method: Colorimetric test (670 nm))
Chol. Cholesterol concentration (mmol/L) (Method: Colorimetric test(540 nm))
Creat. Creatinine concentration (μmol/L) (Method: Two-point rate test (670 nm))
Phos. Phosphorus concentration (mmol/L) (Method: Colorimetric test (680 nm))
Na+ Sodium concentration (mmol/L) (Method: Potentiometric test)
K+ Potassium concentration (mmol/L) (Method: Potentiometric test)
Ca++Calcium concentration (mmol/L) (Method: Colorimetric test (680 nm))
Cl- Chloride concentration (mmol/L) (Method: Potentiometric test)
Tot. Prot. Total Protein concentration (g/L) (Method: Colorimetric test (540 nm))
Alb. Albumin concentration (g/L) (Method: Colorimetric test (630 nm))
A/G Alb/glob ratio (Method: Calculated value)
AST/GOT Aspartate Aminotransferase activity (U/L) (Method: Multiple-point rate test (340 nm))
ALT/GPT Alanine Aminotransferase activity (U/L) (Method: Multiple-point rate test (340 nm))
ALKP Alkaline. Phosphatase – activity (U/L) (Method: Multiple-point rate test (400 nm))
Bile acids (umol/L) (Method: Colorimetric test (546 nm))

Urinalysis
The evaluation of the urine samples were performed as indicated below.
LEU / Leukocyte (Method: Medi-Test Stick 10)
NIT / Nitrite (Method: Medi-Test Stick 10)
pH (Method: Medi-Test Stick 10)
PRO / Protein (Method: Medi-Test Stick 10)
GLU / Glucose (Method: Medi-Test Stick 10)
UBG / Urobilinogen (Method: Medi-Test Stick 10)
BIL / Bilirubin (Method: Medi-Test Stick 10)
KET / Ketones (Method: Medi-Test Stick 10)
BLD / ERY Blood/Erythrocytes (Method: Medi-Test Stick 10)
SG / Specific Gravity (Method: Medi-Test Stick 10)
SED / Sediment (Method: Microscopic examination)
VOL / Volume (Method: Volumetric method)
Colour/appearance (Method: Observation)








Oestrous cyclicity (parental animals):
Not assessed. The female mating index and the female fertility index were calculated.
Sperm parameters (parental animals):
Parameters examined:
Testes and epididymides were weighed individually.
Histopathological evaluation of the male gonads in high dose and control males including testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
The efficiency of suckling was observed by the presence of milk in the pups' stomach.
The pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups.

GROSS EXAMINATION OF DEAD PUPS:
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All males were dosed for 28 days then were euthanized
- Maternal animals: Females were sacrificed on postpartal/ postnatal day 5. Females showing no-evidence of copulation were sacrificed, 25-27 days after the end of the mating period.

GROSS NECROPSY
- Gross necropsy was performed on all animals. Terminally, after completion of the treatment, animals were euthanised under pentobarbital anaesthesia, followed by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the females as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all parental animals were determined:

- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids

Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution.

On completion of the macroscopic examination the following tissues and organs were retained from all animals:

Gross findings, Animal identification (1), Brain (2), Heart (3), Large intestine (4), Lungs with bronchi (5), Adrenal gland, Lymph node (6), Eye with the optic nerve (7), Thyroid with parathyroid gland (7), Spinal cord (cervical lumbar, and thoracic levels), Larynx, Nasopharynx, Aorta (thoracic and abdominal), Ovary; oviduct, Spleen, Oviduct, Sternum with marrow, Clitoral gland / Preputial gland, Pancreas, Stomach, Epididymidis, Pituitary, Testis, Prostate, Thymus, Oesophagus, Salivary gland (10), Femur with marrow incl. joint, Sciatic nerve, Tongue , Seminal vesicle with coagulating gland, Trachea , Kidney, Urinary bladder, Skeletal muscle (quadriceps), Uterus (9), External lacrimal gland, Skin/subcutis with mammary gland area(inguinal), Vagina, Harderian gland, Liver, Small intestine (8).

1. Fixation and preservation only.
2. Cerebral cortex, midbrain, cerebellum and medulla.
3. Section including both ventricles and atria, septum with papillary muscle.
4. Caecum, colon and rectum.
5. Lungs of euthanized animals will be infused with formalin.
6. Mandibular and mesenteric.
7. Parathyroids and optic nerves will be examined histologically only if present in routine sections.
8. Duodenum, ileum and jejunum with Peyer’s patches.
9. Horns, body and cervix.
10. Salivary glands (including mandibular, sublingual and parotid glands)

For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• all macroscopic findings (abnormalities), except of minor order from all animals
• reproductive organs of all animals of the control and high dose group and of all males (testes, epididymides, prostate gland, seminal vesicles with coagulation gland and preputial gland) that failed to sire and all females (uterus, cervix, clitoral gland, and vagina) that failed to deliver healthy pups (this was notified by Memo).

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

As no test item related pathology findings were noted, no additional histopathology evaluation was considered required.


Postmortem examinations (offspring):
SACRIFICE
All pups were culled on PND4.
Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities.

GROSS NECROPSY
Any pups showing abnormalities in structure or behaviour, including the pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination, in order to identify the probable cause of death if possible.

Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Reproductive indices:
Mating and fertility indices were calculated for both males and females. Gestation index was also calculated.
Male mating index = number of males with confirmed mating/total number of males cohabited x 100
Female mating index = number of sperm-positive females/total number of females cohabited x 100
Male fertility index = number of males impregnating a female/total number of males cohabited x 100
Female fertility index = number of pregnant females/number of sperm-positive females x 100
Gestation index = number of females with live born pups/number of pregnant females x 100
Offspring viability indices:
The following indices were calculated:
Survival Index = number of live pups (at designated time)/number of pups born x 100
Pre-implantation mortality = number of corpora lutea - number of implantations/number of corpora lutea x 100
Intrauterine mortality = number of implantations - number of liveborn/number of implantations x 100
Total mortality = number of implantations - number of viable pups (d4)/number of implantations x 100
Sex ratio = number of pups examined - number of males/number of pups examined x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Mortality
There was no mortality during the study.

Clinical observation

No test item-related adverse effects or systemic clinical signs were noted following daily administration of the test item by oral gavage.

Soft or liquid faeces were noted in 3 males in Control, Low and Mid dose groups. In High dose females thin fur of both forelimbs and hind limbs and in thorax ventral area was observed in 1 animal; scar on the neck ventral area was seen in one animal and red discharge from right eye in one animal was also noted during the observation period. These observations were considered to be incidental.

Neurological assessment

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment period.

When compared to Control, there were no toxicologically significant differences in the mean grip strength values of the forelimbs or hind limbs in the Main animals when evaluated on Day 27 (males) or PPD 4 (females). The mean grip strength of the forelimbs was lower than control in the Low dose females at approximately -13%, p<0.01 and for hind limbs in the Low dose males approximately -22%, p<0.05. In the absence of a consistent dose or gender response these variations were regarded as incidental and not to reflect an adverse or a test item related effect.

Increased vocalization was observed on occasion in the animals (1/5, 1/5, 0/5 and 0/5 in males and 0/5, 0/5, 0/5 and 1/5 in females, Control, Low, Mid and High dose, respectively). Slightly decreased righting reflex (in one and two male in the Mid and High dose groups, respectively and in one female in Low dose group) when subjected to the modified Irwin test (functional observation battery). In one High dose male, decreased grip strength score was seen. However, no treatment-related differences to the Control, or dose, or gender related response, were noted, and this signs were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.

During evaluation of motor activity, the total travelled distance was slightly lower in High dose males and females. The differences attaining statistically significance between 25-30 minute (p<0.05) in males. In females, dose dependent decreased activity was seen between 50-55 minutes and overall for the 60 minute duration (p<0.01 and p<0.05, respectively). The mean values were lower than the controls by approximately 63% between 50-55 minutes and by 25% when evaluated for the 60 minute duration. When evaluated as individual values, the control values for overall duration were in the range of 6480 and 9639 cm, while values of the high dose females were in the range of 4774 and 7552 cm; for the period of 50-55 minutes, the control range was 260 – 824 cm, the high dose range was 61 – 368 cm. In the High group there was one female (4502) with markedly low individual value from 35 to 60 minutes. The extended evaluation to the lower doses shows a dose-dependent decreased activity in these time periods. Taking into account historical data, the pattern of acclimatisation to the arena and the other individual SMART parameters, these differences were considered to be incidental or individual findings, which were not related to treatment, or were with no toxicological significance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Body weight and body weight gain

No test item related effects were noted on the mean body weight and body weight gain values following daily administration of PREPOLYMER D at dose levels up to and including 100 mg/kg bw/day, during the treatment period.

A higher body weight gain was recorded for High dose males between Days 0-7 and for Mid dose females between Days 0-14 (p<0.05) compared to the control animals. In the absence of a consistent dose or gender response and comparing data with historical data, these variations were regarded as incidental and not to reflect an adverse or a test item related effect.

Food consumption

There were no test item-related differences in the mean daily food consumption in any test-item treated group when compared to the Control.

Compared to control, differences attaining statistical significance were noted for males at Low dose between Days 21 and 28 (p<0.01). The individual values remained within the normal ranges and the finding was not considered toxicologically significant or to reflect an adverse effect of PREPOLYMER D.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Oestrus cycle, reproductive ability assessment and indices

There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with PREPOLYMER D administration.

In all groups, the mating indices were 100%, the fertility indices 100, 83, 92 and 83% due to 0, 2, 1, 2/12 non-pregnant females, in Groups 1, 2, 3 and 4 (these values are all in the normal historical range). The gestation indices were 100% in all groups.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 4 days of pairing (cohabitation).

Evaluation of the gestation, parturition and post-partal period

No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed.

The mean duration of pregnancy was similar in the control and test item treated groups, all were 22 to 24 days. All the parturitions were normal.

The numbers of corpora lutea and implantation sites were higher than control in all treated groups, attaining statistical significance but without any dose response relationship.

There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 100 mg/kg bw/day.

Compared to the control group, higher pre-implantation mortality (%) was observed in Low dose females (by 40%) without attaining statistically significance. In the absence of the similar effect in the Mid and High dose group, these changes were considered incidental and unrelated to the treatment.

There were no changes in reproductive parameters that were considered to be related to treatment with the test item.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Compared to controls, the absolute and relative weights of liver were slightly increased in all treated groups, both in males (evaluated on Day 28) and females (evaluated on PPD4). The differences at 10 and 30 mg/kg/day were in the normal control range, but the 100 mg/kg/day group increases were in the range of 11-18% and attained statistical significance for absolute and body weight or brain related mean values. The changes were not associated with any findings in clinical pathology or microscopic changes. The changes at 100 mg/kg/day were considered to reflect an adaptive response and not an adverse effect of treatment.

Compared to controls, slightly lower weights of seminal vesicles were recorded for all treated males. The difference was approximately 9-16% and attained statistical significance (p<0.05 or p<0.01 in Low and High dose, respectively) for absolute values, body weight and brain weight related mean values. These differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance. The changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation.

The thymus weight was slightly higher in Low and High dose males, attaining statistical significance for absolute values in Low dose (p< 0.01) and for body weight and brain weight related mean values in Low or High dose male animals (p<0.05 or p<0.01). These findings are considerable associated to the higher body weight at termination and there is not any toxicological consequences.

GROSS PATHOLOGY (PARENTAL ANIMALS)

Macroscopic Findings

No treatment related macroscopic findings could be noted at necropsy.

Changes such as enlarged prostate, small or enlarged testes, small epididymides, dilated vagina, pelvic dilatation in the kidneys, pale focus and enlargement of the spleen, based on the low incidence and distribution in control and dosed animals, was considered as incidental or background.

HISTOPATHOLOGY (PARENTAL ANIMALS)

Microscopic Findings

There was no evidence of test item-related histological findings in the High Dose animals or macroscopic observations from all groups in the reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

Diffuse ductal dilatation (in one High Dose male), marked, bilateral (in 1 Low and 1 High dose males) and minimal, unilateral (in one Control male) tubular degeneration/atrophy in the testes and marked aspermia (in 1 Low and 1 High dose males) in correlation with macroscopic observations, or moderate, intratubular, debris material in the epididymides, focal/multifocal, perivascular, mononuclear cell infiltrate in the prostate in two Control males, minimal congestion/haemorrhage in the thymus, minimal, multifocal tubular basophilia and pelvic dilatation in the kidneys, multifocal, perivascular, mononuclear cell infiltrate and periportal, hepatocellular vacuolation in the liver, minimal/mild extramedullary haematopoiesis and focal necrosis in the spleen, based on the low incidence and/or severity and/or distribution cross control and dosed animals were incidental or regarded as common background.

OTHER FINDINGS (PARENTAL ANIMALS)

Clinical pathology

Haematology
When compared to the controls, there were no differences that were considered toxicologically significant in the treated animals.

Variations were noted in a few parameters, on occasion attaining statistical significance, including lower Prothrombin Time (PTT) up to -12% in all treated males (p<0.01), or Monocytes (Mono %) with -34% in High dose females (p<0.05).

Evaluation of the mean and individual results in comparison with the control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range and of no toxicological significance.

Clinical chemistry
In the animals evaluated at the completion at termination (on Day 28 in males and on PPD5 in females), there were no toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to PREPOLYMER D administration in the conditions of this study.

A few clinical chemistry parameters showed on occasion statistically significant variations, i.e. slightly higher than control Bile Acid concentration (p<0.05) in Mid and High dose males, Total Bilirubin (T-Bil) (p<0.01) in Mid dose females or Urea concentration (Urea) (p<0.05), Glucose concentration (Glucose) (p<0.01) or Calcium concentration (Ca++) (p<0.05) in High dose females. However, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis
There was no effect of treatment noted during urinalysis.

Slight differences which attained statistical significance were record in urine volume in males at 10 mg/kg bw/day (p<0.05) and in females at 10, 30 and 100 mg/kg bw/day (p<0.05, p<0.05 and p<0.01, respectively). Slightly higher pH value was measured at Mid dose males (p<0.05). However these findings were regarded as minor variations and to be of no toxicological importance


Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effects at highest dose tested - 100 mg/kg bw/day. Liver weights were slightly higher at 100 mg/kg/day in both sexes but was attributed to an adaptive change.
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5 at 100 mg/kg bw/day - the highest dose tested.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY AND CLINICAL SIGNS (OFFSPRING)

PREPOLYMER D administered to parental generation at up to 100 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities clearly ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.

The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. A few surviving pups were cold, not suckled, grey or haemorrhagic. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of these findings was low, within the physiological range expected in the population of Wistar rats and considered without toxicological significance and not to reflect any test item or adverse effect.

The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 when evaluated as litter data were comparable to control values at up to and including 100 mg/kg bw/day, or showed minor variations ascribed to individual, biological variability.

Slightly higher viability data in all treated groups was within the normal range and not considered to reflect any treatment related effect.

The sex ratios were similar in the Control and treated groups, with no statistically significant differences observed.

BODY WEIGHT (OFFSPRING)

There were no effects considered adverse on the offspring weight or weight gain following administration of PREPOLYMER D at 10, 30 or 100 mg/kg bw/day to parental generation under the conditions of this study.

When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.

Compared to control, statistical significance was noted in the mean body weights values evaluated for all pups at 100 mg/kg bw/day group (High dose) on PND0 (below control) (p<0.01) and on PND4 (below control) (p<0.01) at 10 and 100 mg/kg bw/day (Low and High dose). These differences are related to higher pup numbers per litter and total litter weights (more pups results in lower weight per pup, this is normal and unrelated to treatment).

Lower mean body weight gain values were measured for all dose groups between PND0 - 4 (p<0.01). All values were in the normal range, no differences were considered to be related to treatment with test item.

There were no effects of treatment on pup weights or weigh gain.

GROSS PATHOLOGY (OFFSPRING)
No macroscopic changes were seen in remaining F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects ascribed to the test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia.
Reproductive effects observed:
not specified

Oestrus cycle, reproductive ability assessment and indices: 

 

Dose (mg/kg bw/day)

Control

10

30

100

Mated males - females

12/12

12/12

12/12

12/12

Paired males - females

12/12

12/12

12/12

12/12

Infertile males – Non-pregnant females (sperm negative)

0/12

0/12

0/12

0/12

Pregnant females, but not delivered

0

2

1

2

Male-female Mating Index (%)

100

100

100

100

Male-female Fertility Index (%)

100

83

92

83

Gestation Index (%)

100

100

100

100

 

Dose (mg/kg bw/day)

Control

10

30

100

Duration of mating period (days)$

1-4

1-4

1-4

1-4

Females showing evidence of copulation (N)

12/12

12/12

12/12

12/12

Females achieving pregnancy (N)

12/12

10/12

11/12

10/12

$ from pairing up to successful coitus/sperm positive and/or vaginal plug

Females

Dose (mg/kg bw/day)

Control

10

30

100

Number of delivered dams/dams with implantations

12/12

10/10

11/11

10/10

Duration of pregnancy (days, mean)

22.50

22.10

22.55

22.30

Number of corpora lutea / dams (mean)

15.17

18.30*

19.18*

18.40*

Number of implantations / dams (mean)

14.17

16.40

17.73*

17.50*

Number of dams with live pups day 0 / number of dams delivered

12/12

10/10

11/11

10/10

Number of dams with live pups day 4 / number of dams delivered

12/12

10/10

11/11

10/10

Level of significance: *= p<0.05

Females

Dose (mg/kg bw/day)

Control

10

30

100

Pre-implantation mortality (%)

7.44

10.45

7.19

4.20

Intrauterine mortality (%)

3.92

4.23

12.08

6.59

Post-natal mortality (%)

6.83

0.63

0.43

1.00

Total mortality (%)

10.55

4.85

12.51

7.49

Offspring (F1) Generation:

 

 

Dose (mg/kg bw/day)

Control

(12 dams)

10#

(10 dams)

30#

(11 dams)

100#

(10 dams)

Number of pups born (total)

164

161

172

165

Number of pups born (mean)

13.67

16.10

15.64

16.50

Number of dead pups (total) PND0

4

3

0

2

Number of live births (total)

164

158

172

164

Number of live births (mean)

13.67

15.80

15.64

16.40

Number of viable pups (total) PND0

160

158

172*

163

Cannibalized pups PND0-4

4

0

1

0

Dead pups (found dead, intact) PND0-4

8

4

0

3

Number of viable pups (total) PND4

152

157**

171**

162**

Number of viable pups (mean) PND4

12.67

15.70

15.55

16.20*

Viability indices (%)

93

98

99

98

Not suckled

1

0

0

2

Survival index PND0

97.99

98.30

100

98.61

Survival index PND4

93.17

97.68

99.57

98.24

#: Dams No. 2502, 2506, 3503, 4508 and 4512 excluded from statistics, due to not delivered.

Level of significance: *= p<0.05; **= p<0.01

 

 

Dose (mg/kg bw/day)

Control

(12 dams)

10#

(10 dams)

30#

(11 dams)

100#

(10 dams)

Sex ratio %, PND0

52.45

51.69

52.01

53.45

Sex ratio %, PND4

56.48

51.98

51.75

53.29

#: Dams No. 2502, 2506, 3503, 4508 and 4512 excluded from statistics, due to not delivered.

Body weight and body weight gain:

 

 

Dose (mg/kg bw/day)

 

Control

10

30

100

 

Litter mean:

 

Mean body weight (g), PND0

12 litters

10 litters

11 litters

10 litters

 

6.79

6.56

6.69

6.47

 

Mean body weight (g), PND4

12 litters

10 litters

11 litters

10 litters

 

11.39

10.43

10.96

10.70

 

Body weight gain (g), PND0-4

4.61

3.88

4.27

4.22

 

Mean Litter Weight PND0

91.47

104.89

103.62

105.09

 

Mean Litter Weight PND4

140.84

161.06

167.95

169.68

 


 

 

 

Dose (mg/kg bw/day)

 

Control

10

30

100

 

Mean for all pups:

 

Mean body weight (g), PND0

164 pups

161 pups

172 pups

165 pups

 

6.69

6.51

6.63

6.37**

U

Mean body weight (g), PND4

152 pups

157 pups

171 pups

162 pups

 

11.12

10.26**

10.80

10.47**

U

Body weight gain (g), PND0-4

4.42

3.75**

4.17**

4.09**

U

 ** = p<0.01;NS = not significant ; U = Mann-Whitney U-test versus Control

Organ weights:

 

Dose (mg/kg bw/day)

 

Control

10

30

100

 

Males, on Day 28

 

Liver weight

absolute (g)

12.48

14.24*

13.53

14.60**

DN

differences %

(14)

(8)

(17)

 

body weight relative

(%)

2.79

2.99

3.01

3.14*

DN

differences %

(7)

(8)

(13)

 

brain weight relative

(%)

566.78

647.94*

613.02

670.26**

DN

differences %

(14)

(8)

(18)

 

Females, on PPD4

 

Liver weight

absolute (g)

11.58

11.99

12.39

12.91**

DN

differences %

(4)

(7)

(11)

 

body weight relative

(%)

3.62

3.73

3.74

4.01**

DN

differences %

(3)

(3)

(11)

 

brain weight relative

(%)

554.26

574.50

589.43

636.13**

DN

differences %

(4)

(6)

(15)

 

 * = p < 0.05      ** = p < 0.01       DN = Duncan's Multiple Range Test

Results of dose formulation analysis:

Date

Nominal concentrationmg/mL

Measured concentrations with the 95% confidence intervals,  mg/mL

Measured concentration in percentage of the nominal

29-30 Oct

2013

5

4.9±0.1

98%

15

14.9±0.1

99%

50

51.7±1.9

103%

19-20 Nov 2013

5

4.5±0.0

90%

15

14.5± 0.2

96%

50

45.3±0.6

91%

10 Dec

2013

5

4.6±0.1

91%

15

14.2± 0.3

94%

50

45.2±0.4

91%

Conclusions:
In summary, daily administration of PREPOLYMER D by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 10, 30, or 100 mg/kg bw/day during the treatment period under the conditions of this study. Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD.

No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5, under the conditions of this study.

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia.

There were no adverse test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex. Liver weights were slightly higher at 100 mg/kg/day in both sexes but was attributed to an adaptive change.
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the toxicity of the test item PREPOLYMER D following repeated daily administration by oral gavage to Wistar rats. The study also included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.

Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD. Male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD4, according to the following Experimental Design:

Gr. No.

Group Designation

Dose Level
(mg/kg bw/day)

Conc. (mg/mL)

Dose volume

(mL/kg bw)

Animal Numbers/ID

Male

Female

1

Control

0

0

2

1001-1012

1501-1512

2

Low Dose

10

5

2001-2012

2501-2512

3

Mid Dose

30

15

3001-3012

3501-3512

4

High Dose

100

50

4001-4012

4501-4512

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs and neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. Pups were examined at euthanasia. At termination of the adults, necropsy with macroscopic examination was performed; weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives. For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

 

In summary, daily administration of PREPOLYMER D by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 10, 30, or 100 mg/kg bw/day during the treatment period under the conditions of this study. Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD.


No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5, under the conditions of this study.

 

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia.

There were no adverse test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex. Liver weights were slightly higher at 100 mg/kg/day in both sexes but was attributed to an adaptive change.

 


Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This study was performed according to OECD guideline and GLP and rated K1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on fertility via oral route:

K1: The study was performed according to OECD guideline and GLP.

Effects on developmental toxicity

Description of key information

There was no evidence of treatment related effect in the OECD422 on the survival of development of the pups. There were no indication of visceral abnormalities during gross post mortem examination. Overall there were no indication of treatment related adverse effects on development.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 October 2013 to 13 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: Young adult rats, approximately 11 weeks old at starting and 13 weeks at mating. The age range within the study was kept to the minimum.
- Weight at study initiation: Males: 351 g – 411 g, Females: 208 g - 242 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment.
- Fasting period before study: Not specified
- Housing: Rodents were group-housed, up to 3 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding
allowed digging and other normal rodent activities (i.e. nesting). Cage type: Type II and/or III polypropylene/polycarbonate. Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
- Water: Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 24.0 °C
- Humidity (%): 32 – 62 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 29 October 2013 (start of treatment) To: 13 December 2013 (last necropsy)
Animal identification:
Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth,
Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations
Analysis of test item formulations for concentration and homogeneity were performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment. One set was taken to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution for concentration measurements.

Analytical method:
To each of the 1 mL formulation sample, 5 mL of Hexane and 1 mL of saturated Sodium-chloride solution were added. The samples were stirredvigorously for 45 minutes.
5 and 15 mg/mL formulations: the Hexane phase was subjected to GC analysis without further dilution.
50 mg/mL formulation: the hexane phase was diluted by 5 fold prior to GC analysis.

Results:
Test item content of the dosing formulations was determined on 3 occasions during the study. The measured concentrations of PREPOLYMER D evaluated for each test item-dose group varied between 90 and 103%. No test item was detected in the control samples. These results were within acceptable ranges (90 % - 110%).
Details on mating procedure:
Details on mating procedure
- M/F ratio per cage: 1/1 for the mating period
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 4 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Sperm positive females were caged individually.
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Duration of treatment / exposure
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 4 days mating period, through gestation and up to the day before the necropsy (at least 4 days post-partum dosing). Females showing no-evidence of copulation were sacrificed, 25-27 days after the end of the mating period.
Frequency of treatment:
Frequency of treatment
Once daily
Duration of test:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.
Females were dosed for 14 days pre-mating, for up to 4 days mating period, through gestation and up to the day before the necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed, 25-27 days after the end of the mating period.

All F1 offspring were terminated on Day 4 post-partum; in order to allow for overnight fasting of dams prior to urine collection on PPD5, offspring were euthanized on PND/PPD 4, and the dams on PPD/PND 5.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 male + 12 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data, formulation and analytical trials and information from previous experimental work, including the results of a preliminary dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 13/227-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. During Phase 2 (14 days’ Repeat Dose Phase) in the DRF, treatment-related mortality (4 out of 6) was observed at 200 mg/kg bw/day in females. There was no mortality in the lower dose groups (100 and 50 mg/kg bw/day). From this information, a dose of 200 mg/kg bw/day caused lethal effects and was hence well in excess of the MTD, so although no toxicity was observed at 100 mg/kg bw/day, this dose level was considered to be the MTD.The oral route was selected as it is a possible route of exposure to the test item in humans.
- Randomization: All parental (P) animals were sorted according to body weight by computer and divided to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomizaton to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC+4.0 software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.
Maternal examinations:
Estrous cyclicity (parental animals)
Not assessed. The female mating index and the female fertility index were calculated.
Sperm parameters (parental animals)
Parameters examined:
Testes and epididymides were weighed individually.
Histopathological evaluation of the male gonads in high dose and control males including testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa.
Fetal examinations:
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
The efficiency of suckling was observed by the presence of milk in the pups' stomach. The pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal, PND0 or 1) and on
PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups.

GROSS EXAMINATION OF DEAD PUPS:
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded.
Statistics:
Statistics
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Indices:
Reproductive indices
Mating and fertility indices were calculated for both males and females. Gestation index was also calculated.
Male mating index = number of males with confirmed mating/total number of males cohabited x 100
Female mating index = number of sperm-positive females/total number of females cohabited x 100
Male fertility index = number of males impregnating a female/total number of males cohabited x 100
Female fertility index = number of pregnant females/number of sperm-positive females x 100
Gestation index = number of females with live born pups/number of pregnant females x 100

Offspring viability indices
The following indices were calculated:
Survival Index = number of live pups (at designated time)/number of pups born x 100
Pre-implantation mortality = number of corpora lutea - number of implantations/number of corpora lutea x 100
Intrauterine mortality = number of implantations - number of liveborn/number of implantations x 100
Total mortality = number of implantations - number of viable pups (d4)/number of implantations x 100
Sex ratio = number of pups examined - number of males/number of pups examined x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related adverse effects or systemic clinical signs were noted following daily administration of the test item by oral gavage.
Soft or liquid faeces were noted in 3 males in Control, Low and Mid dose groups. In High dose females thin fur of both forelimbs and hind limbs and in thorax ventral area was observed in 1 animal; scar on the neck ventral area was seen in one animal and red discharge from right eye in one animal was also noted during the observation period. These observations were considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item related effects were noted on the mean body weight and body weight gain values following daily administration of PREPOLYMER D at dose levels up to and including 100 mg/kg bw/day, during the treatment period.

A higher body weight gain was recorded for High dose males between Days 0-7 and for Mid dose females between Days 0-14 (p<0.05) compared to the control animals. In the absence of a consistent dose or gender response and comparing data with historical data, these variations were regarded as incidental and not to reflect an adverse or a test item related effect.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to the controls, there were no differences that were considered toxicologically significant in the treated animals.

Variations were noted in a few parameters, on occasion attaining statistical significance, including lower Prothrombin Time (PTT) up to -12% in all treated males (p<0.01), or Monocytes (Mono %) with -34% in High dose females (p<0.05).

Evaluation of the mean and individual results in comparison with the control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range and of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the animals evaluated at the completion at termination (on Day 28 in males and on PPD5 in females), there were no toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to PREPOLYMER D administration in the conditions of this study.

A few clinical chemistry parameters showed on occasion statistically significant variations, i.e. slightly higher than control Bile Acid concentration (p<0.05) in Mid and High dose males, Total Bilirubin (T-Bil) (p<0.01) in Mid dose females or Urea concentration (Urea) (p<0.05), Glucose concentration (Glucose) (p<0.01) or Calcium concentration (Ca++) (p<0.05) in High dose females. However, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment noted during urinalysis.

Slight differences which attained statistical significance were record in urine volume in males at 10 mg/kg bw/day (p<0.05) and in females at 10, 30 and 100 mg/kg bw/day (p<0.05, p<0.05 and p<0.01, respectively). Slightly higher pH value was measured at Mid dose males (p<0.05). However these findings were regarded as minor variations and to be of no toxicological importance.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment period.

When compared to Control, there were no toxicologically significant differences in the mean grip strength values of the forelimbs or hind limbs in the Main animals when evaluated on Day 27 (males) or PPD 4 (females). The mean grip strength of the forelimbs was lower than control in the Low dose females at approximately -13%, p<0.01 and for hind limbs in the Low dose males approximately -22%, p<0.05. In the absence of a consistent dose or gender response these variations were regarded as incidental and not to reflect an adverse or a test item related effect.

Increased vocalization was observed on occasion in the animals (1/5, 1/5, 0/5 and 0/5 in males and 0/5, 0/5, 0/5 and 1/5 in females, Control, Low, Mid and High dose, respectively). Slightly decreased righting reflex (in one and two male in the Mid and High dose groups, respectively and in one female in Low dose group) when subjected to the modified Irwin test (functional observation battery). In one High dose male, decreased grip strength score was seen. However, no treatment-related differences to the Control, or dose, or gender related response, were noted, and this signs were considered to be reactions to different type of stimuli or manipulations.

During evaluation of motor activity, the total travelled distance was slightly lower in High dose males and females. The differences attaining statistically significance between 25-30 minute (p<0.05) in males. In females, dose dependent decreased activity was seen between 50-55 minutes and overall for the 60 minute duration (p<0.01 and p<0.05, respectively). The mean values were lower than the controls by approximately 63% between 50-55 minutes and by 25% when evaluated for the 60 minute duration. When evaluated as individual values, the control values for overall duration were in the range of 6480 and 9639 cm, while values of the high dose females were in the range of 4774 and 7552 cm; for the period of 50-55 minutes, the control range was 260 – 824 cm, the high dose range was 61 – 368 cm. In the High group there was one female (4502) with markedly low individual value from 35 to 60 minutes. The extended evaluation to the lower doses shows a dose-dependent decreased activity in these time periods. Taking into account historical data, the pattern of acclimatisation to the arena and the other individual SMART parameters, these differences were considered to be incidental or individual findings, which were not related to treatment, or were with no toxicological significance.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Compared to controls, the absolute and relative weights of liver were slightly increased in all treated groups, both in males (evaluated on Day 28) and females (evaluated on PPD4). The differences at 10 and 30 mg/kg/day were in the normal control range, but the 100 mg/kg/day group increases were in the range of 11-18% and attained statistical significance for absolute and body weight or brain related mean values. The changes were not associated with any findings in clinical pathology or microscopic changes. The changes at 100 mg/kg/day were considered to reflect an adaptive response and not an adverse effect of treatment.

Compared to controls, slightly lower weights of seminal vesicles were recorded for all treated males. The difference was approximately 9-16% and attained statistical significance (p<0.05 or p<0.01 in Low and High dose, respectively) for absolute values, body weight and brain weight related mean values. These differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance. The changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation.

The thymus weight was slightly higher in Low and High dose males, attaining statistical significance for absolute values in Low dose (p< 0.01) and for body weight and brain weight related mean values in Low or High dose male animals (p<0.05 or p<0.01). These findings are considerable associated to the higher body weight at termination and there is not any toxicological consequences.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related macroscopic findings could be noted at necropsy.

Changes such as enlarged prostate, small or enlarged testes, small epididymides, dilated vagina, pelvic dilatation in the kidneys, pale focus and enlargement of the spleen, based on the low incidence and distribution in control and dosed animals, was considered as incidental or background.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was no evidence of test item-related histological findings in the High Dose animals or macroscopic observations from all groups in the reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

Diffuse ductal dilatation (in one High Dose male), marked, bilateral (in 1 Low and 1 High dose males) and minimal, unilateral (in one Control male) tubular degeneration/atrophy in the testes and marked aspermia (in 1 Low and 1 High dose males) in correlation with macroscopic observations, or moder
ate, intratubular, debris material in the epididymides, focal/multifocal, perivascular, mononuclear cell infiltrate in the prostate in two Control males, minimal congestion/haemorrhage in the thymus, minimal, multifocal tubular basophilia and pelvic dilatation in the kidneys, multifocal, perivascular, mononuclear cell infiltrate and periportal, hepatocellular vacuolation in the liver, minimal/mild extramedullary haematopoiesis and focal necrosis in the spleen, based on the low incidence and/or severity and/or distribution cross control and dosed animals were incidental or regarded as common background.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites were higher than control in all treated groups, attaining statistical significance but without any dose response relationship.

There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 100 mg/kg bw/day.

Compared to the control group, higher pre-implantation mortality (%) was observed in Low dose females (by 40%) without attaining statistically significance. In the absence of the similar effect in the Mid and High dose group, these changes were considered incidental and unrelated to the treatment.There were no changes in reproductive parameters that were considered to be related to treatment with the test item.
Dead fetuses:
no effects observed
Description (incidence and severity):
PREPOLYMER D administered to parental generation at up to 100 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities clearly ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. A few surviving pups were cold, not suckled, grey or haemorrhagic. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of these findings was low, within the physiological range
expected in the population of Wistar rats and considered without toxicological significance and not to reflect any test item or adverse effect.

The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 when evaluated as litter data were comparable to control values at up to and including 100 mg/kg bw/day, or showed minor variations ascribed to individual, biological variability.Slightly higher viability data in all treated groups was within the normal range and not considered to reflect any treatment related effect.The sex ratios were similar in the Control and treated groups, with no statistically significant dif
ferences observed.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of pregnancy was similar in the control and test item treated groups, all were 22 to 24 days. All the parturitions were normal.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of pregnancy was similar in the control and test item treated groups, all were 22 to 24 days. All the parturitions were normal.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
In all groups, the mating indices were 100%, the fertility indices 100, 83, 92 and 83% due to 0, 2, 1, 2/12 non-pregnant females, in Groups 1, 2, 3 and 4 (these values are all in the normal historical range). The gestation indices were 100% in all groups.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): ration of PREPOLYMER D at 10, 30 or 100 mg/kg bw/day to parental generation under the conditions of this study.

When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.

Compared to control, statistical significance was noted in the mean body weights values evaluated for all pups at 100 mg/kg bw/day group (High dose) on PND0 (below control) (p<0.01) and on PND4 (below control) (p<0.01) at 10 and 100 mg/kg bw/day (Low and High dose). These differences are related to higher pup numbers per litter and total litter weights (more pups results in lower weight per pup, this is normal and unrelated to treatment).

Lower mean body weight gain values were measured for all dose groups between PND0 - 4 (p<0.01). All values were in the normal range, no differences were considered to be related to treatment with test item.

There were no effects of treatment on pup weights or weigh gain
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 when evaluated as litter data were comparable to control values at up to and including 100 mg/kg bw/day, or showed minor variations ascribed to individual, biological variability.

Slightly higher viability data in all treated groups was within the normal range and not considered to reflect any treatment related effect.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratios were similar in the Control and treated groups, with no statistically significant differences observed.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Lower mean body weight gain values were measured for all dose groups between PND0 - 4 (p<0.01). All values were in the normal range, no differences were considered to be related to treatment with test item.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
examined macroscopically. A few surviving pups were cold, not suckled, grey or haemorrhagic. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of these findings was low, within the physiological range expected in the population of Wistar rats and considered without toxicological significance and not to reflect any test item or adverse effect.
External malformations:
no effects observed
Description (incidence and severity):
No external abnormalities clearly ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Description (incidence and severity):
No macroscopic changes were seen in remaining F1 offspring generation euthanized and examined
externally at scheduled termination on PND 4.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
no
Conclusions:
In summary, daily administration of PREPOLYMER D by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 10, 30, or 100 mg/kg bw/day during the treatment period under the conditions of this study. Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD.

No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5, under the conditions of this study.

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia.
There were no adverse test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex. Liver weights were slightly higher at 100 mg/kg/day in both sexes but was attributed to an adaptive change.
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the toxicity of the test item PREPOLYMER D following repeated daily administration by oral gavage to Wistar rats. The study also included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from

conception to Day 4 post-partum.

Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD. Male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD4, according to the following Experimental Design:

Group No

Group Designation

Dose Level

(mg/kg bw/

day)

Conc. (mg/

mL)

Dose volume

(mL/kg bw)

Animal numbers

 

 

 

 

 

Males

Females

1

Control

0

0

2

1001-1012

1501-1512

2

Low Dose

10

5

2

2001-2012

2501-2512

3

Mid Dose

30

15

2

3001-3012

3501-3512

4

High Dose

100

50

2

4001-4012

4501-4512

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs and neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical

chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. Pups were examined at euthanasia. At

termination of the adults, necropsy with macroscopic examination was performed; weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives. For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

In summary, daily administration of PREPOLYMER D by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 10, 30, or 100 mg/kg bw/day during the treatment period under the conditions of this study. Dose levels were set following

a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/ kg/day was considered to be the MTD.

No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5, under the conditions of this study.

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia.

There were no adverse test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex. Liver weights were slightly higher at 100 mg/ kg/day in both sexes but was attributed to an adaptive change.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The test substance did not cause any adverse effects up to the highest dose tested in an OECD422 study, 100 mg/kg bw/day. Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD and used as the highest dose in the OECD422 study. This study was performed according to OECD guideline and GLP and rated K1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:

K1: The study was performed according to OECD guideline and GLP.

Justification for classification or non-classification

The test substance did not cause any adverse effects up to the highest dose tested in an OECD422 study, 100 mg/kg bw/day. Dose levels were set following a preliminary study, where significant lethality was observed at 200 mg/kg/day hence 100 mg/kg/day was considered to be the MTD and used as the highest dose in the OECD422 study. 

Data for the major consituents, 40% is bis(2-chloroethoxy)methane CAS 111 -91 -1: Development The results from an OECD414 study show that the test substance does not need to be classified for developmental toxicity. The test substance does not result in adverse effects on development, visceral or skeletal malformations or embryotoxicity, of the offspring. Fetotoxicity was observed in the high dose group; higher numbers of dead foetuses and hence post-implantation loss. The NOAEL fetotoxicity is 10 mg/kg bw/day. The effects observed at 30 mg/kg bw/day may be related to sub-clinical maternal toxicity. The high dose of 30 mg/kg bw/day is only half of the dose of 60 mg/kg bw/day which caused 100% mortality in the preliminary study, so sub-clinical maternal toxicity in the current high dose group is probable. There are no signs of specific developmental toxicity or teratogenicity. The test substance is known to interfere with fatty acid metabolism and damage the mitochondria leading to cardiotoxicity and mortality in rats, for details see the section on repeated dose toxicity. Based on these results the substance is not classified for developmental toxicity.

Fertility

There was no evidence of treatment related effect in the OECD422 on fertility at the top dose of 100 mg/kg bodyweight per day. As the test substance is an intermediate handled under strictly controlled conditions, exposure of the workers is not expected. Based on this and the lack of effects in the OECD422 not further testing is required. Based on the the tets substannce is not classified for reproduction toxicity.