Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,15-dichloro-3,5,8,11,13-pentaoxapentadecane; 1-chloro-2-[(2-chloroethoxy)methoxy]ethane; 1-chloro-2-{[2-(2-chloroethoxy)ethoxy]methoxy}ethane

EC Number:

940-783-4

Molecular formula:

C5H10Cl2O2 C10H20Cl2O5 C7H14Cl2O3

IUPAC Name:

1,15-dichloro-3,5,8,11,13-pentaoxapentadecane; 1-chloro-2-[(2-chloroethoxy)methoxy]ethane; 1-chloro-2-{[2-(2-chloroethoxy)ethoxy]methoxy}ethane

impurity 1

Reference substance name:

2-chloroethanol

EC Number:

203-459-7

EC Name:

2-chloroethanol

Cas Number:

107-07-3

Molecular formula:

C2H5ClO

IUPAC Name:

2-chloroethanol

Test material form:

liquid

Details on test material:

Name: PREPOLYMER D
Chemical Name: Reaction mass of Bis (2-chloroethoxy)methane and 1,15-dichloro-3,5,8,11,13-pentaoxa-pentadecane and 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane
Batch/Lot Number:1603101
Purity: 100% (multi constituent substance)
Appearance: Extremely pale yellow liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system 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,

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Results (fetuses)

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

Effect levels (fetuses)

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

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Applicant's summary and conclusion

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.