Registration Dossier

Administrative data

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.  

Data for one of the of the two major consituents, 40% is bis(2-chloroethoxy) methane CAS 111 -91 -1, shows a similar effect with mortality above a certain dose.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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 for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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 oral 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
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
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
Observations and examinations performed and frequency:
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
- Time schedule: 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.

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:
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.

CLINICAL PATHOLOGY
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: Yes
- Time schedule for collection of blood: Immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia.
- Animals fasted: Yes (overnight period of food deprivation, after the litter has been culled).
- How many animals: subgroup B, 5 males and 5 females/group
- 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: Yes
- Time schedule for collection of blood: Immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia.
- Animals fasted: Yes (overnight period of food deprivation, after the litter has been culled).
- How many animals: subgroup B, 5 males and 5 females/group
- 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: Yes
- Time schedule for collection of blood: Immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia.
- Animals fasted: Yes (overnight period of food deprivation, after the litter has been culled).
- How many animals: subgroup B, 5 males and 5 females/group
- 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)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment.
- Dose groups that were examined: 5 males and 5 females/group, “subgroup A”:
- Battery of functions tested: sensory activity / landing foot splay / fore/hind grip strength / motor activity:

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).

OTHER:
Observation of the delivery process and nursing instinct - see IUCLID Chapter 7.8.1 Toxicity to reproduction for details.







Sacrifice and pathology:
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 PATHOLOGY: Yes
- 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: Yes
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.
Other examinations:
See IUCLID Chapter 7.8.1 Toxicity to reproduction for details of reproductive/developmental parameters examined.
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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weights were slightly higher at 100 mg/kg/day in both sexes but was attributed to an adaptive change.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
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.

BODY WEIGHT AND 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.

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

NEUROBEHAVIOUR
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.

ORGAN WEIGHTS
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
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: NON-NEOPLASTIC
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
See IUCLID Chapter 7.8.1 Toxicity to reproduction for details of results of reproductive/developmental parameters examined.
Dose descriptor:
NOAEL
Effect level:
100 other: 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.
Critical effects observed:
not specified

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.

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. 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.

 

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.

 


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1: The study was performed according to OECD guidelines and GLP.

Additional 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. 

Data for one of the of the two major consituents, 40% is bis(2-chloroethoxy)methane CAS 111 -91 -1, shows a similar effect with mortality above a certain dose. Summary fo the effects observed with CAS 111 -91 -1:

In two range finding studies it was shown that the toxicity after oral administration of diformal is influenced by the concentration of the dosing solution and if the animals given the oral doses are fasted or unfasted. Based on the first range finding study it was concluded that 100 mg/kg of formal was lethal at concentrations of 100 mg/ml but essentially non toxic at a concentration of 10 mg/ml.

In the second range finding study the animals were dosed at concentration of 100 mg/ml.

The test substance, administered as a 100 mg/ml solution in corn oil, was initially administered to 60 Sprague-Dawley CD rats (5/sex/group) at dose levels of 20, 40, 50, 60, 80 and 100 mg/kg bw/day. Control animals (5/sex) received the vehicle at the same dose volume as administered to group VII animals (1.0 ml/kg bw/day). Because no signs of toxicity were seen after the first week, the two lower doses were raised from 20 and 40 mg/kg bw/day to 150 and 200 mg/kg bw/day, respectively, for the second week of the study.

Two additional (satellite) studies were performed to 1) clarify the effect of fasting on mortality at the 100 mg/kg dose level and 2) provide information on mortality, clinical signs and clinical laboratory values in animals receiving doses of 120 and 160 mg/kg bw/day (5/sex/dose) for up to seven days.

In the first satelite group all females in group I (fasted) died and all males in group I were moribund. All animals in group II (unfasted) survived.

The second satellite group showed results that are in line with the results found in the main group of the second range finding study.

Based on the apparent effects on blood urea nitrogen levels in females receiving doses as low as 50 mg/kg bw/day and on the elevated alkaline phosphatase level in males receiving 100 mg/kg bw/day, the NOEL for FORMAL in rats under conditions of this study was 40 mg/kg bw/day for females and 80 mg/kg bw/day for males.

In a 90 day oral repeated dose study the test substance, diluted in corn oil at a concentration of 100 mg per ml was administered to 100 Sprague-Dawley CD rats (10/sex/group) at dose levels of 10, 20, 40, 80 and 120 mg/kg/day. Cardiotoxicity was also observed causing mortality at 120 mg/kg bw.

Based on microscopic evidence of renal and hepatic pathology in males receiving 20 mg/kg/day, the no observed effect level (NOEL) for FORMAL when administered orally to rats for three months under conditions of this study was 10 mg/kg/day.

In a 90 day dermal repeated dose study it has been shown that Bis(2-chloroethoxy)methane is cardiotoxic. The Lowest NOAEL for this effect is 100 mg/kg bw. CEM cardiotoxicity is characterized by cytoplasmic vacuolation of myocytes, necrosis, and inflammation. The mechanism of cardiotoxicty has been investigated. It has been shown that CEM damages the mitochondria. The substance is metabolized to thiodiglycolic acid. Fatty acids are a major source of energy in the heart and because thiodiglycolic acid interferes with fatty acid metabolism, CEM mitochondria damage may be due in part to depletion of nutrients. Since heart tissue contains more mitochondria than other tissues due to a high energy demand the heart tissue is more susceptable to damage by CEM.

In a 16 day dermal study it has been shown that after the initial damage induced by CEM or its metabolite, thiodiglycolic acid, protective mechanisms within the heart are initiated, enabling it to cope with the continued exposure to the toxicant while eliminating some damaged myofibers. At day 16 if the study damage seen in the earlier phase of the study was repaired. However this protective mechanism cannot cope with longterm exposure to CEM since at the end of the 90 day study cardiotoxicity is observed.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

No effects were observed in the study however 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 this study.  

 

Repeated dose toxicity: dermal - systemic effects (target organ) cardiovascular / hematological: heart

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 one of the of the two major consituents, 40% is bis(2-chloroethoxy)methane CAS 111 -91 -1, shows a similar effect with mortality above a certain dose. The data for this constituent is relevant for C&L.

 

STOT Single Exp. 1: Based on major constituent, 40% is bis(2-chloroethoxy)methane CAS 111-91-1:Cause of death: degeneration of the myocardium. Only occurs above certain dose level in rats (± 100 mg/kg bw) between 1 and 9 doses.

 

STOT Rep. Exp. 2.: Based on major constituent, 40% is bis(2-chloroethoxy)methane CAS 111-91-1:In a 90 day dermal repeated dose study it has been shown that Bis(2-chloroethoxy) methane is cardiotoxic. The Lowest NOAEL for this effect is 100 mg/kg bw.

Based on microscopic evidence of renal and hepatic pathology in males receiving 20 mg/kg/day, the no observed effect level (NOEL) when administered orally to rats for three months under conditions of this study was 10 mg/kg/day.