3-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]- 2-hydroxypropyl methacrylate and 1-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-3-hydroxypropan-2-yl methacrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 02 January 2009 and 24 November 2009. The in-life phase of the study was conducted between 26 May 2009 (first day of treatment) and 18 July 2009 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-2-hydroxypropyl 2-methylprop-2-enoate and 1-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-3-hydroxypropan-2-yl 2-methylprop-2-enoate

EC Number:

700-043-1

Molecular formula:

C24H56O8Si5

IUPAC Name:

Reaction mass of 3-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-2-hydroxypropyl 2-methylprop-2-enoate and 1-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-3-hydroxypropan-2-yl 2-methylprop-2-enoate

Test material form:

other: liquid

Details on test material:

Sponsor’s identification : OH-mPDMS
Description : clear, colourless liquid
Chemical name : Mono(Hydroxymethacryloxypropoxypropyl)
Polydimethylsiloxane
Lot number : 17554.025
Date received : 03 March 2008
Storage conditions : room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™HsdRccHan™:WIST strain rats were obtained from Harlan Laboratories UK Limited, Oxon, UK.
- Age at study initiation: Approximately twelve weeks old.
- Weight at study initiation: Males weighed 304 to 382 g, the females weighed 190 to 242 g.
- Fasting period before study: Not stated.
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to
their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories UK Limited, Oxon, UK) was used throughout the study period.
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 12 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.
The stability and homogeneity of the test material formulations were determined.
Results show the formulations to be stable for at least nineteen days. Formulations were therefore prepared every two weeks and stored at
approximately 4ºC in the dark.
Samples were taken of each test material formulation and were analysed for concentration of OH-mPDMH. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.


VEHICLE
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary: The concentration of OH-mPDMS in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.
Samples: The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Standards: Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml.
Procedure: The standard and sample solutions were analysed by HPLC using the following conditions:
HPLC: Agilent Technologies 1050 or 1200, incorporating autosampler and workstation.
Column: Luna 5μ C8 (150 x 4.6 mm id) @ 40°C
Mobile phase: Methanol
Flow-rate: 1 ml/min
UV detector wavelength: 210 nm
Injection volume: 25 μl
Retention time: ~ 2.8 mins

Homogeneity Determinations:
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
Stability Determinations: The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for nineteen days.
Verification of Test Material Formulation Concentrations: The test material formulations were sampled and analysed within four days of
preparation.

Method Validation
Linearity: A range of standard solutions covering the concentration range 0 to 0.1560 mg/ml, were prepared and analysed.
The detector response was shown to be linear up to 0.1560 mg/ml.
Specificity: The diluent solvent methanol and a blank Arachis Oil BP (control) were analysed. Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
Accuracy: Samples of Arachis Oil BP were accurately fortified with known amounts of test material and analysed. The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion: The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

Up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females)

Frequency of treatment:

The test material was administered daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

10 males and 10 females per dose group (plus control group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of a preliminary range-finder performed as part of the study.
The rangefinder was performed to establish the maximum tolerated dose level (up to 1000 mg/kg/day) of the test material following repeated oral administration to the Wistar Han™: HsdRccHan™:WIST strain rat, and to provide information for selection of dose levels for use in the main reproductive screening investigation.
Dose levels: 0, 500 and 1000 mg/kg/day.
The test material was administered daily, for 14 consecutive days.
Results:
Mortality- There were no unscheduled deaths during the treatment period.
Clinical Observations- No clinically observable signs of toxicity were detected.
An incident of increased salivation was evident for one male treated with 1000 mg/kg/day immediately after dosing on the final day of treatment. Scab formation was observed for one male treated with 500 mg/kg/day between Days 4 to 10. One female treated with 500 mg/kg/day showed noisy respiration immediately after dosing from Day 9 to Day 11 of treatment. These findings were isolated in nature, and as such were considered
unrelated to treatment.
Bodyweight- No adverse effects on bodyweight was evident.
Necropsy- No macroscopic abnormalities were observed for treated animals at terminal kill.
Conclusion: The dose levels for the main test were chosed, following consultation with the Sponsor, as:
High dose: 1000 mg/kg/day
Intermediate dose: 300 mg/kg/day
Low dose: 100 mg/kg/day
plus a control group treated with vehicle alone (Arachis oil BP)

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

Clinical Observations:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Clinical observations included cage-side visual inspection of anomalies with excreta. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Functional Observations:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments:
Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

Functional Performance Tests.
Motor Activity:
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength:
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex, finger approach.

Bodyweight:
Individual bodyweights were recorded on Day 1 (prior to dosing) and then twice weekly for males until termination and twice weekly for females until pairing. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Bodyweights were also recorded prior to termination.

Food Consumption:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food Efficiency:
Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and during lactation.

Water Consumption:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Laboratory Investigations:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Day 14 (day prior to pairing) and prior to tremination (Day 42 for males and Day 4 post partum for females). Blood
samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at termination. Animals were not fasted prior to sampling.

Haematology:
The following
parameters were measured:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using 0.5 ml blood samples collected into tubes containing sodium citrate solution (0.11 mol/l).

Blood Chemistry:
The following parameters were measured:
Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphate (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl¯), Total bilirubin (Bili)

Sacrifice and pathology:

GROSS PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights:
The following organs, removed from all surviving animals at terminal kill were dissected free from fat and weighed before fixation:
Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Thyroid, Liver.

HISTOPATHOLOGY:
Samples of tissues were preserved from all animals in buffered 10% formalin except where indicated. The tissues shown in bold from the remaining control and 1000 mg/kg/day were also processed. (Please see Any other information on materials and methods section for list of tissues).

All tissues were despatched to the Test Site (Harlan Laboratoties Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland) for processing . The tissues from five selected control and 1000 mg/kg/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg/day were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and
examined.
Since there were indications of treatment-related changes in lungs, examination was subsequently extended to include similarly prepared sections of lungs from five animals per sex from the low and intermediate groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE 84 Pathology computerisation system for tabulation and report production.

Other examinations:

Mating:
Following two weeks of treatment, animals were approximately fourteen weeks of age and were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Litter Data:
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Sex of offspring on Day 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Day 1 and 4 post partum

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Haematology, blood chemistry, absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights

Please see section ' any other information on materials and methods' below for further details.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

Adult Responses:

MORTALITY:
One female treated with 1000 mg/kg/day was killed in extremis due to difficulties during parturition. There were no further unscheduled deaths during the study.

CLINICAL OBSERVATIONS:
No clinically observable signs of toxicity were detected.
One female treated with 1000 mg/kg/day showed clinical signs consisting of prostration, hypothermia, pallor of the extremities, pilo-erection and ptosis on Day 41. This animal was in the late stage of gestation and the clinical signs observed would suggest that the animal was experiencing difficulties in parturition. Due to the severity of these observations, this animal was subsequently terminated on Day 41.
Remaining clinical signs consisted of increased salivation detected for one male treated with 100 mg/kg/day on Day 23 and swollen limbs were observed for another male treated with 100 mg/kg/day from Day 11 onwards. Generalised fur loss was evident for one female treated with 300 mg/kg/day from Day 39 onwards. This observation was also observed for one control female on Day 43 to Day 45. These findings were isolated in
each case, and were not considered to represent systemic toxicity.

FUNCTIONAL OBSERVATIONS:
Behavioural Assessments:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests:
A statistically significant increase in the final 20% of overall activity was observed for males treated with 1000 and 300 mg/kg/day (P<0.01). This was reflected in overall motor activity (P<0.05), with some values for the treated males outside of the normally expected ranges for these parameters.
No such effects were detected for females treated with 1000 or 300 mg/kg/day, or for animals of either sex treated with 100 mg/kg/day.
No treatment-related effects were detected in grip strength performance for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity scores observed for treated animals when compared to controls.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

BODYWEIGHT:
No adverse effects on bodyweights change were detected for treated animals when compared to controls.
Females treated with 1000 mg/kg/day showed a slightly higher overall bodyweight gain when compared to controls during gestation and lactation, although statistical differences were not observed. Statistically significant intergroup differences were detected for females from all treatment groups in comparison to controls between Days 11 to 15 (P<0.05), although a convincing dose-related response was not observed.

Males treated with 1000 and 300 mg/kg/day showed a statistically significant reduction in bodyweight gains when compared to controls between Days 1 to 4 (P<0.01), and the effect was still evident for males treated with 1000 mg/kg/day between Days 4 to 8 (P<0.05).

FOOD CONSUMPTION:
No adverse effect on food consumption was detected for males during the treatment period, or for females during the pre-mating, gestation or lactation phases of the study. Food efficiency (the ratio of bodyweight gain to dietary intake) was not affected for males throughout the treatment period, or for females during the pre-mating phase.

WATER CONSUMPTION:
No treatment-related intergroup differences in water intake were detected for treated animals when compared to controls.

HAEMATOLOGY:
No toxicologically significant effects were detected in the haematological parameters investigated prior to pairing on Day 14 or prior to termination for treated animals when compared to controls.

Females treated with 1000 mg/kg/day showed a statistically significant increase in activated partial prothrombin times (APTT) during the Day 14 assessments (P<0.05), with two individual values outside of the normally expected ranges for this parameter. Assessments were therefore repeated on Day 4 post partum. The final assessments did not reveal any significant intergroup differences, therefore, this initial increase in APTT was considered not to represent a true effect of treatment.

BLOOD CHEMISTRY:
No toxicologically significant effects were detected in the blood chemical parameters investigated prior to pairing on Day 14 or prior to termination for treated animals when compared to controls.

Females treated with 1000 mg/kg/day showed a statistically significant increase in bilirubin levels when compared to controls during the Day 14 assessments (P<0.05). A reduction in blood albumin levels were also evident, but these were observed in the female 300 mg/kg/day dose group only (P<0.05), with two values from this dose group outside of the normally expected range. Blood chemical assessments were therefore repeated prior to termination.

Final blood chemical assessments revealed an increase in potassium (P<0.05) and bilirubin (P<0.01) for males treated with 1000 mg/kg/day when compared to controls. All individual values for treated animals were within the normally expected ranges for these parameters, although one control male showed a higher than expected value. Increases in blood sodium and calcium were also observed for females treated with 1000 and 300 mg/kg/day when compared to controls (P<0.05). Increases in sodium levels were also evident for females treated with 100 mg/kg/day (P<0.05) in comparison to controls. All individual values for treated animals were within the normally expected ranges for these parameters, although one control value for both sodium and calcium were lower than the expected ranges. This may have contributed to the statistically significant
increases in the treated groups, which therefore were not considered to represent true effects of treatment.

PATHOLOGY(adults):
No treatment-related macroscopic abnormalities were detected.
The 1000 mg/kg/day female killed in extremis during parturition, showed pale kidneys and ten dead foetuses in the right uterine horn. Red staining was also recorded around the ano-genital region.
Macroscopic abnormalities for terminal kill animals were confined to small seminal vesicles seen for one control group male.

Organ Weights:
There were no treatment-related effects detected for organ weights from treated animals when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

HISTOPATHOLOGY:
Histopathogical examinations revealed the following treatment-related effects:

LUNGS:
A greater incidence of groups of alveolar macrophages was seen among males treated with 1000 mg/kg/day; alveolar macrophages at this dose level also exhibited foamy/vacuolated cytoplasm in excess of that normally seen as spontaneous change in untreated rats. Alveolar macrophages with foamy cytoplasm or granulomatous appearance were also seen for one female treated with 1000 mg/kg/day and for one male and for two females treated with 300 mg/kg/day.
Remaining histopathological changes seen among surviving control and intermediate dose animals were all considered to be spontaneous in origin and unrelated to treatment. The following conditions warrant specific mention:

ADRENAL GLANDS: Cortical vacuolation was seen in a few control and treated male rats and was of no toxicological significance in this investigation.

BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.

HEART: Focal myocarditis was observed in a few control and treated rats and is a common background lesion in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.

KIDNEYS: Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats. Focal corticomedullary mineralisation is a commonly observed background condition among female rats and was of no toxicological significance in this investigation.

LIVER: Scattered mononuclear cell foci were observed in a few control and treated rats examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.

LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for the majority of animals examined in the study and is not indicative of respiratory disease.

OESOPHAGUS: Inflammatory cell infiltrates in the peripheral musculature is a commonly observed change that is considered to be related to the physical trauma of gavage dosing.

SKELETAL MUSCLE: Mononuclear cell foci are commonly observed in the skeletal muscle of laboratory maintained rats and are of no toxicological significance at the incidences seen in this investigation.

SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.

THYROID: Follicular cell hypertrophy is commonly seen among untreated rats of either sex and there was no indication of a relationship to treatment in this study.

THYMUS: Lymphoid atrophy is frequently seen among pregnant and lactating female rats and there was no evidence of a treatment related group distribution of incidence or severity in this study.

REPRODUCTIVE TRACT AND RELATED ORGANS

PITUITARY: No treatment-related changes were seen.

TESTIS/EPIDIDYMIS: No treatment-related changes were seen.

SEMINAL VESICLES/COAGULATING GLAND: No treatment-related changes were seen.

PROSTATE: No pathological changes were seen.

MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of the majority of female rats examined. The appearance of the mammary tissue is consistent with pregnancy and lactation.

OVARY: No pathological changes were seen.

UTERUS: Areas of haemorrhage and fibrosis were seen in the myometrium and adjacent connective tissue of the uterus in the majority of female animals examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat.

VAGINA: No treatment-related changes were seen.


All other morphological changes in the above and remaining tissues were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without t oxicological significance.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment-related effects at 1000 and 300 mg/kg/day considered not to represent an adverse effect of treatment, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day.

2

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed at the lowest dose level employed, hence a 'No Observed Effect Level' (NOEL) was established at 100 mg/kg/day.

3

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed for reproduction.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Discussion:

The oral administration of OH-mPDMS to rats for a period of fourty-two days for males and up to fifty-four days for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of up to 1000 mg/kg/day resulted in treatment-related effects at 1000 and 300 mg/kg/day.

No clinically observable signs of toxicity were observed during the daily clinical observations and no treatment-related effects were observed during the weekly open field arena observations. Grip strength and sensory reactivity assessments performed at the end of the in-life phase did not reveal any differences for treated groups when compared to controls. Motor activity assessments, however showed an increase in motor activity over the final 20% of assessment, known as the asymptotic period (Reiter and Macphail, 1979), which was reflected in the overall activity values for males treated with 1000 and 300 mg/kg/day. The majority of the individual values were outside of the normally expected ranges for these parameters. In the absence of supporting effects observed during the daily clinical observations or sensory reactivity assessments conducted around the same time, and also an absence of such effects observed for females treated at these dose levels, these increases were considered not to represent a true neurotoxic effect, and in isolation, were considered as non-adverse in nature.

Post-mortem findings did not reveal any effects considered to be attributable to treatment, although histopathological examinations revealed treatment-related effects of the lungs. These consisted of an increased number of groups of alveolar macrophages exhibiting foamy or vacuolated cytoplasm for males treated with 1000 mg/kg/day and for one female treated at this dose level. The effects were also evident for one male and two females treated with 300 mg/kg/day. Groups of alveolar macrophages are seen as a spontaneous background change among laboratory maintained rats and the group distribution can be variable. However, a relationship to treatment in this investigation could not be excluded for males treated with 1000 mg/kg/day. Although the incidence of groups of alveolar macrophages in the 300 mg/kg/day groups was not appreciably greater than for control groups an effect of treatment similarly cannot be excluded in view of cytological changes. Such changes are occasionally seen when the test material enters the respiratory tract during gavage administration and the sporadic occurrence of the lesion within groups would tend to support this. These findings, in the absence of any inflammatory or degeneration changes in this study were considered to be a direct local effect as a consequence of partial aspiration of the test material, which is occasionally observed in oral gavage studies. The macrophage activity represents a normal biological response in order to clear the foreign material. This finding therefore did not represent systemic toxicity or an adverse effect of treatment.

No treatment-related effects in the reproductive parameters were observed. All treated and control females showed an equal number of litters at termination on Day 5 post partum and no treatment-related effects were observed for offspring growth or development. One female treated with 1000 mg/kg/day was killed in extremis following clinical signs of prostration, hypothermia, pallor of the extremities, ptosis and piloerection. These signs were considered to represent difficulties in parturition. Pregnancy was confirmed by the presence of ten foetuses in utero during the post-mortem examination performed on this animal. Difficulties in parturition are occasionally observed in reproductive studies and, in isolation, did not represent an effect of treatment in this study.

Applicant's summary and conclusion

Conclusions:

The oral administration of OH-mPDMS to rats by gavage, at dose levels of 1000, 300 and 100 mg/kg/day, resulted in treatment-related effects at 1000 and 300 mg/kg/day. These effects were considered not to represent an adverse effect of treatment, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day. No treatment-related effects were observed at the lowest dose level employed, hence a 'No Observed Effect Level' (NOEL) was established at 100 mg/kg/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996). The study was also designed to comply with the Commission Regulation No 440/2208 of 30 May 2008.

Methods.

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses:

Mortality. One female treated with 1000 mg/kg/day was killed in extremis due to difficulties during parturition. There were no further unscheduled deaths during the study.

Clinical Signs. No clinically observable signs of toxicity were detected. One female showed clinical signs to suggest difficulties encountered in parturition, resulting in the early sacrifice of this animal on Day 41.

Behavioural Assessment. No treatment-related effects were detected.

Functional Performance Tests. Increases in overall activity, specifically in the final 20% of motor activity assessment was observed for males treated with 1000 and 300 mg/kg/day. No treatment-related effects were detected for grip strength measurements.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Bodyweights. No adverse effects on bodyweights change were detected.

Food Consumption and Food Efficiency. No adverse effect on food consumption or food efficiency was detected.

Water Consumptions. No treatment-related intergroup differences in water intake were detected for treated animals when compared to controls.

Haematology. No toxicologically significant effects were detected in the haematological parameters investigated.

Blood Chemistry. No toxicologically significant effects were detected in the blood chemical parameters investigated.

Reproductive Performance:

Mating and Fertility. No treatment-related effects were detected in mating performance.

Gestation Length. No treatment-related effects were detected in the length of gestation.

Litter Responses:

Offspring Litter Size and Viability. No significant differences were detected in litter sizes and viability for treated groups when compared to controls.

Offspring Growth and Development. There were no differences in litter weights or mean offspring bodyweights between control and treated animals. No obvious clinical signs of toxicity were detected.

Pathology:

Necropsy. No treatment-related macroscopic abnormalities were detected for interim death or terminal kill animals.

Organ Weights. No treatment-related effects were detected.

Histopathology. Histopathogical examinations revealed the following treatment-related effects:

LUNGS: A greater incidence of groups of alveolar macrophages was seen among males treated with 1000 mg/kg/day; alveolar macrophages at this dose level also exhibited foamy/vacuolated cytoplasm in excess of that normally seen as spontaneous change in untreated rats. Alveolar macrophages with foamy cytoplasm or granulomatous appearance were also seen for one female treated with 1000 mg/kg/day and for one male and for two females treated with 300 mg/kg/day.

Conclusion. The oral administration of OH-mPDMS to rats by gavage, at dose levels of 1000, 300 and 100 mg/kg/day, resulted in treatment-related effects at 1000 and 300 mg/kg/day. These effects were considered not to represent an adverse effect of treatment, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day. No treatment-related effects were observed at the lowest dose level employed, hence a 'No Observed Effect Level' (NOEL) was established at 100 mg/kg/day.

No treatment-related effects were observed for reproduction, therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 1000 mg/kg/day.