9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 20 September 2016 and 21 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD guideline 422 without deviation and in compliance with GLP guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

EC Number:

289-390-3

EC Name:

9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Cas Number:

88127-84-8

Molecular formula:

C21H44O11Si

IUPAC Name:

9-[2-(2-methoxyethoxy)ethoxy]-9-{3-[(oxiran-2-yl)methoxy]propyl}-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Test material form:

liquid

Details on test material:

Batch number: 84246753
Expiry Date: 14 August 2017

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification : 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE
Physical State/Appearance : Clear colorless liquid
Chemical Name : Organosilane
Purity : 60-100%
Batch Number : ADC393100
Label : FK RAM 1087 ADF0021200
Date Received : 07 April 2016
Storage Conditions : Room temperature, in the dark
Expiry Date : 17 January 2018
No correction for purity was made.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 291 to 340g, the females weighed 199 to 226g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing 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.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

IN-LIFE DATES: From: 27 September 2016 To: 20 November 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for approximately three hours when stored at ambient conditions. Formulations were therefore prepared daily.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Vaginal plug and sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SEE "Any other information" Section below

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for approximately three hours when stored at ambient conditions. Formulations were therefore prepared daily.
As the test item formulations were prepared on a daily basis, samples of five test item formulations were taken and analyzed for concentration of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations is below. In the majority of instances, test item concentrations in the dosing formulations were retrospectively found to be outside the acceptance criteria of ±20%. With the exception of animals dosed at 1000 mg/kg bw/day which were treated at a lower concentration than intended on one occasion and were found to be within the acceptance criteria during one other analysis point. It can be concluded that animals from all dose groups were generally dosed at higher levels than intended throughout the majority of this study. A review of the formulation and analytical data did not identify a specific reason for these results. As the NOAEL for this study was considered to be 1000 mg/kg bw/day (high dose level) it can be concluded that these higher dose levels did not affect the outcome or integrity of this study.

Duration of treatment / exposure:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Once a day for the duration of the study.

Details on study schedule:

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 or 45.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day

No. of animals per sex per dose:

12 per sex per dose of test item plus 12 per sex in control group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work, including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study (Study Number: CD65DS), where dose levels up to 1000 mg/kg bw/day were well tolerated. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Serial Observations
General Observations/Measurements

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing 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 post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Due to an oversight, food consumption measurements were started on Day 3 post partum for the following animals; 23, 39, 42, 45, 71, 87, 89, 92, 93 and 95. This meant that food consumption data was collected for one day only. As such it has been excluded from the mean food consumption calculations as it was considered not to accurately reflect the groups as a whole.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral 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.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

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 on each occasion (at least two hours after dosing), 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, Reiter and Macphail, 1979).

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. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Reproductive Performance
Mating
Animals 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 estrus 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. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible 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

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte 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 samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Litter observations:

Litter Data
On completion of parturition (Day 0 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 Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum (this was not conducted for litters from females 15, 38, 44, 61 and 94 in error)
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (Day 4 post partum weights were not conducted in error for litters from females 15, 38, 44, 61 and 94, litter weights were calculated retrospectively from this data)

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

Postmortem examinations (parental animals):

Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 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 (Salewski 1964). The corpora lutea were also counted.
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 were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals Pituitary (post-fixation)
Brain Prostate and Seminal Vesicles
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals Muscle (skeletal)
Aorta (thoracic) Ovaries
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary
Brain (including cerebrum, cerebellum and pons) Prostate
Caecum Rectum
Coagulating gland Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including peyer’s patches) Testes
Jejunum Thyroid/Parathyroid
Kidneys Trachea
Liver Thymus
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix
Mammary gland Vagina
Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye,Suffolk, IP23 7PX) for processing). The tissues from five selected control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes in the spleens of females, examination was subsequently extended to include similarly prepared sections of spleens from female animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK.

Data Evaluation
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.

Statistics:

SEE "Any other information on materials and methods" below

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility Indices
Mating index (%) : Number animals mated / Animals paired × 100
Pregnancy Index (%) (%) : Number pregnant females / Number of animals mated × 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index
The following was calculated for each group:
Parturition Index (%) : Number of females delivering live offspring / Number of pregnant females x100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
Implantation losses
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) : No. corpora lutes - No. implantaion sites / No. corpora lutes x 100
Post-implantation loss (%) : No. implantation sites - total no. offspring born / No. implantation sites x 100
Live Birth and Viability Indices
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Sex ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Percentage of males : Number of male offspring/ Total number of offspring x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were generally no findings observed that indicated any adverse systemic toxicity of the test item in animals from any treatment group.
Animals of either sex (12 males and 12 females) treated with 1000 mg/kg bw/day showed incidences of increased salivation from Day 2 until Day 43 (males) and Day 45 (females). Two males treated with 300 mg/kg bw/day exhibited intermittent instances of increased salivation from Days 16 to 43. One female treated with 1000 mg/kg bw/day exhibited noisy respiration on Day 44 only. Such instances of increased salivation are frequently observed when animals are dosed via the oral gavage route and are generally considered to be associated with distaste or slight irritancy of the test item formulations rather than indicating any systemic effect of treatment. Noisy respiration can also be observed following the oral administration of a slightly irritant test item formulation and in isolation was considered not to be of toxicological significance. One female treated with 1000 mg/kg bw/day exhibited pilo-erection from Days 36 to 37. Observations of this nature are not uncommon around the time of parturition and in isolation was considered unrelated to treatment. One female animal treated with 100 mg/kg bw/day exhibited an abnormal gait (unable to put pressure on left forelimb) from Days 7 to 21. This was considered to be the result of a physical injury and unrelated to treatment. One control male animal exhibited noisy respiration on Day 39 and a further control male exhibited generalized fur loss from Days 38 to 45. As these animals were not treated with the test item these findings are of no toxicological significance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals from all treatment groups generally exhibited reduced body weight gains during the treatment period resulting in overall reductions in absolute body weight gain in a dose related manner. Reductions in body weight gains achieved statistical significance in male animals treated with 1000 mg/kg bw/day during Weeks 1 and 4 (p<0.05) and Week 6 (p<0.01). Statistical significance was achieved during Week 4 (p<0.01) and during Week 6 (p<0.01) for animals treated with 300 mg/kg bw/day.
There was considered to be no adverse effect on body weight development for female animals treated with 100, 300 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was considered to be no significant effect of treatment with the test item up to a dose level of 1000 mg/kg bw/day on dietary intake for animals of either sex throughout the treatment period.
Female animals treated with 1000 mg/kg bw/day exhibited slight reductions in food consumption during the first two weeks of maturation but without achieving statistical significance. This slight reduction in food consumption continued for the first two weeks of gestation, achieving statistical significance (p<0.05) during the first week. No such effects were noted during the lactation phase of the study.
Minor fluctuations were noted in food conversion efficiency, however, these differences were deemed to be reflective of intergroup differences in body weight gains and/or dietary intake.

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles didn't reveal any intergroup differences in water consumption

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Female animals treated with 1000 mg/kg bw/day showed statistically significant increases (p<0.01) in mean corpuscular hemoglobin and mean corpuscular volume, increases appeared to be dose related. The majority of animals exhibited values that were within the historical control data range. However, these hematological findings may be linked to the minor increases in splenic hematopoiesis noted in these females, although no such in-life changes were noted in females treated with 100 or 300 mg/kg bw/day. As such, the changes noted in these hematological parameters are considered to be of equivocal significance, and could be considered as non-adverse.
There were considered to be no treatment-related effects detected in the hematological parameters examined for male animals from any treatment group.
Male animals from all dose groups showed statistically significant reductions in mean corpuscular hemoglobin concentration in a dose related manner (p<0.05 (low and intermediate) and p<0.01 (high)). Male animals treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total leukocyte count and lymphocytes and a statistically significant increase (p<0.05) in activated partial thromboplastin time. Eosinophils were statistically significantly reduced (p<0.05) in all treated male animals, however, a true dose-related response was not evident. With the exception of two isolated instances in animals treated with 1000 mg/kg bw/day (one showing decreased mean corpuscular hemoglobin concentration and one exhibiting decreased lymphocytes) all values were within the historical control data range and as there were no corresponding histopathological correlates these findings were considered not to be related to systemic toxicity of the test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were considered to be no treatment-related effects detected in the blood chemical parameters examined.
Male animals from all treatment groups showed a statistically significant reduction (p<0.01) in glucose levels. In contrast, blood glucose was statistically significantly higher (p<0.05) in female animals treated with 300 mg/kg bw/day. Chloride concentrations were statistically higher (p<0.05) for male animals treated with 300 mg/kg bw/day and 1000 mg/kg bw/day, although a true dose-related response was not evident.
With the exception of male animals that were treated with 300 mg/kg bw/day which exhibited chloride concentrations which were higher than the historical control data range, the majority of blood chemical values lay within the historical control ranges and as there were no corresponding histopathological correlates these findings were considered to be incidental and unrelated to treatment of the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at 100, 300 or 1000 mg/kg bw/day.
Functional Performance Tests
There were no toxicologically significant changes in functional performance considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.
Females from all treatment groups showed a statistically significant reduction (p<0.05) in hindlimb grip strength. The intergroup differences were confined to one out of the three tests and a true dose-related response was not evident. In the absence of any clinical signs of neurotoxicity evident on the study, the intergroup differences were considered not to be of toxicological importance.
Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Spleen
There was a minor increase in hematopoiesis in the spleen of treated female animals. This occurred in two females treated with 100 mg/kg bw/day (minimal), three females treated with 300 mg/kg bw/day (1 minimal, 2 mild) and in four females treated with 1000 mg/kg bw/day (2 minimal, 2 mild). This also occurred at a marked severity in a further female treated with 300 mg/kg bw/day, this was considered to be due to a macroscopic abnormality in the stomach (ulceration).
No other changes were noted to account for findings seen in-life or variations in organ weights.
Non-Productive Mating
The following pairings did not produce a litter despite positive signs of mating:
Animals 17F and 5M (Control) showed no histological changes to account for the lack of pregnancy.
Animals 48F and 36M (Group 2) showed no histological changes to account for the lack of pregnancy.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

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

Fertility
No treatment-related effects were detected in fertility.
One control female and one female treated with 100 mg/kg bw/day were not pregnant following positive evidence of mating. No histological changes were noted to account for the lack of pregnancy, therefore this was considered to be incidental.

Gestation Length
Gestation lengths were generally between 22 and 23½ days. One female treated with 100 mg/kg bw/day had a gestation length of 24 days and a female treated with 300 mg/kg bw/day exhibited a gestation length of 24½ days but ultimately had a total litter loss. Mean gestation lengths for females treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in gestation length when compared to control. However, all gestation lengths were within the historical control range, and as such the intergroup variations were considered not to be of toxicological significance.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOEL

Effect level:

ca. 10 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, weak, no milk in the stomach, physical injury, missing, cannibalized or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.
For animals treated with 1000 mg/kg bw/day, actual litter weights were slightly lower than controls on Days 1 and 4 post partum, which is an obvious effect of the slightly lower litter size apparent at this dosage as a result of one female with only three offspring born. Individual male and female mean offspring body weights were generally comparable to control.

Gross pathological findings:

no effects observed

Details on results (F1)

Offspring Litter Size, Sex Ratio and Viability
With the exception of one female treated with 1000 mg/kg bw/day which only gave birth to three offspring, of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were comparable to controls.
In general, the number of corpora lutea and implantations were similar across all treatment groups when compared to control and as such there could be considered to be no significant treatment-related effects. Statistical analysis of the data did not reveal any significant intergroup differences. However, animals treated with 1000 mg/kg bw/day exhibited an increase in both pre and post-implantation losses and a slightly lower litter size (but without achieving statistical significance in any parameter) when compared to control. The post-implanation loss and slightly lower litter sizes were, however, considered to be mainly due to one female animal which only gave birth to three offspring. A further two animals from this dose group also gave birth to only six offspring, however, this litter size is within the historical control data range and as such these differences could be considered to be due to normal biological variation. Offspring viability were comparable to control in all treatment groups. Total litter losses were noted in one control female and another was noted in one female treated with 300 mg/kg bw/day. In the absence of a similar effect seen at 1000 mg/kg bw/day, these total litter losses were considered unrelated to treatment.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements 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).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day.  A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.  

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 pairing phase, and for five selected parental females from each dose group on Day 4 post partum.  Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.  

Adult males were terminated on Day 44 or 45, followed by the termination of all females and offspring on Day 5 post partum.  Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were generally no findings observed that indicated any adverse systemic toxicity of the test item in animals from any treatment group.

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters at 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests

There were no toxicologically significant changes in functional performance considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

Male animals from all treatment groups generally exhibited reduced body weight gains during the treatment period resulting in overall reductions in absolute body weight gain in a dose related manner.  

There was considered to be no adverse effect on body weight development for female animals treated with 100, 300 or 1000 mg/kg bw/day.

Food Consumption

There was considered to be no significant effect of treatment with the test item up to a dose level of 1000 mg/kg bw/day on dietary intake for animals of either sex throughout the treatment period.  Minor fluctuations were noted in food conversion efficiency, however, these differences were deemed to be reflective of intergroup differences in body weight gains and/or dietary intake.

Water Consumption

Visual inspection of water bottles didn't reveal any intergroup differences in water consumption.

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

No treatment-related effects were detected in fertility.

Gestation Lengths

Gestation lengths were generally between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum, sex ratio and viability were generally comparable to controls.

Offspring Growth and Development

No obvious affect of treatment was noted in offspring growth and development.  In general, offspring body weight gain and litter weights on Days 1 and 4 post partum were comparable to control litters.  Surface righting was also generally comparable to controls.

Laboratory Investigations

Hematology

There were considered to be no treatment-related effects detected in the hematological parameters examined for male animals from any treatment group.

Female animals treated with 1000 mg/kg bw/day exhibited increases in mean corpuscular hemoglobin and mean corpuscular volume.  No such effects were detected in females treated with 300 or 100 mg/kg bw/day.

Blood Chemistry

There were considered to be no treatment-related effects detected in the blood chemical parameters examined.

Pathology

Necropsy

Macroscopic examination at terminal necropsy did not reveal any findings that were considered to be treatment related in animals of either sex up to a dose level of 1000 mg/kg bw/day.

Organ Weights

There were considered to be no treatment-related effects detected in animals of either sex up to a dose level of 1000 mg/kg bw/day.

Histopathology

There was a minor increase in hematopoiesis in the spleen of treated female animals.  This occurred in two females treated with 100 mg/kg bw/day (minimal), three females treated with 300 mg/kg bw/day (1 minimal, 2 mild) and in four females treated with 1000 mg/kg bw/day (2 minimal, 2 mild).

This also occurred at a marked severity in a further female treated with 300 mg/kg bw/day.  This was considered to be a consequence of a separate abnormality (marked ulceration of the stomach).

Conclusion

The oral administration of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects.  The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.