Dimethoxymethylsilane

Endpoint summary

Description of key information

Oral (OECD 408), rat: NOAEL (systemic) >= 300 mg/kg bw/day; NOAEL (local) = 125 mg/kg bw/day

Dermal (non-guideline supporting study), rabbit: NOAEL (systemic) = 171 mg/kg bw/day; LOAEL (local) = 0.051 mg/cm²

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany GmbH
- Age at study initiation: 41 to 47 days
- Weight at study initiation: Males 221 to 293 g; Females 151 to 212 g
- Housing: two or three animals of the same sex in polycarbonate cages with stainless steel mesh lids and wood based bedding.
- Diet: Teklad 2014C, pelleted diet, ad libitum (removed overnight prior to blood sampling and during the period of urine collection).
- Water: Potable water from the public supply, ad libitum (except for during the period of urine collection).
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: No known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

dried and de-acidified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in a glove box under nitrogen.
A series of formulations at the required concentrations were prepared by adding a volume of test item to a volume of corn oil.
Conversion factor for preparation of formulations: The test item had a density of 0.86 g/mL so that 1 g of test item is 1.16 mL.
The headspace within the formulation containers was minimized as much as possible and filled with an inert gas. The formulations were provided in bottles sealed with a rubber bung. A stirring flea was added to each formulation bottle (except vehicle control) prior to being sealed. Formulations were prepared daily for the 10 mg/kg bw/day group and twice weekly for the 100 and 300/500 mg/kg bw/day groups. All formulations were stored in a refrigerator. Before and throughout the dosing procedure, formulations were stirred using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics.
- Concentration in vehicle: 8.7, 36.25 and 145 µL/mL (7.5, 31.25 and 125 mg/mL, respectively). The top concentration was reduced to 87 µL/mL (75 mg/mL) from Day 39 of the study.
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the formulations was determined during Week 1 and Week 12 of the study. The formulation analysis results were not available on issuance of the current draft report. The information will be included when available from the testing laboratory.

Duration of treatment / exposure:

13 weeks (control and test groups)
13 weeks and 4 week recovery period (satellite control and high-dose group)

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Dose level reduced to 300 mg/kg bw/day from Day 39

No. of animals per sex per dose:

10 (main study)
5 (satellite control and high-dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for this study were selected based on the results of a 3-week preliminary study conducted at the test laboratory (Labcorp Study No. 8470250). In the study, doses of 750 or 1000 mg/kg bw/day were not tolerated, with clinical signs including respiratory irregularities, underactivity, piloerection, hunched and prostrate or flattened posture resulted in premature sacrifice of several individuals. Macroscopic findings in the decedents comprised thickening and abnormal content of the gastrointestinal tract, pallor and blocked nasal turbinates. Respiratory irregularities persisted to the end of the treatment period in some surviving animals receiving 750 or 1000 mg/kg bw/day. The terminal investigations revealed increased liver weights in both sexes. Due to the adverse clinical signs and premature deaths, these dose levels were considered to be above the maximum tolerated dose.
There were no treatment-related clinical signs or effects on body weight gain or food intake in animals receiving 250 or 500 mg/kg bw/day and there were no macroscopic findings at necropsy. The evaluation of organ weights at the end of the 24-day treatment period revealed higher liver weights, compared with control.
It was therefore considered that 500 mg/kg bw/day was a suitable high dose level for administration in the 13-week study. The lowest dose of 10 mg/kg bw/day was selected with an aim to obtain a clear no-observed-adverse-effect level (NOAEL) and the intermediate dose of 100 mg/kg bw/day was selected to assess the dose-responsiveness of any findings.

- Fasting period before blood sampling for clinical biochemistry: yes (overnight)
- Rationale for selecting satellite groups: to evaluate recovery of any effects observed during the treatment period.
- Post-exposure recovery period in satellite groups: 4 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
- Signs Associated with Dosing: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation; One to two hours after completion of dosing all groups; As late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity.
Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the treatment and recovery periods and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, the remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the treatment and recovery periods.

WATER CONSUMPTION: Yes
- Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and Week 12 of the study.
- Dose groups that were examined: Control and high dose groups.
- The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 (all main study animals); Week 4 of recovery (all control and high dose recovery group animals).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters checked: Haematocrit, Haemoglobin concentration, Erythrocyte count, Absolute reticulocyte count, Mean cell haemoglobin, Mean cell volume, Mean cell haemoglobin concentration, Red cell distribution width, Total leucocyte count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelet count, Prothrombin time, Activated partial thromboplastin time.
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (all main study animals); Week 4 of recovery (all control and high dose recovery group animals).
- Animals fasted: Yes
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Glucose, Bilirubin - total, Cholesterol - total, Cholesterol - LDL, Cholesterol - HDL, Creatinine, Urea, Blood urea nitrogen, Total protein, Albumin, Albumin/globulin ratio, Sodium, Potassium

SERUM HORMONES: Yes
- Time of blood sample collection: At necropsy
- Animals fasted: No
- How many animals: All main and recovery study animals (samples from recovery animals were not analyzed).
- Parameters checked: Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 (all main study animals); Week 4 of recovery (all control and high dose recovery group animals).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Clarity, Colour, Volume, pH, Specific gravity, Bile pigments, Urobilinogen, Blood pigments, Protein (total and concentration), Glucose (total and concentration)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and once prior to dosing in Week 12.
- Dose groups that were examined: All main study and recovery animals.
- Battery of functions tested:
Sensory reactivity and grip strength: Approach response, Grip strength: forelimb and hindlimb, Startle reflex (auditory), Tail pinch response, Pinna reflex (touch response)

Motor activity: Before commencement of treatment and during Week 12 of treatment (before dosing), the motor activity of all animals was measured using a Rodent Activity Monitoring System. In addition, all recovery phase males were tested during Week 4 of recovery.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity, respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing

OTHER:
Vaginal smears for assessment of estrous cycles:
- All females (including recovery phase females) except premature decedents.
- Using pipette lavage for 4 days before each scheduled termination. The last smear was taken on the morning of necropsy within 1 hour of blood collection for thyroid hormone analyses.
- Smears assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) at termination. These observations assist in the histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1: Any other information on results incl. tables).
- Method of termination: Exsanguination following blood sampling (under terminal anesthesia).
- Bone marrow smears were prepared at termination from all animals killed, immediately following death.
- Organ weights: All main study and recovery animals. For bilateral organs, left and right organs were weighed together unless otherwise specified on the pathology procedures table.

HISTOPATHOLOGY: Yes (see Tables 1 and 2: Any other information on results incl. tables).

Optional endpoint(s):

Optional endpoints: Yes
- Sperm analysis: All males killed after 13 weeks of treatment (not decedents) or 4 weeks of recovery.
- A sperm sample was taken from the left vas deferens at necropsy of all males (except premature decedents) killed after 13 weeks of treatment and assessed for motility using a computer assisted sperm analyzer (CASA).
- The residual sperm samples taken from the left vas deferens were retained in neutral buffered formalin. The left testis and left cauda epididymis were also weighed and retained deep-frozen (-10 to -30°C).
As no treatment-related changes were identified from organ weight or histopathology investigations, an assessment of sperm count and sperm morphology was not performed and all retained samples were discarded when the report was finalized. The results of the sperm motility assessment were retained in the raw data but not reported.

Statistics:

See field 'Any other information on materials and methods incl. tables'.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs observed were related to impaired breathing (principally rales) and were generally confined to animals receiving 500 mg/kg bw/day. The findings were first observed from the end of Week 2 but showed no trend with time. The incidence of affected animals was typically low (1 or 2 on each occasion).

The high dose level was lowered from 500 to 300 mg/kg bw/day from Day 39 of dosing. After the dose was lowered, signs of rales persisted in two males and one female receiving 300 mg/kg bw/day. One high dose male displayed signs of rales from Day 80.

Signs of rales and chin rubbing were occasionally reported in association with dose administration.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were seven unscheduled deaths among animals given 500 mg/kg bw/day that were considered test item-related. Four males and three females were humanely sacrificed due to clinical signs.

Male No. 14 (main study) administered 500 mg/kg bw/day, was euthanized for welfare reasons on Day 13 after displaying signs of rales, impaired breathing, sneezing and piloerection. The macroscopic examination revealed gas in the cecum, ileum, jejunum and stomach and distension of the stomach. At microscopic evaluation necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. In addition, inflammatory cell infiltrate in the liver and dilatation of the lumen of the stomach were observed. The reason for dispatch was marked breathing impairment and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

Male No. 16 (main study) administered 500 mg/kg bw/day, was euthanized for welfare reasons on Day 14 due to poor clinical condition, with ante mortem signs comprising wet rales and abdominal distension. The macroscopic examination was unremarkable. At microscopic evaluation, necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. In addition, basophilia of the proximal tubules and a cortical infarct in the kidney and dilatation of the ventricles of the brain were noted. The reason for dispatch was poor clinical condition and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

Female No. 115 (main study) administered 500 mg/kg bw/day, was euthanized for welfare reasons on Day 14 due to signs of rales, rapid breathing and a thin build. The macroscopic examination revealed blocked nasal turbinates and a mass in the liver associated with a diaphragmatic hepatic hernia. At microscopic evaluation necrosis of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. In addition, herniated hepatic tissue in the diaphragm and basophilia of the proximal tubules in the kidney were noted. The reason for dispatch was poor clinical condition and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

Female No. 142 (recovery phase) administered 500 mg/kg bw/day, was euthanized for welfare reasons on treatment Day 13 due to rales, gasping and piloerection. The macroscopic examination revealed gaseous content of the cecum, ileum and jejunum and distension of the cecum. At microscopic evaluation necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. The reason for dispatch was marked breathing impairment and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

Male No. 13 (main study) administered 500 mg/kg bw/day, was euthanized for welfare reasons on Day 26 showing gasping and breathing impairment. The macroscopic examination revealed gaseous distension of the cecum, colon, duodenum, ileum, jejunum and stomach. At microscopic evaluation necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. In addition, dilatation of the lumen of the esophagus, chronic cardiomyopathy, contraction of the spleen and dilatation of the lumen of the stomach were noted. The reason for dispatch was marked breathing impairment and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

Female No. 113 (main study) administered 500 mg/kg bw/day, was euthanized for welfare reasons on Day 35 after showing rales, gasping and piloerection. The macroscopic examination was unremarkable. At microscopic evaluation necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. In addition, corticomedullary mineralization in the kidney was noted. The reason for dispatch was marked breathing impairment and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

Male No. 11 (main study) administered 500 mg/kg bw/day, was euthanized for welfare reasons on Day 36 after displaying rales and labored breathing. The macroscopic examination revealed fluid in the nasal cavities and thickened pancreas. At microscopic evaluation necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate of the nasal cavities were observed. In addition, basophilia of the proximal tubules in the kidney, hyperplasia of the urothelium and mononuclear inflammatory cells in the urinary bladder were noted. The reason for dispatch was poor clinical condition and the main factor contributing to death was considered as the lesions of the nose/turbinates, likely due to reflux.

The findings observed in the nasal cavities (necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate) are considered adverse since they were up to marked in severity grading and were considered as the main factor contributing to death. These findings are indicative of a damage due to the reflux associated with the gavage dosing procedure (owing to administration of a high volume of a low viscosity/volatile test material and/or mechanically upon removal of the gavage tube), which may result in accidental aspiration of test formulation and/or gastric content into the nasal cavity (Damsch et al. 2011). They are not considered as a primary effect of the test item.

There was one unscheduled death that was not considered test item-related. Female No. 151 (main study) receiving 500 mg/kg bw/day was found dead before dosing on Day 28 with no clinical signs recorded ante mortem. The macroscopic examination revealed a perforated lower esophagus and abnormal contents in the thoracic cavity. Moderate neutrophilic inflammation with embedded vegetal fragments was observed in the esophagus, epicardium of the heart and pleura of the thoracic cavity, at microscopic examination. In addition, moderate increased apoptosis was observed in the cortex of the thymus. This was concluded to be a procedural error.

There were no deaths after the dose level was lowered to 300 mg/kg bw/day

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain was unaffected by treatment.

During the recovery phase, body weight gain was lower than control in both sexes previously given 500/300 mg/kg bw/day, however there was a high degree of individual variation, with the lower gain being attributed to one or two individuals and, as such, this was not attributable to previous treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food intake.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No significant effect was observed by daily visual observation.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmoscopic findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examination performed in Week 13 revealed, compared with control, statistically significantly lower monocyte counts in females receiving 125 or 500/300 mg/kg bw/day, with the majority of individual values at 500/300 mg/kg bw/day being at or just below the lower end of the background control range (0.05x10E+9 - 0.33x10E+9/L (n=112)). There was, however, no effect on any other leucocyte subpopulation and no similar finding in males. These findings were not apparent at the end of the four-week recovery period, indicating that full recovery had occurred.

All other differences from controls, including those attaining statistical significance, were minor or lacked dose-response, and, as such, were considered to be of no toxicological significance. These included a trend towards shorter prothrombin time in both sexes at all dose levels and of activated partial thromboplastin time at all dose levels in females, where statistical significance was not attained, and such reductions are considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed in Week 13 revealed, compared with controls, statistically significantly lower creatinine concentration in males receiving 300/500 mg/kg bw/day, though all individual values were within the background control range (19-47 µmol/L (n=116)). Full recovery was evident for this finding.

In females, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) concentrations were statistically significantly higher than control at 500/300 mg/kg bw/day but there was no clear dose-response and all individual values were within the background control range (total cholesterol: 1.03-3.72 mmol/L (n=117); HDL: 0.95-2.37 mmol/L (n=30); LDL: 0.10-0.30 mmol/L (n=30)). Total protein concentration was statistically significantly higher than control at 500/300 mg/kg bw/day, with three individuals having values above the background control range (61-79 g/L (n=117)). Albumin concentration was unaffected, resulting in a lower albumin to globulin ratio, with the majority of values being below the background control range (1.29-1.90 (n=98)). This indicated that the increase to protein was due to a higher globulin fraction. These findings were not present at the end of the recovery period, indicating that full recovery had occurred.

All other differences from controls, including those attaining statistical significance, were minor or lacked dose-response, and, as such, were considered to be of no toxicological significance. Such differences included the statistically significant lower bilirubin concentration in females receiving 30, 125 or 500/300 mg/kg bw/day where none of the values were abnormal.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T3, T4 and TSH, cervix histopathology, coagulating gland histopathology, epididymis histopathology, epididymis weight, oestrus cyclicity, liver weight, mammary gland histopathology (males and females), ovary histopathology, ovary weight, prostate histopathology, prostate weight, seminal vesicles histopathology, seminal vesicles weight, testis histopathology, testis weight, thyroid histopathology, thyroid weight, uterus histopathology, uterus weight, vagina histopathology, vaginal smears, adrenals histopathology, adrenals weight, brain weight, pituitary histopathology and pituitary weight. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The analysis of urine performed in Week 13 of treatment revealed, compared with controls, high protein output and concentration in females receiving 500/300 mg/kg bw/day. The increase of protein output and concentration was partly due to one individual with a particularly high value (No. 118, protein output: 16.356 mg; concentration: 3.48 g/L) but with this animal excluded the mean value remained higher than controls. In addition to this animal, No. 119 also had a value above the background range (0.287-2.062 mg (n=57)).

Glucose output and concentration were higher than control in females receiving 500/300 mg/kg bw/day, with statistical significance being attained for the output value, though all but one of the individual values (No. 119) were within the background control range (1.578-5.799 µmol (n=30)).

There was a marginal, but statistically significant, increase of pH in females receiving 150 or 500/300 mg/kg bw/day. All individual values were within or slightly below the background control range (5.6-6.9 (n=66)).

There were no treatment-related findings in males.

All other inter-group differences from controls, including those attaining statistical significance, were minor or lacked dose response and were therefore attributed to normal biological variation. Such differences included the statistically significantly low glucose concentration in males receiving 30 mg/kg bw/day, which was the result of a higher urine volume.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Sensory Reactivity and Grip Strength:
Sensory reactivity and grip strength were unaffected by treatment.

Motor Activity:
The number of low (cage-floor) and high (rearing) beam breaks tended to be lower than control in males receiving 30, 125 or 500/300 mg/kg bw/day, with statistical significance being attained at the 18, 36, 42, 48 and 54-minute intervals at 125 and/or 500/300 mg/kg bw/day (and on one occasion at 30 mg/kg bw/day). There was, however, not always a trend with dose, the effect was not evident at each timepoint and although there was a reduction of total beam breaks, which was treatment-related for the number of high beam breaks, statistical significance was not attained for the total values. At the end of the four-week recovery period, (cage-floor) and high (rearing) beam breaks were higher than controls in males previously given 500/300 mg/kg bw/day, indicating recovery from any effect of treatment.

There was no effect in females at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher than control absolute, adjusted and relative to body weight mean liver weights were observed in both sexes receiving 125 or 500/300 mg/kg bw/day at the scheduled termination (see Table 4 ‘Any other information on results incl. tables').

No test-item related differences in organ weights were seen at the end of the recovery phase.

All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: the magnitude was considered small; inconsistency between sexes; presence only in absolute weight or in relative to body weight ratios but not both; and/or lack of a dose relationship or correlative findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related macroscopic findings were seen at necropsy at the scheduled termination or at the end of the recovery phase.

All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls) and/or were as expected for Sprague-Dawley rats of this age. Therefore, they were considered not test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related microscopic findings were seen at the scheduled termination or at the end of the recovery phase.

All microscopic findings at scheduled termination were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls) and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Measurement:
The analysis of serum thyroid hormone levels revealed no effect on treatment on thyroid stimulating hormone concentration (TSH).
Mean TSH concentration was higher than control in both sexes receiving 500/300 mg/kg bw/day, but there was a high degree of variation in all dose groups, statistical significance was not attained, and all individual values were within the historical control data range (see Table 3 ‘Any other information on results incl. tables').

The results for T3 and T4 analysis will be included when available from the testing laboratory.

Estrous Cycles:
Estrous cyclicity was unaffected by treatment.

Details on results:

It is concluded that the oral administration of dimethoxy(methyl)silane to Sprague Dawley rats caused local toxicity to the nasal epithelium at 500 mg/kg bw/day (necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria) which resulted in adverse respiratory clinical findings and premature mortality. This effect was attributed to local irritation caused by reflux associated with the physiochemical properties of the test item and gavage dosing procedure and not systemic toxicity. The high dose level was lowered to 300 mg/kg bw/day from Day 39, which prevented further mortality, but respiratory clinical signs persisted until the end of the study. There were no adverse systemic findings but there was evidence of a non-adverse, adaptive effect on the liver and a non-adverse reduction of motor activity in males, the cause of which was undetermined.
The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was therefore considered to be 500/300 mg/kg bw/day. Due to the adverse local effect on the nasal epithelium and the associated clinical signs and mortality, the overall NOAEL was considered to be 125 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic findings observed at this dose level

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse local findings observed at this dose level

Critical effects observed:

no

Table 3: Summary of thyroid hormone concentrations (pg/mL)

	Male				Female
Dose (mg/kg bw/day)	0	30	125	500/300†	0	30	125	500/300†
TSH	1210.59	844.05	1068.14	1532.84	519.45	450.70	605.33	930.25

TSH – thyroid stimulating hormone

†Dose level reduced to 300 mg/kg bw/day from Day 39.

 

Table 4: Test Article-Related Effects on Liver Weights - Difference from Control at Scheduled Termination

	Male				Female
Dose (mg/kg bw/day)	0	30	125	500/300†	0	30	125	500/300†
Absolute weight (g)	17.5	x1.09	x1.13	x1.07	10.26	x1.03	x1.12	x1.19
Adjusted for body weight (g)	17.6	x1.06	x1.12*	x1.10**	10.6	x0.95	x1.1*	x1.14
Relative to body weight (%)	2.95	x1.06	x1.12	x1.09	3.31	x0.99	x1.1*	x1.15**

* p<0.05

** p<0.01

†Dose level reduced to 300 mg/kg bw/day from Day 39.

Note: Values for absolute weight and ratio of organ weights (relative to body weight) for dosed groups expressed as X-fold control mean value.

Conclusions:

The test item was tested for subchronic oral toxicity according to OECD TG 408 and in compliance with GLP. The systemic NOAEL was determined to be >= 300 mg/kg bw/day for male and female rats, as no adverse systemic effects were observed at any dose level. Due to the adverse local effect on the nasal epithelium and the associated clinical signs and mortality, the overall NOAEL was considered to be 125 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.6, of Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rabbits were exposed dermally (occlusive) to the test substance for 9 doses over a time of 11 days. Animals were euthanized at the end of exposure period. 5 animals from high-dose and control group were kept as satellite groups for 2 weeks of recovery. Observations were made for clinical effects, gross and macroscopic lesions, haematology, clinical chemistry and urinalysis.

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Products, Inc., Denver
- Age at study initiation: 17 - 19 weeks old
- Weight at study initiation: 3.0 - 4.0 kg
- Housing: individually in stainless steel cages with wire mesh floors
- Diet: Agway Pro-Lab Certified Rabbit Formula (Agway Inc. Waverly, NY), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 21 °C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Type of wrap if used: polyethylene sheeting, secured with waterproof tape before treatment; after application of test substance usig a syringe with gavage needle, entire treatment area was overwrapped with VETRAP bandaging tape
- Area of exposure: no data
- % coverage: no data
- Time intervals for shavings or clipplings: no details given

REMOVAL OF TEST SUBSTANCE
- Washing: no washing, area was cleaned by wiping with a dry cloth
- Time after start of exposure: 6 h

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

11 days, and 2 weeks post exposure (satellite control and high dose groups)

Frequency of treatment:

5 consecutive days of exposure, followed by a 2 days rest period, followed by 4 consecutive days of exposure
6 hours per day

Dose / conc.:

0.05 other: mL/kg bw (nominal)

Dose / conc.:

0.1 other: mL/kg bw (nominal)

Dose / conc.:

0.2 other: mL/kg bw (nominal)

Dose / conc.:

43 mg/kg bw/day (nominal)

Dose / conc.:

85 mg/kg bw/day (nominal)

Dose / conc.:

171 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per gender
5 per gender for 43 mg/kg dose group

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before dosing

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily before dosing

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1, 8 and 12 during dosing and weekly during recovery period

FOOD CONSUMPTION:
- Food consumption for each animal determined throughout the study

WATER CONSUMPTION: Yes
- Time schedule for examinations: determined throughout the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of study
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at termination of study
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked in table [No.1] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (selected tissues, not further specified, wet weights of brain, liver, kidneys, heart, spleen, adrenals and testes)
HISTOPATHOLOGY: Yes (selected tissues, not further specified but including skin)

Other examinations:

scanning electron microscopy/energy-dispersive x-ray analysis (SEM/EDX) was performed on treated skin of one high-dose rabbit per gender from the recovery group to determine the nature of the pigmented material observed in the skin by light microscopy.

Statistics:

The data for quantitative continuous variables were intercompared for the three treatment groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA) and t-test. The t-test was used when the F-value from the ANOVA was significant. When Levene's test indicated simialr variances and the ANOVA was significant a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogenous variances, all groups were compared by an ANOVA for unequal variances followed when necessary by a separate variance test for pairwise comparison.
Nonparametric data were statistically evaluated using the Kruskal-Wallis test followed by Mann-Whitney U test when appropriate. Incidence data were compared using the Fisher's exact test. For all statistical tests, the probability value of p<0.05 (two-tailed) was used as the critical level of significance.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

moderate to severe skin irritation, females more affected than males, lesion severity correlated with dose

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 specified

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

no treatment-related effects, except decrease in monocytes for high dose group males, which may have been due to sequestration of circulating leukocytes at the site of skin irritation

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

the only finding were lesions to the skin

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

skin lesions were found

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Treatment-related clinical lesions were limited to the the treated skin. Females tended to be affected slightly more than males. There was moderate to severe skin irritation as evidenced by exfoliation, fissures, and ecchymoses for both genders in the high-dose group, with ulceration and necrosis found only in high-dose group females. In addition, Draize scoring of skin irritation revealed barely perceptible to well-defined erythema and edema in the mid- and high-dose groups of male animals and barely perceptible to moderate erythema in all dose groups of female animals. In general, lesion severity correlated with dose.

HAEMATOLOGY
In animals euthanised after a 2 week recovery period, the only noteworthy finding was a statistically significant decrease in monocytes for high-dose group males, which may have been due to sequestration of circulating leukocytes at the site of skin irritation.

GROSS PATHOLOGY
The only gross lesions found involved the treated skin of rabbits of both genders from all exposure groups at both necropsies. Necropsy findings were similar to clinical observations. The most common lesion was exfoliation or scaling, which was present in most intermediate and high-dose group rabbits, and five of five females and one of five males from the low-dose group from the primary necropsy, as well as in four of five high-dose group rabbits/gender from the recovery group necropsy. Discoloration of the skin, either erythema or ecchymoses, was observed sporadically (more frequently in females) in intermediate and high-dose group rabbits euthanized on day 12, but not observed in the recovery group animals. Lesions indicative or more serious skin irritation, such as ulceration and necrosis (females only), or fissures (both sexes), were noted sporadically in the high-dose group animals euthanized on day 12. Four intermediate dose group females also had fissures and one had ulceration as well.

HISTOPATHOLOGY: NON-NEOPLASTIC
Statistically significant treatment-related microscopic lesions were observed in the treated skin of MDMS-exposed rabbits from all three dose groups of female animals and from the interrnediate and high-dose groups of male animals euthanized immediately following the exposure period. Lesions indicative of treatment-induced irritation of the skin included hyperkeratosis (corresponding to the exfoliation reported grossly), acanthosis, dermal congestion, edema, hemorrhage, epidermitis, ulceration, dermatitis, and dermal fibrosis. The only lesions seen in low-dose group rabbits were hyperkeratosis and dermatitis. Slight dermatitis (lymphocytic infiltrates) was also seen in a few control group rabbits. Acute inflammation in the intermedite and high-dose group animals was often most intense at the epidermal junction, even when the overlying epidermis was intact. Dermal fibrosis was associated with phagocytosed foreign material within macrophages, which appeared as a brown crystalline substance (dermal pigmentation) on H & E-stained sections. Significant residual lesions of treated skin were also present in all high-dose group rabbits euthanized after the 2-week recovery period. Lesions included hyperkeratosis, dermal fibrosis, and granulomatous dermatitis, in which phagocytic giant cells containing pigmented foreign material were prominent.
Scanning electron microscopy of the superficial dermis revealed numerous electron-dense deposits that were proven by elemental analysis to contain silicon. This material is believed to represent a polymer derived from absorbed MDMS or its breakdown products.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

171 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic effects observed at the highest dose tested, the only abnormal finding involved the treated skin

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

43 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

0.051 mg/cm² per day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

table1: Summary of clinical observations of rabbits

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
Treated skin
total number examined	10	5	10	10	10	5	10	10
Number affected
Exfoliation	0	1	10	10	0	5	10	10
Ecchymoses	0	0	0	3	0	0	3	9
Ulceration	0	0	0	0	0	0	0	2
Fissures	0	0	0	8	0	0	4	10
Necrosis	0	0	0	0	0	0	0	3

table2: Necropsy Findings for rabbits euthanized on day 12

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	5	10	5	5	5	10	5
Number affected
Exfoliation	0	1	9	5	0	5	10	5
Excoriation	1	0	0	1	0	0	0	0
Erythema	0	0	1	1	0	0	4	0
Ecchymoses	0	0	0	1	0	0	2	2
Ulceration	0	0	0	0	0	0	0	1
Fissures	0	0	0	2	0	0	4	5
Necrosis	0	0	0	0	0	0	1	2

table3: Necropsy Findings for rabbits euthanized on day 29

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	-	-	5	5	-	-	5
number affected
Exfoliation	0	-	-	4	0	-	-	4
Papule	0	-	-	1	0	-	-	0

table4: Microscopic Findings for rabbits euthanized on day 12

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	5	10	5	5	5	10	5
examined, unremarkable	3	0	0	0	4	0	0	0
number affected
Hyperkeratosis	0	2	10**	5**	0	5**	10**	5**
Acanthosis	1	0	10**	5*	0	1	7*	5**
Congestion	0	0	4	0	0	0	6*	4*
Epidermitis	0	0	7*	4*	0	0	9**	5**
Ulceration	1	0	3	2	0	0	3	4*
Dermal edema	0	0	0	0	0	0	1	3
Dermal hemorrhage	0	0	4	3	0	0	7*	5**
Dermatitis	2	5	10*	5	1	4	10**	4
Dermal fibrosis	0	0	10**	4*	0	0	8**	5**
Dermal pigmentation	0	0	10**	4*	0	0	9**	5**

Significantly different from control group, *p<0.05, **p<0.01

table5: Microscopic Findings for rabbits euthanized on day 29

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	0	0	5	5	0	0	5
examined, unremarkable	4	-	-	0	1	-	-	0
number affected
Hyperkeratosis	0	-	-	5**	0	-	-	5**
Acanthosis	0	-	-	0	0	-	-	2
Ulceration	0	-	-	0	1	-	-	0
Dermal hemorrhage	0	-	-	0	1	-	-	1
Dermatitis	1	-	-	5*	3	-	-	4
Granulomatous dermatitis	0	-	-	4*	0	-	-	5**
Dermal fibrosis	0	-	-	2	0	-	-	5**
Dermal pigmentation	0	-	-	5**	0	-	-	5**

Significantly different from control group, *p<0.05, **p<0.01

Conclusions:

Repeated dermal application of the test substance did not result in systemic toxicity. The only abnormal findings involved the treated skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

171 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rabbits were exposed dermally (occlusive) to the test substance for 9 doses over a time of 11 days. Animals were euthanized at the end of exposure period. 5 animals from high-dose and control group were kept as satellite groups for 2 weeks of recovery. Observations were made for clinical effects, gross and macroscopic lesions, haematology, clinical chemistry and urinalysis.

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Products, Inc., Denver
- Age at study initiation: 17 - 19 weeks old
- Weight at study initiation: 3.0 - 4.0 kg
- Housing: individually in stainless steel cages with wire mesh floors
- Diet: Agway Pro-Lab Certified Rabbit Formula (Agway Inc. Waverly, NY), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 21 °C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Type of wrap if used: polyethylene sheeting, secured with waterproof tape before treatment; after application of test substance usig a syringe with gavage needle, entire treatment area was overwrapped with VETRAP bandaging tape
- Area of exposure: no data
- % coverage: no data
- Time intervals for shavings or clipplings: no details given

REMOVAL OF TEST SUBSTANCE
- Washing: no washing, area was cleaned by wiping with a dry cloth
- Time after start of exposure: 6 h

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

11 days, and 2 weeks post exposure (satellite control and high dose groups)

Frequency of treatment:

5 consecutive days of exposure, followed by a 2 days rest period, followed by 4 consecutive days of exposure
6 hours per day

Dose / conc.:

0.05 other: mL/kg bw (nominal)

Dose / conc.:

0.1 other: mL/kg bw (nominal)

Dose / conc.:

0.2 other: mL/kg bw (nominal)

Dose / conc.:

43 mg/kg bw/day (nominal)

Dose / conc.:

85 mg/kg bw/day (nominal)

Dose / conc.:

171 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per gender
5 per gender for 43 mg/kg dose group

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before dosing

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily before dosing

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1, 8 and 12 during dosing and weekly during recovery period

FOOD CONSUMPTION:
- Food consumption for each animal determined throughout the study

WATER CONSUMPTION: Yes
- Time schedule for examinations: determined throughout the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of study
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at termination of study
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked in table [No.1] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (selected tissues, not further specified, wet weights of brain, liver, kidneys, heart, spleen, adrenals and testes)
HISTOPATHOLOGY: Yes (selected tissues, not further specified but including skin)

Other examinations:

scanning electron microscopy/energy-dispersive x-ray analysis (SEM/EDX) was performed on treated skin of one high-dose rabbit per gender from the recovery group to determine the nature of the pigmented material observed in the skin by light microscopy.

Statistics:

The data for quantitative continuous variables were intercompared for the three treatment groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA) and t-test. The t-test was used when the F-value from the ANOVA was significant. When Levene's test indicated simialr variances and the ANOVA was significant a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogenous variances, all groups were compared by an ANOVA for unequal variances followed when necessary by a separate variance test for pairwise comparison.
Nonparametric data were statistically evaluated using the Kruskal-Wallis test followed by Mann-Whitney U test when appropriate. Incidence data were compared using the Fisher's exact test. For all statistical tests, the probability value of p<0.05 (two-tailed) was used as the critical level of significance.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

moderate to severe skin irritation, females more affected than males, lesion severity correlated with dose

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 specified

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

no treatment-related effects, except decrease in monocytes for high dose group males, which may have been due to sequestration of circulating leukocytes at the site of skin irritation

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

the only finding were lesions to the skin

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

skin lesions were found

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Treatment-related clinical lesions were limited to the the treated skin. Females tended to be affected slightly more than males. There was moderate to severe skin irritation as evidenced by exfoliation, fissures, and ecchymoses for both genders in the high-dose group, with ulceration and necrosis found only in high-dose group females. In addition, Draize scoring of skin irritation revealed barely perceptible to well-defined erythema and edema in the mid- and high-dose groups of male animals and barely perceptible to moderate erythema in all dose groups of female animals. In general, lesion severity correlated with dose.

HAEMATOLOGY
In animals euthanised after a 2 week recovery period, the only noteworthy finding was a statistically significant decrease in monocytes for high-dose group males, which may have been due to sequestration of circulating leukocytes at the site of skin irritation.

GROSS PATHOLOGY
The only gross lesions found involved the treated skin of rabbits of both genders from all exposure groups at both necropsies. Necropsy findings were similar to clinical observations. The most common lesion was exfoliation or scaling, which was present in most intermediate and high-dose group rabbits, and five of five females and one of five males from the low-dose group from the primary necropsy, as well as in four of five high-dose group rabbits/gender from the recovery group necropsy. Discoloration of the skin, either erythema or ecchymoses, was observed sporadically (more frequently in females) in intermediate and high-dose group rabbits euthanized on day 12, but not observed in the recovery group animals. Lesions indicative or more serious skin irritation, such as ulceration and necrosis (females only), or fissures (both sexes), were noted sporadically in the high-dose group animals euthanized on day 12. Four intermediate dose group females also had fissures and one had ulceration as well.

HISTOPATHOLOGY: NON-NEOPLASTIC
Statistically significant treatment-related microscopic lesions were observed in the treated skin of MDMS-exposed rabbits from all three dose groups of female animals and from the interrnediate and high-dose groups of male animals euthanized immediately following the exposure period. Lesions indicative of treatment-induced irritation of the skin included hyperkeratosis (corresponding to the exfoliation reported grossly), acanthosis, dermal congestion, edema, hemorrhage, epidermitis, ulceration, dermatitis, and dermal fibrosis. The only lesions seen in low-dose group rabbits were hyperkeratosis and dermatitis. Slight dermatitis (lymphocytic infiltrates) was also seen in a few control group rabbits. Acute inflammation in the intermedite and high-dose group animals was often most intense at the epidermal junction, even when the overlying epidermis was intact. Dermal fibrosis was associated with phagocytosed foreign material within macrophages, which appeared as a brown crystalline substance (dermal pigmentation) on H & E-stained sections. Significant residual lesions of treated skin were also present in all high-dose group rabbits euthanized after the 2-week recovery period. Lesions included hyperkeratosis, dermal fibrosis, and granulomatous dermatitis, in which phagocytic giant cells containing pigmented foreign material were prominent.
Scanning electron microscopy of the superficial dermis revealed numerous electron-dense deposits that were proven by elemental analysis to contain silicon. This material is believed to represent a polymer derived from absorbed MDMS or its breakdown products.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

171 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic effects observed at the highest dose tested, the only abnormal finding involved the treated skin

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

43 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

0.051 mg/cm² per day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

table1: Summary of clinical observations of rabbits

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
Treated skin
total number examined	10	5	10	10	10	5	10	10
Number affected
Exfoliation	0	1	10	10	0	5	10	10
Ecchymoses	0	0	0	3	0	0	3	9
Ulceration	0	0	0	0	0	0	0	2
Fissures	0	0	0	8	0	0	4	10
Necrosis	0	0	0	0	0	0	0	3

table2: Necropsy Findings for rabbits euthanized on day 12

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	5	10	5	5	5	10	5
Number affected
Exfoliation	0	1	9	5	0	5	10	5
Excoriation	1	0	0	1	0	0	0	0
Erythema	0	0	1	1	0	0	4	0
Ecchymoses	0	0	0	1	0	0	2	2
Ulceration	0	0	0	0	0	0	0	1
Fissures	0	0	0	2	0	0	4	5
Necrosis	0	0	0	0	0	0	1	2

table3: Necropsy Findings for rabbits euthanized on day 29

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	-	-	5	5	-	-	5
number affected
Exfoliation	0	-	-	4	0	-	-	4
Papule	0	-	-	1	0	-	-	0

table4: Microscopic Findings for rabbits euthanized on day 12

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	5	10	5	5	5	10	5
examined, unremarkable	3	0	0	0	4	0	0	0
number affected
Hyperkeratosis	0	2	10**	5**	0	5**	10**	5**
Acanthosis	1	0	10**	5*	0	1	7*	5**
Congestion	0	0	4	0	0	0	6*	4*
Epidermitis	0	0	7*	4*	0	0	9**	5**
Ulceration	1	0	3	2	0	0	3	4*
Dermal edema	0	0	0	0	0	0	1	3
Dermal hemorrhage	0	0	4	3	0	0	7*	5**
Dermatitis	2	5	10*	5	1	4	10**	4
Dermal fibrosis	0	0	10**	4*	0	0	8**	5**
Dermal pigmentation	0	0	10**	4*	0	0	9**	5**

Significantly different from control group, *p<0.05, **p<0.01

table5: Microscopic Findings for rabbits euthanized on day 29

	Males				Females
group [mg/kg bw]	0	43	85	171	0	43	85	171
treated skin
total number examined	5	0	0	5	5	0	0	5
examined, unremarkable	4	-	-	0	1	-	-	0
number affected
Hyperkeratosis	0	-	-	5**	0	-	-	5**
Acanthosis	0	-	-	0	0	-	-	2
Ulceration	0	-	-	0	1	-	-	0
Dermal hemorrhage	0	-	-	0	1	-	-	1
Dermatitis	1	-	-	5*	3	-	-	4
Granulomatous dermatitis	0	-	-	4*	0	-	-	5**
Dermal fibrosis	0	-	-	2	0	-	-	5**
Dermal pigmentation	0	-	-	5**	0	-	-	5**

Significantly different from control group, *p<0.05, **p<0.01

Conclusions:

Repeated dermal application of the test substance did not result in systemic toxicity. The only abnormal findings involved the treated skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

0.051 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP.

Additional information

In the 13-week toxicity study according to OECD TG 408 and in compliance with GLP, dimethoxy(methyl)silane was given orally by gavage to Sprague Dawley rats at dose levels of 30, 125 and 500 mg/kg bw/day, the top dose level was reduced to 300 mg/kg bw/day from Day 39 due to excessive mortality (Labcorp, 2023, Draft). The control animals received the vehicle dried and de-acidified corn oil. To evaluate the potential reversibility of any findings, a 4-week treatment-free period was included for the control and high-dose groups.

The initial high dose of 500 mg/kg bw/day was not tolerated and resulted in seven premature deaths due to signs of respiratory difficulties (principally rales). The cause of these signs, and therefore death, was determined to be necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria and serocellular exudate. These findings were considered indicative of a damage due to the reflux associated with the gavage dosing procedure, which may have resulted in accidental aspiration of test formulation and/or gastric content into the nasal cavity and were not considered as a primary effect of the test item. No further mortality occurred after the highest dose was reduced to 300 mg/kg bw/day from Day 39, however clinical signs of rales persisted in some individuals until the end of the treatment period.

Body weight gain, food intake and sensory reactivity and grip strength were unaffected by treatment.

The number of low (cage-floor) and high (rearing) beam breaks tended to be lower than control in males receiving 30, 125 or 500/300 mg/kg bw/day. At the end of the four-week recovery period, (cage-floor) and high (rearing) beam breaks were higher than controls in males previously given 500/300 mg/kg bw/day, indicating that recovery occurred.

There were no treatment-related ophthalmoscopic findings.

At the haematological examination in Week 13, monocyte counts were low in females receiving 125 or 500/300 mg/kg bw/day. This was not present after 4 weeks of recovery, indicating that the change was reversible.

The following treatment-related findings were identified at the biochemical examination of the blood plasma performed in Week 13: low creatinine concentration in males receiving 300/500 mg/kg bw/day; high total cholesterol, high-density lipoprotein and low density lipoprotein concentrations in females receiving 500/300 mg/kg bw/day; high total protein concentration in females receiving 500/300 mg/kg bw/day, with low albumin to globulin ratio, indicating that the increase was due to globulin. These findings were not present at the end of the recovery period, indicating that full recovery had occurred.

The analysis of urine performed in Week 13 of treatment revealed, compared with controls: high protein output and concentration in females receiving 500/300 mg/kg bw/day; high glucose output and concentration females receiving 500/300 mg/kg bw/day; high pH in females receiving 125 or 500/300 mg/kg bw/day. It was not possible to assess recovery for these findings. There were no treatment-related findings in males.

Thyroid stimulating hormone concentration was unaffected by treatment.

Estrous cyclicity was unaffected by treatment.

After 13 weeks of treatment, liver weights were higher than control in both sexes receiving 125 or 500/300 mg/kg bw/day. This effect was not present at the end of the recovery period and there was no histopathological correlate.

No test item-related macroscopic or microscopic findings were seen after 13 weeks of treatment or at the end of the recovery phase.

It was concluded that the oral administration of dimethoxy(methyl)silane to Sprague Dawley rats caused local toxicity to the nasal epithelium at 500 mg/kg bw/day (necrosis and inflammation of the respiratory epithelium, degeneration of the olfactory epithelium, fibrosis of the lamina propria) which resulted in adverse respiratory clinical findings and premature mortality. This effect was attributed to local irritation caused by reflux associated with the physiochemical properties of the test item and gavage dosing procedure and not systemic toxicity. The high dose level was lowered to 300 mg/kg bw/day from Day 39, which prevented further mortality, but respiratory clinical signs persisted until the end of the study. There were no adverse systemic findings but there was evidence of a non-adverse, adaptive effect on the liver and a non-adverse reduction of motor activity in males, the cause of which was undetermined. Full recovery occurred after the 4-week treatment free period.

The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was therefore considered to be 500/300 mg/kg bw/day. Due to the adverse local effect on the nasal epithelium and the associated clinical signs and mortality, the overall NOAEL was considered to be 125 mg/kg bw/day.

 

In the available dermal repeated dose toxicity study (Losco, 1996), New Zealand White rabbits of both sexes were exposed to dimethoxy(methyl)silane (CAS 16881-77-9) under occlusive conditions for 9 doses over a time of 11 days. The study is considered as supporting only, as although it meets generally accepted scientific principles, it was not conducted according to an appropriate OECD TG and was not compliant with GLP.

The animals received the test item at doses of 43, 85, and 171 mg/kg bw/day. At the end of exposure period the animals were euthanised. 5 animals from high-dose and control group were kept as satellite groups for 2 weeks of recovery. Observations were made for clinical effects, gross and microscopic lesions, haematology, clinical chemistry and urinalysis. Repeated dermal application of the test substance did not result in systemic toxicity. The only abnormal findings involved the treated skin.

Clinical observations included mild to moderate irritation of the treated skin, affecting mainly high dose group animals euthanized immediately after the exposure period, with females being slightly more sensitive. Significant gross and microscopic lesions were seen in the treated skin of animals receiving 85 mg/kg bw/day and higher. Gross lesions consisted of erythema, ecchymosis, exfoliation, excoriation, fissures, ulceration and necrosis. Microscopic lesions included hyperkeratosis, acanthosis, congestion, haemorrhage, epidermitis, dermatitis and ulceration. Dermal fibrosis and prominent granulomatous inflammation, associated with pigmented granular foreign material, was found in the superficial dermis. After the 2 week recovery period, exfoliation was the only gross skin lesion found in high dose group animals. Microscopic skin lesions consisted of marked granulomatous dermatitis and a fibrotic reaction associated with the foreign material, as well as residual lesions of surface irritation.

Based on the outcome of the study, the NOAEL for systemic toxicity was set at 171 mg/kg bw/day. Due to skin irritating effects seen in this study already at the low dose level of 43 mg/kg bw/day, the LOAEL for local effects was set at 0.051 mg/cm².

Justification for classification or non-classification

The available data on repeated dose toxicity of dimethoxy(methyl)silane do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and is therefore conclusive but not sufficient for classification.