Registration Dossier

Administrative data

Description of key information

Oral: OECD 422 draft, rat: NOAEL (systemic) = not available yet

Based on the preliminary study results, the read-across to the structural analogue substance triethoxy(2,4,4 -trimethylpentyl)silane (CAS 35435 -21 -3) cannot be justified and classification as STOT RE2 might be warranted. Therefore, the decision on the need to conduct a subchronic toxicity study, according to OECD 408, is still under consideration for trimethoxy(2,4,4 -trimethylpentyl)silane. As soon as this decision has been made, this section will be updated accordingly.

Dermal: No data available.


Inhalation (OECD 412): NOAEC = 2890 mg/m³ air (male/female)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10. January to 4. April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
food consumption was not measured in recovery males, clinical observation was not conducted on two days in females.
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit- und Lebensmittelsicherheit
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 13-15 weeks
- Weight at study initiation: males: 324 - 378 g, females: 203 - 250 g
- Fasting period before study: no
- Housing: 5 animals per sex per cage during pre-mating and post-mating period. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared at least once every 10 days which is with the stability frame.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s
- Concentration in vehicle: 0, 12.5, 37.5, 75 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of formulation concentrations of the test substance was based on a GC-FID method. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.
Duration of treatment / exposure:
males: 28 days
females: up to 63 days
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
main study: 10 animals per sex per dose
recovery group: 12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level of 300 mg/kg bw/day was chosen on the basis of a dose range finding study, where mortality was observed after repeated oral administration at a dose of 1000 mg/kg bw/day (later reduced to 600 mg/kg bw/day). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations: health condition, morbidity, moratility

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, 9 and 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment for the main group; for the recovery group additionally in the last week of the recovery period
- Dose groups that were examined: main study: in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated); recovery study: all males and females

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: No
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment period and in all recovery group males and females at the end of the recovery period as part of the sacrifice of the animals
- Parameters checked: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Animals fasted: No
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment period and in all recovery group males and females at the end of the recovery period as part of the sacrifice of the animals
- Parameters checked: alanine aminotransferase (ALAT); aspartate-aminotransferase (ASAT); alkaline phosphatase (AP); creatinine (Crea); total protein (TP); albumin (Alb); urea; total bile acids (TBA); total cholesterol (Chol); glucose (Gluc); sodium (Na); potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked: specific gravity; nitrite; pH-value (pH); protein; glucose; ketone bodies (Ket); urobilinogen (UBG); bilirubin (BIL); erythrocytes (Ery); leukocytes (Leuc); urine colour and appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week before the first treatment and during the last week of the treatment for main study group; animals of the recovery groups were examined once before the first exposure, during the last week of treatment as well as in the last week of the recovery period.
- Dose groups that were examined: 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. For the recovery groups all males and females were examined.
- Battery of functions tested: sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature

IMMUNOLOGY: No

OTHER: From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, and from all adult males at termination, blood samples were collected. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary, based on the fact that no major histopathological finding was observed in thyroid/ parathyroid gland of selected male and female adult animals and no effect was observed on hormone levels of males and day 13 pups.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents (see table 1)

HISTOPATHOLOGY: Yes: see table 1








Other examinations:
Organ weights: testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thymus, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), liver, kidneys (paired weight), spleen, adrenal (paired weight), brain, pituitary gland, heart
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Dunn’s Test, Dunnett’s Test or Student’s T-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).
Dose descriptor:
NOAEL
Remarks on result:
other: no final report available yet
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was according an apropriate OECD test guideline, and in compliance with GLP.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
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:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar (Hoe: WISKf(SPF71))
- Source: Hoechst AG, Pharma-Forschung-Toxikologie, Kastengrund (breeding under SPF conditions)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males: 137-150 g (mean = 143.3± 3.74), females: 137-146 g (mean = 141.0±1.29)
- Fasting period before study: no
- Housing: individually during exposure, 5 per Makrolon cage (type 4) after exposure
- Diet: Altromin 1324-pellets (Altromin GmbH, Lage/Lippe, Germany) ad libitum except during exposure
- Water: tap water ad libitum except during exposure


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 50±20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 (7.00-19.00)

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: Particle size (mean): 99.98% of the particle in 0.3 mg/L group, 99.98% of the particle in 1.5 mg/L group and 99.87% of the particle in 3.0 mg/L group are below 6 µm.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Animals were exposed to the test atmosphere in nose-only inhalation units
- Exposure apparatus: The inhalation chambers used in the study were stainless steel/glass cylindrical columns placed in a hood with a volume of approximately 4 m³. Each column had a volume of 80 L and consisted of a top assembly with the inlet of the test atmosphere, one rodent tube section and the bottom, the base assembly with the exhaust port.
- Method of holding animals in test chamber: Nose only; cylindrical tubes.
- Method of conditioning air: Compressed air (4 bar), oil separation filter/absolute filter; to achieve requested air humidity moistened air was feeded directly in the chamber
- System of generating particulates/aerosols: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing test material through special nozzles. The operating pressure was 4 bar.
- Temperature, humidity: 20.0-23.5°C, 31.5-60.6%
- Continuous measurement of CO-, CO2- and O2-concentration during exposure in exposure chamber; CO: 0 ppm; CO2: 3800-7900 ppm; O2: 19.8-20.6 Vol %
- Air flow rate: Air inlet 800 L/h; 1100 L/h exhausted
- Method of particle size determination: The particle size distribution was measured using an APS 33 Aerodynamik Particle Sizer from TSI Inc., St Paul. The aerodynamic diameter range measured with this analyser covered 0.486 to > 15.4 micrometer. Measurements were performed daily 30 minutes, 2 and 4 hours respectively after starting the exposure. After the end of the exposure period, the arithmetic means together with the standard deviations based on the 3 points in time were calculated using a statistics program. The statistics program was used further to calculate an overall arithmetic mean for each dose group based on the arithmetic means for each single day of exposure.
- Treatment of exhaust air: Escaped particles were exhausted by a gas purifying plant and neutralised. Additionally, there was an exhaust device at the bottom of each inhalation chamber to remove the aerosol by filtering over a Buehler-Filter and a gas-washing bottle.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric verification of concentrations using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (Sartorius Membranfilter GmbH, Goettingen). Samples were taken daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.
Chemical verification of concentrations was performed using a gas chromatograph. Sampling was performed on days 1, 8, 15, 22, and 27 of the treatment.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetric verification of concentrations
Gravimetric measurements were performed using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (sartorius Membranfilter GmbH, Goettingen). The air flow rate was 3 L/min, which is equivalent to an intake velocity of 1.25 m/sec. Gravimetric measurements were performed daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.

Chemical verification of concentrations
Within 60 minutes 31 liter aerosol from the exposure chambers was pumped through 3 gas-washing bottles filled with acetone (PESTANAL, RIEDEL DE HAEN) connected in series and standing in a cool trap. Thereof aliquots were taken and analysed using a gas chromatograph. The concentration of isooctyltrimethoxysilane in the exposure chambers was calculated based on the results of the GC and considering the aerosol- and acetone volumina. Sampling was performed on days 1, 8, 15, 22 and 27 of the treatment.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.32 mg/L air (analytical)
Dose / conc.:
1.54 mg/L air (analytical)
Dose / conc.:
2.89 mg/L air (analytical)
Dose / conc.:
0.3 mg/L air (nominal)
Dose / conc.:
1.5 mg/L air (nominal)
Dose / conc.:
3 mg/L air (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Five animals of each sex and group were used to assess recovery from treatment-related effects
- Post-exposure recovery period in satellite groups: 15 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Mortality, behaviour and general state of health (before and after exposure and during exposure; one time per day during weekend); Neurological disorders, opacity of eyes, damage of oral mucosa, disorder of tooth growth (weekly)

BODY WEIGHT: Yes
- Time schedule for examinations: Before exposure began and then twice per week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Twice per week

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All dose groups and control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of serum: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex (Nembutal narcosis)
- Parameter checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure period
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations (neurological disorders): Once per week
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
None
Statistics:
The following parameters have been checked inter-collective concerning statistical significance (p = 0.05) in accordance with internal SOP: body weight, body weight gain; haematology (excluding differential blood count, heinz bodies, reticulocytes); clinical chemistry (excluding bilirubin direct, methaemoglobin, g-glutamyltranspeptitase); serum electrophoresis, relative organ weights, pH-value urine.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)
Mortality:
mortality observed, treatment-related
Description (incidence):
staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
increased for high dose males on day 7, high dose females on days 7-21, mid dose males on days 14 until recovery, and mid dose females on days 14-29
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
3 males and 1 female of the high dose group had signs of minimal intense irritation in the alveoli
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
Following exposure each animal of the high dose group had a staggering gait, which was resolved by the next day. One animal additionally showed ruffled fur and retracted belly flanks on day 10.
In the mid dose group, animals that showed a lack of coordination following exposure were recovered within two hours after exposure. The low dose and control group animals did not have any clinical signs.
No clinical signs were observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN:
On days 1 and 3 males of the high and mid dose group had a slightly increased mean body weight (10 animals each dose group, experimental period) in comparison to the control animals. Females of the high dose group had a slightly reduced mean body weight on day 14 and days 21-29 (10 animals, experimental period) and days 29-31 (5 animals, recovery period) of the recovery period.

FOOD CONSUMPTION:
Males from high and mid dose groups on days 3-29 (experimental period) and females on days 3-29 (experimental period) and days 29-31 (recovery period) had slight reductions in feed intake.

WATER CONSUMPTION:
In high dose males there was an increased water consumption on day 7 until the end of the experimental period. Females of this group had elevated water consumption on days 7-29 (experimental period). In the mid dose group males, water consumption was increased from day 14 until start of recovery period, and in females of this group from day 14-29 (experimental period).

OPHTHALMOSCOPIC EXAMINATION:
No effects on the eyes.

HAEMATOLOGY:
Statistically significant effects one day after the end of exposure were as follows: increased albumin in mid dose females, increased alpha2 globulin in high dose males, decreased alpha3 globulin in mid and high dose females, increased albumin to globulin ratio in mid dose females.
Statistically significant effects 15 days after the end of exposure (recovery group) were as follows: increased albumin in low dose males and high dose males and females, decreased beta1 globulin in low and high dose males, decreased g1 globulin in low dose male and females and high dose females, and increased albumin to globulin ratio in low and high dose males. However, all values were in the normal range and were not considered to be toxicologically relevant.

URINALYSIS:
No treatment-related effects.

NEUROBEHAVIOUR:
Not examined but there were no neurological disorders observed in the clinical observations.

ORGAN WEIGHTS:
There were statistically significant increases in lung weight in mid dose males one day after exposure, and lung weight in high dose females sacrificed 15 days after exposure. However, all values were within the normal range, and there were no corresponding histopathological findings. Therefore, the effect was considered non-adverse.

GROSS PATHOLOGY:
There were no treatment-related macroscopic changes identified.

HISTOPATHOLOGY:
There were no treatment related findings in the control, low and mid dose groups. In the high dose group three males and one female, which were sacrificed one day after the last exposure, had signs of minimal intense irritation in the alveoli (isolated and small clusters of foam cells). There were no other treatment-related effects.
Dose descriptor:
NOAEC
Effect level:
ca. 3 000 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.
Dose descriptor:
NOAEC
Effect level:
2 890 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.
Critical effects observed:
no
Conclusions:
The test item was tested for subacute inhalation toxicity according to OECD TG 412 and in compliance with GLP. The NOAEC was determined to be 3000 mg/m³ (nominal). No specific target organ toxicity was observed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 890 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was according an apropriate OECD test guideline, and in compliance with GLP.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
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:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar (Hoe: WISKf(SPF71))
- Source: Hoechst AG, Pharma-Forschung-Toxikologie, Kastengrund (breeding under SPF conditions)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males: 137-150 g (mean = 143.3± 3.74), females: 137-146 g (mean = 141.0±1.29)
- Fasting period before study: no
- Housing: individually during exposure, 5 per Makrolon cage (type 4) after exposure
- Diet: Altromin 1324-pellets (Altromin GmbH, Lage/Lippe, Germany) ad libitum except during exposure
- Water: tap water ad libitum except during exposure


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 50±20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 (7.00-19.00)

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: Particle size (mean): 99.98% of the particle in 0.3 mg/L group, 99.98% of the particle in 1.5 mg/L group and 99.87% of the particle in 3.0 mg/L group are below 6 µm.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure: Animals were exposed to the test atmosphere in nose-only inhalation units
- Exposure apparatus: The inhalation chambers used in the study were stainless steel/glass cylindrical columns placed in a hood with a volume of approximately 4 m³. Each column had a volume of 80 L and consisted of a top assembly with the inlet of the test atmosphere, one rodent tube section and the bottom, the base assembly with the exhaust port.
- Method of holding animals in test chamber: Nose only; cylindrical tubes.
- Method of conditioning air: Compressed air (4 bar), oil separation filter/absolute filter; to achieve requested air humidity moistened air was feeded directly in the chamber
- System of generating particulates/aerosols: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing test material through special nozzles. The operating pressure was 4 bar.
- Temperature, humidity: 20.0-23.5°C, 31.5-60.6%
- Continuous measurement of CO-, CO2- and O2-concentration during exposure in exposure chamber; CO: 0 ppm; CO2: 3800-7900 ppm; O2: 19.8-20.6 Vol %
- Air flow rate: Air inlet 800 L/h; 1100 L/h exhausted
- Method of particle size determination: The particle size distribution was measured using an APS 33 Aerodynamik Particle Sizer from TSI Inc., St Paul. The aerodynamic diameter range measured with this analyser covered 0.486 to > 15.4 micrometer. Measurements were performed daily 30 minutes, 2 and 4 hours respectively after starting the exposure. After the end of the exposure period, the arithmetic means together with the standard deviations based on the 3 points in time were calculated using a statistics program. The statistics program was used further to calculate an overall arithmetic mean for each dose group based on the arithmetic means for each single day of exposure.
- Treatment of exhaust air: Escaped particles were exhausted by a gas purifying plant and neutralised. Additionally, there was an exhaust device at the bottom of each inhalation chamber to remove the aerosol by filtering over a Buehler-Filter and a gas-washing bottle.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric verification of concentrations using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (Sartorius Membranfilter GmbH, Goettingen). Samples were taken daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.
Chemical verification of concentrations was performed using a gas chromatograph. Sampling was performed on days 1, 8, 15, 22, and 27 of the treatment.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetric verification of concentrations
Gravimetric measurements were performed using a membrane filter, pore size 0.65 micrometer, 50 mm diameter (sartorius Membranfilter GmbH, Goettingen). The air flow rate was 3 L/min, which is equivalent to an intake velocity of 1.25 m/sec. Gravimetric measurements were performed daily in the exposure chambers 30 minutes, 2 and 4 hours respectively after starting the exposure.

Chemical verification of concentrations
Within 60 minutes 31 liter aerosol from the exposure chambers was pumped through 3 gas-washing bottles filled with acetone (PESTANAL, RIEDEL DE HAEN) connected in series and standing in a cool trap. Thereof aliquots were taken and analysed using a gas chromatograph. The concentration of isooctyltrimethoxysilane in the exposure chambers was calculated based on the results of the GC and considering the aerosol- and acetone volumina. Sampling was performed on days 1, 8, 15, 22 and 27 of the treatment.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.32 mg/L air (analytical)
Dose / conc.:
1.54 mg/L air (analytical)
Dose / conc.:
2.89 mg/L air (analytical)
Dose / conc.:
0.3 mg/L air (nominal)
Dose / conc.:
1.5 mg/L air (nominal)
Dose / conc.:
3 mg/L air (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Five animals of each sex and group were used to assess recovery from treatment-related effects
- Post-exposure recovery period in satellite groups: 15 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Mortality, behaviour and general state of health (before and after exposure and during exposure; one time per day during weekend); Neurological disorders, opacity of eyes, damage of oral mucosa, disorder of tooth growth (weekly)

BODY WEIGHT: Yes
- Time schedule for examinations: Before exposure began and then twice per week

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Twice per week

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All dose groups and control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of serum: One day after the last exposure five animals of each sex, then 15 days after last exposure the remaining five animals of each sex (Nembutal narcosis)
- Parameter checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure period
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations (neurological disorders): Once per week
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
None
Statistics:
The following parameters have been checked inter-collective concerning statistical significance (p = 0.05) in accordance with internal SOP: body weight, body weight gain; haematology (excluding differential blood count, heinz bodies, reticulocytes); clinical chemistry (excluding bilirubin direct, methaemoglobin, g-glutamyltranspeptitase); serum electrophoresis, relative organ weights, pH-value urine.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)
Mortality:
mortality observed, treatment-related
Description (incidence):
staggering gait (high dose group, reversible within 1 day), lack of coordination (mid dose, reversible within 2 hours)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
increased for high dose males on day 7, high dose females on days 7-21, mid dose males on days 14 until recovery, and mid dose females on days 14-29
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
3 males and 1 female of the high dose group had signs of minimal intense irritation in the alveoli
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
Following exposure each animal of the high dose group had a staggering gait, which was resolved by the next day. One animal additionally showed ruffled fur and retracted belly flanks on day 10.
In the mid dose group, animals that showed a lack of coordination following exposure were recovered within two hours after exposure. The low dose and control group animals did not have any clinical signs.
No clinical signs were observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN:
On days 1 and 3 males of the high and mid dose group had a slightly increased mean body weight (10 animals each dose group, experimental period) in comparison to the control animals. Females of the high dose group had a slightly reduced mean body weight on day 14 and days 21-29 (10 animals, experimental period) and days 29-31 (5 animals, recovery period) of the recovery period.

FOOD CONSUMPTION:
Males from high and mid dose groups on days 3-29 (experimental period) and females on days 3-29 (experimental period) and days 29-31 (recovery period) had slight reductions in feed intake.

WATER CONSUMPTION:
In high dose males there was an increased water consumption on day 7 until the end of the experimental period. Females of this group had elevated water consumption on days 7-29 (experimental period). In the mid dose group males, water consumption was increased from day 14 until start of recovery period, and in females of this group from day 14-29 (experimental period).

OPHTHALMOSCOPIC EXAMINATION:
No effects on the eyes.

HAEMATOLOGY:
Statistically significant effects one day after the end of exposure were as follows: increased albumin in mid dose females, increased alpha2 globulin in high dose males, decreased alpha3 globulin in mid and high dose females, increased albumin to globulin ratio in mid dose females.
Statistically significant effects 15 days after the end of exposure (recovery group) were as follows: increased albumin in low dose males and high dose males and females, decreased beta1 globulin in low and high dose males, decreased g1 globulin in low dose male and females and high dose females, and increased albumin to globulin ratio in low and high dose males. However, all values were in the normal range and were not considered to be toxicologically relevant.

URINALYSIS:
No treatment-related effects.

NEUROBEHAVIOUR:
Not examined but there were no neurological disorders observed in the clinical observations.

ORGAN WEIGHTS:
There were statistically significant increases in lung weight in mid dose males one day after exposure, and lung weight in high dose females sacrificed 15 days after exposure. However, all values were within the normal range, and there were no corresponding histopathological findings. Therefore, the effect was considered non-adverse.

GROSS PATHOLOGY:
There were no treatment-related macroscopic changes identified.

HISTOPATHOLOGY:
There were no treatment related findings in the control, low and mid dose groups. In the high dose group three males and one female, which were sacrificed one day after the last exposure, had signs of minimal intense irritation in the alveoli (isolated and small clusters of foam cells). There were no other treatment-related effects.
Dose descriptor:
NOAEC
Effect level:
ca. 3 000 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.
Dose descriptor:
NOAEC
Effect level:
2 890 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A No Observed Toxic Effect level (NTEL) of 3 mg/L was defined. Only minor and reversible effects have been observed at 3 mg/L. Therefore the NTEL is identical to the NOAEL.
Critical effects observed:
no
Conclusions:
The test item was tested for subacute inhalation toxicity according to OECD TG 412 and in compliance with GLP. The NOAEC was determined to be 3000 mg/m³ (nominal). No specific target organ toxicity was observed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 890 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was according an aapropriate OECD test guideline, and in compliance with GLP.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

A combined repeated dose oral toxicity and reproduction/developmental toxicity screening test, according to OECD 422 and GLP, with trimethoxy(2, 4, 4-trimethylpentyl)silane in male and female Wistar rats tested at dose levels of 50, 150, and 300 mg/kg bw/day (BSL, 2019) is available. The study was only recently completed at — Eurofins BioPharma Product Testing, Germany — in November 2019. That being so, the final draft of the OECD 422 study report was not available in time to be included in this dossier submission. Nevertheless, the available preliminary results were reviewed, and it is evident that they are severe and significant and would perhaps fulfill the requirement to classify the substance as STOT-RE Cat 2, with the hazard statement 'H373: May cause damage to organs according to Regulation (EC) 1272/2008. A final judgment on the need for this classification will be made once the final report is available from the laboratory. If it does turn out, that the registration substance will need to be classified - STOT-RE Cat 2- then a sub-chronic toxicity study will not be required according to the standard information requirements in the REACh Regulation Annex IX Section 8.6.2. If, on the other hand, the registration substance does not need to be classified, then an OECD 408 study on the registration substance will be initiated. Also, the preliminary results from the OECD 422 study with trimethoxy(2,4,4-trimethylpentyl)silane (CAS 34396-03-7) and the OECD 408 study on triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3) were compared to determine whether or not the read-across from the source substance triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3) could be defended as a conservative approach. The conclusion is that the long-term systemic toxicity profiles of these substances are not alike and most importantly the read-across of the OECD 408 from the triethoxy(2,4,4 -trimethylpentyl)silane (CAS 35435 -21 -3) to trimethoxy(2,4,4-trimethylpentyl)silane (CAS 34396-03-7) cannot be defended as a conservative approach.

As soon as a decision has been made on the need for a classification, the dossier will be updated accordingly. Therefore, the outcome will be either that an OECD 408 study with the registration substance will be contracted based on the final decision TPE-D-2114426294 -52 -01/F or the subchronic study will be waived according to Annex IX Section 8.6.2 of the Reach Regulation.

Repeated dose toxicity: dermal

No data available.

Repeated dose toxicity: inhalation

In the available key study (Hoechst, 1986b) the test item was tested for repeated dose toxicity after subacute inhalative treatment. The test was conducted according to the OECD TG 412 and in compliance with GLP. Groups of ten male and ten female Wistar rats were exposed to a respirable aerosol of the test item at concentrations of 0.32, 1.54 and 2.89 mg/L (mean actual concentration) for 28 days (6 h/day, 5 days/week). After the exposure period five male and five female rats of each group were kept during a 14 d recovery period before necropsy. Following exposure each animal of the high dose group had a staggering gait, which was resolved by the next day. In the mid dose group, animals that showed a lack of coordination following exposure were recovered within two hours after exposure. There were sporadic changes to body weights in comparison to the controls. The low dose and control group animals did not have any clinical signs. There were no treatment-related effects on haematology, clinical chemistry, urinalysis, organ weights, and gross pathology. There were signs of minimal intense alveolar irritation (isolated and small clusters of foam cells) in several high dose animals, which is considered to be the predominant effect. Overall, the NOAEC for this study was considered to be 2.89 mg/L.

 


Justification for classification or non-classification

A final conclusion on classification for target organ toxicity after repeated exposure will be made as soon as the final report data is available for the OECD 422.