1,1,1,3,3,3-hexamethyldisilazane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a repeated dose inhalation toxicity with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP with the registered substance 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5), there were no treatment related-effects observed in any of the reproductive parameters evaluated in male and female animals up to the highest concentration. Therefore the NOAEC for reproductive toxicity was considered to be equal to or greater than 400 ppm (2640 mg/m3) in rats (Dow Corning Corporation 2008).

There is a testing proposal for an oral extended one-generation reproductive toxicity study on trimethylsilanol (CAS 1066 -40 -6), the hydrolysis product of the registered substance, to be performed according to OECD Test Guideline 443 and in compliance with GLP, after approval from ECHA.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.09.2007-14.11.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc, Portage, MI
- Age at study initiation: Minimum of nine weeks
- Weight at study initiation: Males: 291.8 to 354.8 g; Females: 200.5 to 254.9 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages elevated over bedding except during mating when animals were cohoused in the home cage of the male.
- Diet (e.g. ad libitum): Ad libitum, except during exposure period
- Water (e.g. ad libitum): Ad libitum, except during exposure period
- Acclimation period: Seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.04-22.86
- Humidity (%): 33-66
- Air changes (per hr): 11.8-14.3
- Photoperiod (hrs dark / hrs light):


IN-LIFE DATES: From: 27.09.2007 To: 05.03.2008

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2000 litre stainless steel and glass Rochester-style inhalation chambers.
- Method of holding animals in test chamber: four levels of 20 individual animal compartments. The animal cage position assignment within each chanber was rotated daily.
- Source and rate of air: Building air (rate: no data)
- Method of conditioning air: Air from a Nash Air Compressor was passed through a series of filters to remove contaminants. This conditioned air was then passed through HEPA and activated charcoal filters before delivery to the chmabers.
- Temperature, humidity, pressure in air chamber: 22 ±3oC, 50 ±20% humidity, no data on pressure
- Air flow rate: No data
- Air change rate: 10-15 chamber volume air changes per hour
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Automated sampling system designed so that test atmosphere continuously pulled from the chamber and delivered to analyser. Analysis was done using a gas chromatograph equipped with a flame ionisation detector to determine the actual chamber concentrations of test substance.
- Samples taken from breathing zone: No data

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to seven days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged (how): Individually. Pregnant females were moved into shoebox cages.
- Any other deviations from standard protocol: None evident

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Automated sampling system designed so that test atmosphere continuously pulled from the chamber and delivered to analyser. Analysis was done using a gas chromatograph equipped with a flame ionisation detector to determine the actual chamber concentrations of test substance.

Duration of treatment / exposure:

Exposure period: Males (14 days premating and mating up to 28 days): Females (premating 14 days, mating, gestation, and postpartum days 1-3 up to 42 days
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: Males 28 days; Females up to 42 days

Frequency of treatment:

6 hours/day, 7 days/week

Details on study schedule:

Screening study only, so F1 matings were not conducted.

Dose / conc.:

0 ppm (nominal)

Remarks:

Group 1 - Control Group

Dose / conc.:

25 ppm (nominal)

Remarks:

Group 2 (0.16 mg/L)

Dose / conc.:

100 ppm (nominal)

Remarks:

Group 3 (0.66 mg/L)

Dose / conc.:

400 ppm (nominal)

Remarks:

Group 4 (2.66 mg/L)

No. of animals per sex per dose:

10 males and 20 females (including 10 for toxicity group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:Based on previous study.
- Rationale for animal assignment (if not random): Random

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality, and daily for clinical signs of toxicity (including abnormalities in nesting)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and then weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: First day of dosing, and at least weekly thereafter, and on day of sacrifice.

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice as a terminal procedure.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: All males and toxicity phase females. Details reported in Section 7.5.3

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice as a terminal procedure.
- Animals fasted: Yes, overnight
- How many animals: All males and toxicity phase females. Details reported in Section 7.5.3

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, details reported in Section 7.5.3

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Testes were weighed and examined microscopically.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No, all animals were killed in Day 4 post-partum for exminations.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: mating, pregnancy, duration of gestation, mean litter size, mean live litter size, mean litter weight, mean ratio of live births/litter size, and sex ratio.  The number of corpora lutea and the number of uterine implantation sites were determined for all females.


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 29 days treatment.
- Maternal animals: All surviving animals on day 4 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: See Section 7.5.3

Postmortem examinations (offspring):

SACRIFICE
- All offspring on day 4 post-partum
- These animals were subjected to gross necropsy examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not conducted

Statistics:

All data analysis was carried out using SAS version  8.2 or 9.1.3. Descriptive toxicology endpoints:  Parametric data was tested using Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant); nonparametric data was tested by Kruskal-Wallis Test followed by Wilcoxon.  Repeated measures ANOVA was applied to data sets of multiple measurements  across time.  Histomorphology:  Cochran-Armitage Trend test for severity with Fisher's Exact Test for  pairwise comparisons.  Kruskal-Wallis was used to test for shifts in  grade with the Wilcoxon test used to test pairwise comparisons.   Functional observational battery:  ANCOVA (baseline as covariate), repeated measures ANOVA, Mixed modeling, and Jonckeere-Terpstra test (categorical endpoints).

Reproductive indices:

Mating and fertilty indices

Offspring viability indices:

None

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs attributed to test article included uncoordinated gait (not beyond day 10 or 11) and decreased activity noted immediately after exposure in both sexes from the highest dose group. These clinical signs persisted for some time following the exposure period with animals returning to normal prior to the next day's exposure.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant decrease in absolute body weights for females (-15%) with a corresponding decrease in total weight gain compared to controls for both sexes (62 and 25% for females and males, respectively) in the highest dose group. Absolute body weights for the mid dose group females were identified as statistically decreased (-7%) from controls, however, total and percent weight gain were not different.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Total food consumption was statistically decreased for the highest dose group males and females.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased glucose along with increased cholesterol levels, were noted in the highest dose group females with cholesterol and sodium levels statistically increased in the highest dose group males.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the neurobiological function as evaluated through FOB and motor activity evaluations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of tissues and organs for control and high exposure animals demonstrated an increased incidence of centrilobular hypertrophy in the liver of high dose group females. Evaluation of intermediate dose levels confirmed this finding was limited to high dose group females. There were no other test article-related histopathological findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The fertility rate was high resulting in at least 9 litters per group. At all concentrations, there were no treatment-related effects on the mean duration of gestation, number of implantations and mean number of corpora lutea per dam.

Dose descriptor:

NOAEC

Remarks:

General Toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEC

Remarks:

Reproductive toxicity

Effect level:

>= 400 ppm

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

A Group 2-female delivered a normal size litter which included one dead, edematous foetus. This was considered an isolated, spontaneous finding with no additional occurences observed within this or any other exposure group.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

>= 400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1 Summary of reproductive parameter findings*

Parameter	Control	25ppm	100ppm	400ppm
Length of gestation	21	22	22	22
Male pups	8	7	7	7
Female pups	7	8	9	8
Sex ratio	1.2	0.9	0.9	1.0
Total pups (mean)	15	15	15	15
Initial litter weight (g)	88.1	94.5	98.1	96.2
Initial average pup weight (g)	6.2	6.5	6.4	6.5
Implantation sites	16	16	16	15
Number of corpora lutea	17	17	17	16
Number pregnant	10	9	10	10

*There were no statistically significant differences found.

Conclusions:

In a repeated dose inhalation toxicity with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP with the registered substance 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5), there were no treatment related-effects observed in any of the reproductive parameters evaluated in males and females animals up to the highest concentration. Therefore the NOAEC for reproductive toxicity was considered to be equal to or greater than 400 ppm (2.66 mg/L) in rats.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 640 mg/m³

Study duration:

subacute

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the key repeated dose inhalation toxicity with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP, the registered substance 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) was administered to Sprague-Dawley rats via whole-body vapour inhalation for six hours/day, seven days/week at target concentrations of 0, 25, 100 and 400 ppm.

Each exposure group consisted of 10 male and 20 female rats. Females were further divided into a toxicity and reproductive group consisting of 10 animals each (details regarding the repeated dose toxicity elements of the study are presented in Section 7.5.2). Males were used to evaluate toxicity and reproductive parameters. Males were treated for 29 days, and reproductive group females were treated for 14 days prior to mating, throughout mating, gestation and up to and including postpartum day 3. The maximum number of exposures was 42 days. Animals were observed throughout the exposure period for mortality and signs of toxicity. Body weights and food consumption were monitored. Reproductive and developmental parameters evaluated included evidence of mating, pregnancy, duration of gestation, mean litter size, mean live litter size, mean litter weight, mean ratio of live births/litter size and sex ratio. All surviving dams and pups were sacrificed on postpartum day 4 and examined for external gross lesions. The number of corpora lutea and the number of uterine implantation sites were determined for all reproductive phase females. There were no adverse effects on any of the reproductive and developmental toxicity parameters investigated, giving a NOAEC of greater than or equal to 400 ppm for these endpoints

(equivalent to 2640 mg/m³). The NOAEC for repeated dose toxicity for the parents was concluded to be 100 ppm, based on clinical observation,

body weight changes and histological findings (see Section 7.5.2) (Dow Corning Corporation 2008).

1,1,1,3,3,3-Hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) hydrolyses very rapidly in contact with water (<0.04 minutes, <0.5 minutes and <0.1 minutes at pH 4, 7 and 9 and 1^oC) generating trimethylsilanol and ammonia. Reaction rate also increases with temperature, so hydrolysis at physiologically relevant temperatures and pH 7 (relevant for conditions in the lung following inhalation administration) will be even faster. Therefore read-across from the hydrolysis product, trimethylsilanol, to 1,1,1,3,3,3-hexamethyldisilazane for the supporting inhaled OECD 422 study is considered valid. No effects on sexual function and fertility have been noted for the non-silanol hydrolysis product, ammonia (OECD SIDS, 2007).

In an inhalation study conducted according to the same OECD Test Guideline 422 and in compliance with GLP, on the three groups of Sprague-Dawley rats were exposed, via whole-body inhalation to vapour atmospheres of the hydrolysis product, trimethylsilanol (CAS 1066 -40 -6) for 6 hours/day, 7 days/week for 4 weeks, at 0, 60, 300 and 600 ppm. Test substance-related effects were limited to changes in haematology (lower eosinophil and lymphocyte counts for males) and serum chemistry (higher alanine aminotransferase for males and toxicity phase females) at 600 ppm. These changes occurred in the absence of correlating histologic changes and were not considered adverse. There were no treatment related effects observed in any of the reproductive parameters evaluated in male and female animals. Therefore, a NOAEC for reproductive toxicity was concluded to be greater than or equal to 600 ppm under the conditions of this screening study (WIL Research Laboratories, 2008).

Effects on developmental toxicity

Description of key information

In the key prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP with the hydrolysis product, trimethylsilanol (CAS 1066-40-6), the foetal NOAELs was concluded to be 150 mg/kg bw/day based on reduced foetal weight, delayed ossification and increased incidence of some cartilaginous variations noted in foetuses at 450 mg/kg bw/day. There were no maternal developmental effects but general toxicity such as reductions in food consumption and body weight gain were noted in dams at 450 mg/kg bw/day. The maternal NOAEL for general toxicity was therefore considered to be 150 mg/kg bw/day (Harlan, 2014).

Trimethylsilanol as test substance instead of the registered substance 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) for the pre-natal developmental toxicity study via the oral route in the rat is considered valid and in line with ECHA final decision number CCH-D-0000002289-68-04/F.  

There is a testing proposal for an oral pre-natal developmental toxicity study in a second species (rabbit) on trimethylsilanol (CAS 1066-40-6), to be performed according to OECD Test Guideline 414 and in compliance with GLP, after approval form ECHA.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April 2013 to 19 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted according to an appropriate guideline and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD IGS

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 12 weeks
- Weight at study initiation: 230 to 316g
- Fasting period before study: no
- Housing: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of mating and afterwards individually in Makrolon type-3 cages with wire mesh tops with sterilized standard softwood bedding (‘Lignocel’) with paper enrichment (ISO-BLOX).
- Diet (ad libitum): Pelleted standard Harlan Teklad 2018C
- Water (ad libitum): community tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2 - 23.5
- Humidity (%): 27.2 - 70.3
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 April 2013 To: 23 May 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Trimethylsilanol was weighed into a glass beaker on a tared precision balance and the vehicle (corn oil) was added. Using an appropriate homogenizer, a homogeneous suspension was pre-pared. Separate formulations were prepared for each concentration
Dose formulations were devided into daily aliquots.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 10, 30, 90 mg/mL
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each concentration were taken from the middle only to confirm the stability (4 hrs at room temperature and 8 days in refrigerator). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the person responsible for formulation analysis and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by Headspace GC-Method The test item was used as the analytical standard.

Duplicates were taken of all samples and were stored.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation observed
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post coitum
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Day 6 to Day 20 post coitum

Frequency of treatment:

daily

Duration of test:

21 days

Dose / conc.:

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

Remarks:

Group 1 - Control Group

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

22 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Sprague Dawley rats using dose levels of 0, 50, 150 and 600/400 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 times daily during treatment

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: recoreded at 3-day intervals, days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION: No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- liver weight recorded

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction and skeletal examination data:

• Means and standard deviations of various data were calculated and included in the report.

• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Historical control data:

Data from test facility derived during 1992 to 2005 included

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs such as uncoordinated movement, decreased activity and/or prostrate appearance and/or ruffled fur occurred after dosing the animals with dose levels of 150 and/or 450 mg/kg bw/day during the treatment period. These were considered to be related to the treatment with the test item. Due to the full recovery of the animals, slight severity grade and frequency at 150 mg/kg bw/day, this dose level was considered to cause treatment-related but not adverse effects in pregnant rats. Due to the nature, frequency and severity of the findings and the observation that one animal did not fully recover from the symptoms, the dose level of 450 mg/kg bw/day was considered to cause treatment-related and adverse effects in pregnant rats.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 450 and 150 mg/kg bw/day, mean absolute body weight and body weight gain were decreased statistically significantly and were considered to be related to the treatment with the test item. At 450 mg/kg bw/day, the corrected body weight gain was also affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 450 and 150 mg/kg bw/day, test item-related statistically significant decreases were observed for mean food consumption.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Uncoordinated movement, decreased activity with dose levels of 150 and/or 450 mg/kg bw/day during the treatment period. These were considered to be related to the treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects on liver weights were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were observed at any dose level during macroscopical examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The relevant reproduction data (pre-and post-implantation loss and number of fetuses per dam) were not affected by treatment with the test item.
Mean numbers of corpora lutea and implantation sites were similar across all groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

Statistically significantly decreased mean food consumption, body weight, body weight gain and corrected body weight gain were affected by the treatment with the test
item and were considered to be adverse maternal toxic effects.

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal General Toxicity

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day, there were statistically significantly decreased fetal body weights both on litter and individual basis and this was related to the treatment with the test item.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related effects on the sex ratio of the fetuses were noted in any group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg bw/day, there were statistically significantly decreased fetal body weights both on litter and individual basis and this was related to the treatment with the test item.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test item-related findings were noted during skeletal examination of the fetuses. Non-ossified cervical vertebral bodies (1 - 7), some non-ossified caudal vertebrae, incompletely ossified sternebra 5, non-ossified metatarsalia 1 and non-ossified digits on fore and hind limbs were seen in fetuses at 450 mg/kg bw/day. These findings indicated a slight delay in ossification at 450 mg/kg bw/day which correlated with the lower fetal body weight in this dose group and this was related to the treatment with the test item.
At 450 mg/kg bw/day, statistically significantly increased incidence of supernumerary rudimentary ribs, long ventral plate and long or interrupted costal cartilage occurred both on an individual
and litter basis and these indicated a slight disturbance in development.
These observations were only variations with high spontaneous incidence of this strain.

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The mean fetal body weight was statistically significantly lower at 450 mg/kg bw/day and was considered to be related to the treatment with the test item. In correlation with the lower fetal weights a slight delay in ossification was noted in the high dose group (450 mg/kg bw/day). An increased incidence of long or interrupted costal cartilages or long ventral plate indicated a slight disturbance in development at this dose level.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other:

Remarks on result:

other: Effects seen are considered secondary to maternal toxicity or variations only

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: forelimb

skeletal: rib

skeletal: vertebra

skeletal: hindlimb

Description (incidence and severity):

An increased incidence of long or interrupted costal cartilages or long ventral plate indicated a slight disturbance in development at this dose level. However these observations are variations only, and not reported as malformations.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Results for Formulation Analysis

Dose Group	Sample taken from	Nominal Conc (mg/mL)	Actual Conc (mg/mL)	Relative to Nominal (%)	Mean (%)
Control	vehicle	0.0	ND	-	-
2	top middle bottom	10.0 10.0 10.0	9.273 9.830 9.721	92.7 98.3 97.2	96.1
3	top middle bottom	30.0 30.0 30.0	31.61 29.51 29.25	105.4 98.4 97.5	100.4
4	top middle bottom	90.0 90.0 90.0	89.91 90.59 90.59	99.9 100.7 100.7	100.4

ND = not detected

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature and eight days at in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

Table 2 - Summary of Performance of Mated Females

Group Dose (mg/kg bw/day)	1 (0)	2 (50)	3 (150)	4 (450)
Female numbers	1 - 22	23 - 44	45 - 66	67 - 88
Number of mated females	22	22	22	22
Non-pregnant females (A)	1	0	1	1
Number of pregnant females	21	22	21	21
Number of females with live fetuses at termination*	21	22	21	21

*    Only dams with at least one live fetus at caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

(A) Female no. 10 from group 1 (control), female no. 57 from group 3 and female no. 79 from group 4 were not pregnant.

Conclusions:

In the pre-natal developmental toxicity study conducted according to OECD Test Guideline 414 and in compliance with GLP, administration of trimethylsilanol (CAS 1066-40-6) by oral gavage to Sprague-Dawley rats from Day 6 to 20 of pregnancy at 0, 50, 150 or 450 mg/kg/day resulted in clinical signs and reduced food consumption and body weight gain in dams at 150 and 450 mg/kg bw/day. The effects on food consumption and body weight were considered to be adverse at 450 mg/kg/day, therefore the maternal NOAEL for general toxicity was considered to be 150 mg/kg/day. There was no maternal developmental effects observed but reduced foetal weight, delayed ossification and increased incidence of some cartilaginous variations were noted in foetuses at 450 mg/kg/day. The skeletal findings are reported to be variations only and are not considered to be adverse effects as such. The reduced foetal weight is considered secondary to maternal toxicity. Therefore the developmental NOEL is considered to be 150 mg/kg bw/day based on findings observed in the pups.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04.09.2007-14.11.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: OECD 422

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc, Portage, MI
- Age at study initiation: Minimum of nine weeks
- Weight at study initiation: Males: 291.8 to 354.8 g; Females: 200.5 to 254.9 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages elevated over bedding except during mating when animals were cohoused in the home cage of the male.
- Diet (e.g. ad libitum): Ad libitum, except during exposure period
- Water (e.g. ad libitum): Ad libitum, except during exposure period
- Acclimation period: Seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.04-22.86
- Humidity (%): 33-66
- Air changes (per hr): 11.8-14.3
- Photoperiod (hrs dark / hrs light):


IN-LIFE DATES: From: 27.09.2007 To: 05.03.2008

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2000 litre stainless steel and glass Rochester-style inhalation chambers.
- Method of holding animals in test chamber: four levels of 20 individual animal compartments. The animal cage position assignment within each chanber was rotated daily.
- Source and rate of air: Building air (rate: no data)
- Method of conditioning air: Air from a Nash Air Compressor was passed through a series of filters to remove contaminants. This conditioned air was then passed through HEPA and activated charcoal filters before delivery to the chmabers.
- Temperature, humidity, pressure in air chamber: 22 ±3oC, 50 ±20% humidity, no data on pressure
- Air flow rate: No data
- Air change rate: 10-15 chamber volume air changes per hour
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Automated sampling system designed so that test atmosphere continuously pulled from the chamber and delivered to analyser. Analysis was done using a gas chromatograph equipped with a flame ionisation detector to determine the actual chamber concentrations of test substance.
- Samples taken from breathing zone: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Automated sampling system designed so that test atmosphere continuously pulled from the chamber and delivered to analyser. Analysis was done using a gas chromatograph equipped with a flame ionisation detector to determine the actual chamber concentrations of test substance.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to seven days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged (how): Individually. Pregnant females were moved into shoebox cages.
- Any other deviations from standard protocol: None evident
- Verification of same strain and source of both sexes: yes

Duration of treatment / exposure:

Males (14 days premating and mating up to 28 days): Females (premating 14 days, mating, gestation, and postpartum days 1-3 up to 42 days

Frequency of treatment:

6 hours/day, 7 days/week

Duration of test:

42 days

No. of animals per sex per dose:

10 males and 20 females (including 10 females for toxicity group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results in a previous study
- Rationale for animal assignment (if not random): Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality, and daily for clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and then weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: First day of dosing, and at least weekly thereafter, and on day of sacrifice.

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice as a terminal procedure.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: All males and toxicity phase females. Details reported in Section 7.5.3

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice as a terminal procedure.
- Animals fasted: Yes, overnight
- How many animals: All males and toxicity phase females. Details reported in Section 7.5.3

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to start of dosing, and during fourth week of exposure prior to initiation of the daily exposure.
- Dose groups that were examined: on all adult males and all toxicity phase females. Details reported in Section 7.5.3

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

Only a gross necropsy was performed.

Statistics:

All data analysis was carried out using SAS version  8.2 or 9.1.3. Descriptive toxicology endpoints:  Parametric data was tested using Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant); nonparametric data was tested by Kruskal-Wallis Test followed by Wilcoxon.  Repeated measures ANOVA was applied to data sets of multiple measurements  across time.  Histomorphology:  Cochran-Armitage Trend test for severity with Fisher's Exact Test for  pairwise comparisons.  Kruskal-Wallis was used to test for shifts in  grade with the Wilcoxon test used to test pairwise comparisons.   Functional observational battery:  ANCOVA (baseline as covariate), repeated measures ANOVA, Mixed modeling, and Jonckeere-Terpstra test (categorical endpoints).

Indices:

Mating and fertilty indices only - see Section 7.8.1
Pup viability

Historical control data:

None presented

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs attributed to test article included uncoordinated gait (not beyond day 10 or 11) and decreased activity noted immediately after exposure in both sexes from the highest dose group. These clinical signs persisted for some time following the exposure period with animals returning to normal prior to the next day's exposure.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences through gestation day 20 in absolute body weights or body weight gains compared to control across the reproductive group females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reproductive group females demonstrated a statistically significant decrease in average daily food consumption during pre-mating week 2 for group 3 and pre-mating week 1 and gestational week for group 4 females

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

not examined

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

A Group 2-female delivered a normal size litter which included one dead, edematous foetus. This was considered an isolated, spontaneous finding with no additional occurences observed within this or any other exposure group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

At all concentrations, there were no treatment-related effects on the mean duration of gestation, number of implantations and mean number of corpora lutea per dam.

Changes in number of pregnant:

not examined

Description (incidence and severity):

One animal in group 2 which demonstrated positive evidence of mating was found non-gravid and did not deliver

Other effects:

not specified

Dose descriptor:

NOAEC

Remarks:

Maternal general toxicity

Effect level:

>= 100 ppm

Based on:

test mat.

Basis for effect level:

clinical signs

food consumption and compound intake

Key result

Dose descriptor:

NOEC

Remarks:

Maternal developmental toxicity

Effect level:

>= 400 ppm

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOAEC

Effect level:

>= 400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1 Summary of developmental toxicity parameter data*
 

Parameter	Control	25ppm	100ppm	400ppm
Day 4 viable pups	15	14	15	15
Final viable/total	0.97	0.93	0.98	0.99
Final litter weight (g)	141.7	144.6	152.0	145.3
Final average pup weight (g)	10.1	10.7	10.2	9.9
Final sex ratio	1.2	0.9	0.9	1.0
Grossly visible, external, soft tissue and skeletal abnormalities	0	0	0	0

*No statistically significant differences.

Conclusions:

In a repeated dose inhalation toxicity with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP with the registered substance 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5), there were no treatment related-effects observed in any of the developmental parameters in maternal and foetal animals up to the highest concentration. Therefore the NOAEC for developmental toxicity was considered to be greater than or equal to 400 ppm (2.66 mg/L) in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 640 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

1,1,1,3,3,3-Hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) hydrolyses very rapidly in contact with water (<0.04 minutes, <0.5 minutes and <0.1 minutes at pH 4, 7 and 9 and 1^oC) generating trimethylsilanol and ammonia. As the hydrolysis reaction may be acid or based catalysed and can be assumed to be first order, the rate of reaction is expected to be slowest at pH 7 and increase as the pH is raised or lowered. Reaction rate also increases with temperature, so hydrolysis at physiologically relevant temperatures and pH 2 (relevant for conditions in the stomach following oral administration) or pH 7 (relevant for conditions in the lung following inhalation administration) will be even faster. Therefore read-across from the hydrolysis product, trimethylsilanol, to 1,1,1,3,3,3-hexamethyldisilazane for the key oral developmental toxicity study and supporting inhaled study is considered valid. No effects on developmental parameters have been noted for the non-silanol hydrolysis product, ammonia (OECD SIDS, 2007).

In the key developmental toxicity study conducted according to the OECD Test Guideline 414 and in compliance with GLP , oral administration of the hydrolysis product, trimethylsilanol (CAS 1066-40-6) to rats resulted in clinical signs (including uncoordinated movement and/or decreased activity) and reduced food consumption and body weight gain at the highest dose tested, 450 mg/kg bw/day, in the maternal animals.

The reductions in food consumption and body weight gain were considered to be adverse so the maternal No-Observed-Adverse-Effect-Level for general toxicity was considered to be 150 mg/kg bw/day. There were no developmental effects in maternal animals. Reduced foetal weight, delayed ossification and increased incidence of some cartilaginous variations were noted in foetuses at 450 mg/kg bw/day and considered to be adverse, therefore the foetal NOAEL for developmental effects was considered to be 150 mg/kg bw/day (Harlan, 2014).

In a supporting repeated dose inhalation toxicity with the reproduction/developmental toxicity screening test with the same test substance trimethylsilanol (CAS 1066-40-6), conducted according to OECD Test Guideline 422 and in compliance with GLP, three groups of Sprague-Dawley rats were exposed, via whole-body inhalation to vapour atmospheres of the test article for 6 hours/day, 7 days/week for 4 weeks, at 0, 60, 300 and 600 ppm. Test substance-related effects were limited to changes in haematology (lower eosinophil and lymphocyte counts for males) and serum chemistry (higher alanine aminotransferase for males and toxicity phase females) at 600 ppm (2214 mg/m3). These changes occurred in the absence of correlating histologic changes and were not considered adverse. There were no treatment related effects observed in any of the reproductive parameters evaluated in male and female animals. There were no developmental effects in maternal and in the offspring. Therefore, the maternal and foetal NOAECs for developmental toxicity were concluded to be both greater than or equal to 600 ppm under the conditions of this screening study (WIL Research Laboratories, 2008).

In another supporting combined repeated dose inhalation toxicity study with the reproduction/developmental toxicity screening conducted according to OECD Test Guideline 422 and in compliance with GLP, Sprague-Dawley rats were exposed, whole body, to vapour atmospheres of the registered substance 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) for 6 hours/day, 7 days/week for 4 weeks at 0, 25, 100 or 400 ppm. Clinical signs, limited to uncoordinated gait and decreased activity immediately after exposure, reduced body weight gain and centrilobular hypertrophy were noted at 400 ppm (2640 mg/m³). There were no effects on developmental parameters noted (Dow Corning Corporation, 2008).

Justification for classification or non-classification

Based on the available data 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) does not require classification for reproductive or developmental toxicity according to Regulation (EC) 1272/2008.