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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April to 27 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with an appropriate guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

(relative humidity of chamber atmosphere)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

yes

Remarks:

(relative humidity of chamber atmosphere)

Principles of method if other than guideline:

For technical reasons, the relative humidity value of the chamber atmosphere was below the range given by the guidelines. The reason is the dried, pressurized air used for vapor generation, in combination with heating the test item in the nebulizer to 68 - 76 °C. This deviation from the guidelines has no influence on the results of the study, based on the absence of any findings in the respiratory tract and the circumstance that dry air generally worsens any findings in the respiratory tract due to loss of the protective mucus layer on the lining epithelium.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

213-668-5

EC Name:

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

Cas Number:

999-97-3

Molecular formula:

C6H19NSi2

IUPAC Name:

bis(trimethylsilyl)amine

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): 1,1,1,3,3,3-Hexamethyldisilazane

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 268-297g (males); 163-200g (females)
- Fasting period before study: no
- Housing: Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding (Lignocel), including paper enrichment (Enviro-dri)
- Diet (ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba AG, Switzerland),
- Water (ad libitum): community tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 17 April to 27 August 2013

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only, flow-past exposure chamber
- Method of holding animals in test chamber: restraint tubes
- Source and rate of air:
- Method of conditioning air:
- System of generating vapour: 1,1,1,3,3,3-Hexamethyldisilazane was pumped into a glass flask. Compressed, filtered, and dried air was supplied into the glass flask through a metal nebulization tube. The glass flask was kept between a temperature of 68 and 76 °C with a thermal regulating device set to facilitate the process of vaporization.
- Temperature, humidity, pressure in air chamber: 19.9 to 22.6 degrees C: 0.0 to 4.0% ; slight positive pressure to outside
- Air flow rate: 0.75-1.0L/min
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: nominal - wieighing of test susbtance; chemical - on-line GC
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of 1,1,1,3,3,3-Hexamethyldisilazane was measured at least once per hour of exposure per on-line GC for groups 1 to 4.

Analytical concentrations were determined by GC. The method of analysis was performed according to the conditions listed below.
- Column: HP-5MS (30 m, 0.25 mm, 0.25 μm)
- Injector: 250 °C
- Oven: 40 °C for 1 min, then 40 °C/min to 100 °C for 0 min, then 50 °C/min to 230 °C for 1 min
- Detector: 280 °C

- Calibration:
A calibration curve consisting of at least 6 points and ranging between concentrations of approximately 10 ppm to approximately 500 ppm were constructed from the test item in gas bags as part of the technical trials. The calibration gas bags were prepared at each concentration and at least two independent gas bags were used.

- Acceptance Criteria:
The coefficients of variation were below 3.1% (acceptance criteria of >10%) for all calibration gas bag samples at each concentration. The correlation coefficient of the used regression was >0.9989 (acceptance criteria of >0.985). Accordingly, the acceptance criteria were met.

Daily standards constructed from the test item in gas bags were sampled at one of the chamber-lines and used to check the integrity of the sampling line and check the GC calibration. Plots of the peak height used for the calibration were used to assess trends regarding system stability. The acceptance criterion for standard samples was an accuracy of 90 - 110% of the theoretical value. The acceptance criteria for standard samples were met for each exposure session.

The test item was used as the analytical standard.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days/week for 13 weeks, followed by 4 week recovery period for sub-group of Control and high dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Group 1 - Control Group

Doses / concentrations 2

Dose / conc.:

25 ppm (nominal)

Remarks:

Group 2 (165 mg/m3)

Doses / concentrations 3

Dose / conc.:

75 ppm (nominal)

Remarks:

Group 3 (495 mg/m3)

Doses / concentrations 4

Dose / conc.:

400 ppm (nominal)

Remarks:

Group 4 (2640 mg/m3)

No. of animals per sex per dose:

20 Control and high dose, 10 low and intermediate dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose concentrations based on previous OECD 422 inhalation study
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during dosing period, once daily during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily during dosing period, once weekly during recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly during dosing period, once weekly during recovery period

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before dosing started and Week 13
- Dose groups that were examined: all (not recovery animals in Week 13)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy at end of dosing or recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 10 sex/group
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy at end of dosing or recovery period
- Animals fasted: Yes
- How many animals: 10 sex/group
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 18 hour period prior to necropsy at end of dosing or recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No. 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

The following statistical methods were used to analyze the food consumption, body weight, ophthalmoscopic examinations, macroscopic findings, organ weights and ratios, as well as clini¬cal laboratory data:

• The two-sided 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.

• The two-sided Fisher's exact-test was applied to the ophthalmo-scopic and macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ataxia and decreased activity were noted in all animals in the high dose from treatment start onwards. Ataxia was recorded until weeks 8 and 11 in males and females, respectively, and decreased activity was recorded until week 3 for both sexes. Based on adaptation the animals showed during the treatment and on the complete reversibility of the findings during recovery, these findings are considered to be non-adverse.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the course of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body weight gain for high dose animals in both sexes during treatment period. An increased body weight gain was recorded in males during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower for high dose males during treatment period and for high dose females during first two weeks of treatment period. An increased food intake was noted during recovery.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmoscopic findings were observed which were considered to be attributable to exposure with the test item

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Total red blood cells and hematocrit were slightly but statistically significantly higher at the end of the treatment period in females at 400 ppm. These findings had completely reversed at the end of the recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Sodium, potassium and chloride levels were higher at the end of the treatment period in females at 400 ppm. In addition, total protein was lower for those animals. These findings had completely reversed at the end of the recovery period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The pH value was lower at the end of the treatment period in males at 400 ppm. This finding had completely reversed at the end of the recovery period.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weights were higher in females at 400 ppm. In the absence of any histopathological findings in the livers and based on the reversibility
that was observed during the recovery period, this finding is considered to be adaptive.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In comparison to controls the incidence and severity of increased intra-epithelial hyaline droplets and focal or multifocal basophilic tubules was greater in all the male treated groups. In addition in 2/10 males given 400 ppm granular casts were present at the cortico-medullary junction of the kidneys. These findings were all considered to be consistent with a diagnosis of alpha-2µ-nephropathy and therefore of no toxicological significance to humans.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

>= 400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 ppm (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Any other information on results incl. tables

Table 1 - Exposure Conditions

Group	Temperature [°C]	Relative Humidity [%]	Oxygen Concentration [%]
1	22.4 ± 0.2 (n=66)	0.0 ± 0.0 (n=66)	20.2 ± 0.0 (n=66)
2	22.2 ± 0.1 (n=66)	3.0 ± 0.1 (n=66)	20.2 ± 0.0 (n=66)
3	22.1 ± 0.2 (n=66)	3.0 ± 0.5 (n=66)	20.0 ± 0.0 (n=66)
4	22.2 ± 0.1 (n=66)	3.9 ± 0.1 (n=66)	20.2 ± 0.1 (n=66)

For technical reasons, the relative humidity value of the chamber atmosphere was below the range given by the guidelines. The reason is the dried, pressurized air used for vapor generation, in combination with heating the test item in the nebulizer to 68 – 76 °C. This deviation from the guidelines has no influence on the results of the study, based on the absence of any findings in the respiratory tract and the circumstance that dry air generally worsens any findings in the respiratory tract due to loss of the protective mucus layer on the lining epithelium.

Table 2 - Test Atmosphere Concentrations

Group	Chemically Determined AtmosphereConcentration [ppm]	TargetAtmosphereConcentration [ppm]	Atmosphere ConcentrationsRelative to Target
2	25.0 ± 0.5 (n=66, CV=1.8%)	25	100.0%
3	75.0 ± 1.6 (n=66, CV=2.2%)	75	100.0%
4	400 ± 4 (n=66, CV=1.1%)	400	100.0%

 

Applicant's summary and conclusion

Conclusions:

In a repeated dose toxicity study conducted according to OECD Test Guidelines 413 and in compliance with GLP, nose-only inhalation exposure of 1,1,1,3,3,3-hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) at concentrations of 25, 75 or 400 ppm to Sprague-Dawley rats for 6 hours per day, 5 days per week for 13 weeks resulted in minor and reversible changes at the highest concentration. Renal effects in male rats were observed at all concentrations, but these findings were consistent with alpha-2u-nephropathy and therefore of no toxicological significance to humans. Based on the results in this study, the NOAEC was considered to be higher than or equal to 400 ppm, the highest concentration tested.