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Toxicological information

Repeated dose toxicity: inhalation

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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:
other: 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
(relative humidity of chamber atmosphere)
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: 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 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 25, 75, 400 ppm (0, 165, 495, 2640 mg/m3)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 25, 75, 400 ppm (0, 165, 495, 2640 mg/m3)
Basis:
analytical conc.
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
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS: Ataxia and decreased activity wereas noted in all animals in the high dose from treatment start onwards. Ataxia was recored until weeks 8 and 11 in males and females, respectively, and decreased activity was recorded until week 3 for both sexes.

BODY WEIGHT AND WEIGHT GAIN: Lower weight gain for high dose animals during treatment period.

FOOD CONSUMPTION: Lower for high dose males during treatment period and for high dose females during first two weeks of treatment period.

OPHTHALMOSCOPIC EXAMINATION: no effects noted

HAEMATOLOGY: Total red blood cells and hematocrit were higher at the end of the treatment period in females at 400 ppm.

CLINICAL CHEMISTRY: 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

URINALYSIS: The pH value was lower at the end of the treatment period in males at 400 ppm.

ORGAN WEIGHTS: Absolute and relative liver weights were higher in females at 400 ppm.

GROSS PATHOLOGY: no effects noted

HISTOPATHOLOGY: NON-NEOPLASTIC: 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.

Effect levels

Dose descriptor:
NOAEC
Effect level:
2 640 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

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:
Nose-only inhalation exposure of 1,1,1,3,3,3-Hexamethyldisilazane at concentrations of 25, 75 or 400 ppm to Sprague-Dawley rats for 6 hours a day, 5 days a week for 13 weeks resulted in minor changes at the highest concentration and renal effects in males at all concentrations. None of the effects were considered to be adverse and therefore the No-Observed-Adverse-Effect-Concentration was considered to be 400 ppm, the highest concentration tesed.
Executive summary:

Sprague-Dawley rats were exposed by nose-only flow past inhalation for 6 hours a day, 5 days a week for 13 weeks, followed by a 4 -week recovery period, to 1,1,1,3,3,3 -Hexamethyldisilazane at 0, 25, 75 or 400 ppm with minor effects recorded at 400 ppm and renal changes in males recorded 25, 75 and 400 ppm.

The decreased activity and ataxia was recorded after dose administration but not the following day and was therefore considered not to be adverse. The body weight and food consumption effects were slight and also considered not to be adverse. The changes in some haematology, clinical chemistry and/or urinalysis parameters were slight and/or only noted in one sex and/or without pathological correlate and had completely reversed by the end of the recovery period so were considered not to be adverse.

The increase in liver weight was in the absence of any histopathological changes in the liver and was reversible during the recovery period and so was considered to be adaptive and therefore not adverse.

The renal changes, including increased incidence and severity of intra-epithelial hyaline droplets or multifocal basophilic tubules in males at all doses was considered to be consistent with alpha-2u-nephropathy and therefore of no toxicological relevance to humans.

The No-Observed-Adverse-Effect-Concentration (NOAEC) was therefore considered to be 400 ppm (the highest concentration tested).