Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

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.