Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from August 28 to December 12, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Designation Grilbond IL-6 100%
Chemical name N,N’-(methylenedi-p-phenylene)bis[hexahydro-oxo-1H-azepine-1-carboxamide]
CAS no. 54112-23-1
EC no. 258-981-8
Molecular formula C27H32N4O4
Batch no. 9114761/024
Receipt no. 51302
Date of receipt August 03, 2012
Characteristics White powder
Water solubility Insoluble (25°C)
Storage conditions At room temperature
Manufacturer EMS-GRITECH CH-7013 Domat/Ems
Production date July 12, 2012
Stability (expiry date) July 11, 2013
Purity 99.0%
Particle size Particle size parameter provided by EMS-GRILTECH:
D10 = 4 µm
D50 = 20 µm
D95 = 45 µm

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Animals
Species / Strain / Stock: Rat (Rattus norvegicus) / CD / Crl:CD(SD)
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7 97633 Sulzfeld Germany
Age: (at start of exposure): Males: approx. 8 weeks Females: approx. 9 weeks
Body weight shortly before start of the exposure: Males: 262 - 267 g; Females: 222 - 231 g
Selection of species: The rat is a commonly used rodent species for such studies.
Identification of animals: By coloured tail marks and cage label
Number of animals: 10 (5 males and 5 females)
Groups: 1 concentration - Limit test
Concentrations: nominal: 6.67 mg/L air (mean achieved: 5.07 mg/L air, SD = 0.03 mg/L air)
Duration of experiment: at least 5 adaptation days 1 test day, 2 recovery weeks

Housing
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL (see Appendix 2: Limitation for contaminants in the bedding material).
During the 14-day observation period the animals were kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus) at a room temperature of 21.8 °C - 25.0 °C and a relative humidity of 48% ±68%. The ranges are given with 22 °C ±3 °C (maximum range) and a relative humidity of 55% ±15% (maximum range). Deviations from the maximum range caused for example during cleaning procedures are dealt with in SOPs.
The rooms were lit (150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.
Drinking water in bottles was offered ad libitum. Feeding was discontinued approx. 16 hours before administration; only tap water was then available ad libitum.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Route of administration: By inhalation (nose-only exposure)
Duration of exposure to the test item: 4 hours/animal
Exposure concentration: 5.07 mg/L air (mean actual concentration)
Exposure system
The study was carried out using a dynamic inhalation chamber (air changes/h (≥ 12 times)) with a nose-only exposure of the animals according to KIMMERLE & TEPPER. The apparatus consists of a cylindrical exposure chamber (volume 40 L) which holds the animals in pyrex tubes at the edge of the chamber in a radial position.
The dust of the test material was generated with a rotating brush dust generator.
The generator was fed with compressed air (5.0 bar) from a compressor (air was taken from the surrounding atmosphere of the laboratory room and filtered using an in-line disposable gas-filter).
At the bottom of the exposure chamber, the air was sucked off at a lower rate than created by the dust generator in order to produce a homogenous distribution and a positive pressure in the exposure chamber (inflow 900 L/h, outflow 800 L/h).
A manometer and an air-flow meter were used to control the constant supply of compressed air and the exhaust, respectively. Flow rates were checked hourly and corrected if necessary.
The oxygen content in the inhalation chamber was 21% v/v. It was determined at the beginning and at the end of the exposure with a DRÄGER Oxygen-analysis test set (DRÄGER Tube Oxygen 67 28 081).
The whole exposure system was mounted in an inhalation facility to protect the laboratory staff from possible hazards.
The exhaust air was sucked through gas wash-bottles. Exposure started by locating the rats into the exposure chamber after equilibration of the chamber concentration for 15 minutes.
Analysis of the dust concentration
The actual dust concentration in the inhalation chamber was measured 4 times gravimetrically with an air sample filter (Minisart SM 17598; 0.45 µm) and pump controlled by a rotameter. Dust samples were taken once every hour during the exposure. For that purpose, a probe was placed close to the animals' noses in the inhalation chamber and air was sucked through the air sample filter at a constant flow of air of 5 L/min for 1 minute. The filters were weighed before and after sampling on an analytical balance (accuracy 0.1 mg).
Temperature and humidity: The temperature (22 °C ±3 °C) and humidity was checked and noted once every hour during the exposure period of the experiment.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Concentrations:
a single dose of 5.07 mg/L air
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
After completion of exposure, the animals were observed for a period of 14 days. During and following exposure, observations were made and recorded systematically; individual records were maintained for each animal. A careful clinical examination was made at least once daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily to minimize loss of animals to the study.
Cageside observations included but were not limited to: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, as well as somatomotor activity and behaviour pattern.
Particular attention was directed to observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Individual body weights of animals were determined before the exposure on test day 1 and on test days 8 and 15. Changes in weight were calculated and recorded when survival exceeds one day. At the end of the test, all animals were weighed and sacrificed.
Pathology
Necropsy of all animals was carried out and all gross pathological changes were recorded.
No microscopic examination was carried out as no pathological findings were noted at necropsy.
Due to lack of prematurely deceased animals no LC50 could be calculated.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: None of the animals died prematurely after exposure of test article. All animals gained the expected body weight.
Mortality:
None of the animals died prematurely.
Clinical signs:
other: Slight ataxia, slight tremor, slightly reduced muscle tone and slight dyspnoea immediately until 30 minutes or 3 hours after end of exposure in all 5 of 5 male and 5 of 5 female animals. No clinical observations were observed during the further 14-day obs
Body weight:
All animals gained the expected body weight. No treatment-related body weight effects were seen neither for males nor females over the 14-day period as all animals gained the expected body weight.
Gross pathology:
No pathological findings were noted at necropsy.
Other findings:
no data

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available data in this study, LC50(rat, 4 hours) of test article was greater than 5 mg/L air.
Executive summary:

This acute study was performed to assess the acute inhalation toxicity of test item administered to rats for a single 4-hour period at a mean actual concentration of 5.07 mg/L air using a dynamic nose-only exposure chamber. The particle size distribution was determined with a cascade impactor. Under the present test conditions, a 4-hour exposure to Grilbond IL-6 100% at the concentration of 5.07 mg/L air revealed slight ataxia, slight tremor, slightly reduced muscle tone and slight dyspnoea immediately until 30 minutes or 3 hours after end of exposure in all 5 of 5 male and 5 of 5 female animals. None of the animals died prematurely. All animals gained the expected body weight. No pathological findings were noted at necropsy.

Therefore, LC50 of test item for rats is greater than 5.07 mg/L air for 4 hour.