Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-27 to 2018-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
2009-09-07
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2017-05-08
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid, white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, container kept tightly closed and stored in a dry and well-ventilated place

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at start of administration: males: approx. 8 weeks; females: approx. 9 weeks
- Weight at start of administration: males: approx. 300 - 330 g; females: 250 - 258 g
- Fasting period before study: feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: kept singly in MAKROLON cages (type III plus); bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): Commercial diet, ssniff® R/M-H V1534 ((ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55 % ± 10 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
1.68 µm
Geometric standard deviation (GSD):
1.99
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: study was carried out using a dynamic inhalation apparatus (RHEMA-LABORTECHNIK, 65719 Hofheim/Taunus, Germany) (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 (inner diameter: 23.9 cm; height: 63 cm; volume 28.5 L) which holds the animals in pyrex tubes at the edge of the chamber in a radial position.

- System of generating particulates/aerosols: dust of the test material was generated with a rotating brush dust generator (RBG 1000, PALAS GmbH Partikel und Lasermesstechnik, 76229 Karlsruhe, Germany).
The generator was fed with compressed air (5.0 bar) from a compressor (ALUP Kompressorenfabrik, 73257 Köngen, Germany).
At the bottom of the exposure chamber, the air was sucked off at a lower flow rate than it was 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 (ROTA Yokogawa GmbH & Co. KG, 79664 Wehr/Baden, Germany) were used to control the constant supply of compressed air and the exhaust, respectively. Flow rates were checked hourly and corrected if necessary.
The exhaust air was drawn through gas wash-bottles.

- Method of particle size determination: analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor (Cascade impactor 6.0 L/min (Article No. 700800-CI-060), TSE Systems GmbH, 61352 Bad Homburg, Germany).
The dust from the exposure chamber was drawn through the cascade impactor for 1 minute at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance (SARTORIUS, type 1601 004, precision 0.1 mg). Deltas of slides’ weight were determined.
The mass median aerodynamic diameter (MMAD) was estimated by means of non-linear regression analysis. The 10.6 µm particle size range and the filter (particle size range < 0.55 µm) were not included in the determination of the MMAD in order not to give undue weight to these values.
The Geometric Standard Deviation (GSD) of the MMAD was calculated from the quotient of the 84.1%- and the 50%-mass fractions, both obtained from the above mentioned non-linear regression analysis.
In addition, a sample of approx. 10 g test material was taken from the exposure chamber to determine the median physical particle size with a Malvern Mastersizer 2000 by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, pressure in air chamber, oxygen content and carbon dioxide content: oxygen content in the inhalation chamber was 21%. 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). Carbon dioxide concentration did not exceed 1%.
Temperature (20.6 °C ± 0.2 °C) and humidity (63.9 % ± 0.2 %) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

Exposition started by locating the animals into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes.

Before initiating the study with the animals, a pre-test was carried out with the exposure system in order to verify that under the experimental settings chosen, the limit concentration of 5 mg/L air could be achieved by gravimetric analysis.

TEST ATMOSPHERE
- Brief description of analytical method used: actual dust concentration in the inhalation chamber was measured gravimetrically with an air sample filter (Minisart SM 17598 0.45 µm) and pump (Vacuubrand, MZ 2C (Membrane Pump, Vacuubrand GmbH + Co. KG, 97877 Wertheim/Main, Germany)) 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 and air was drawn 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 (accuracy 0.1 mg).
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
1.680 µm (GSD: 1.99)
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
- actual concentration: 5.06 ± 0.02 mg/L air
- nominal concentration: 22.22 mg/L air
No. of animals per sex per dose:
3 males / 3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: during and following exposure, observations were made. Clinical examinations were made at least once daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily with appropriate actions taken to minimize loss of animals to the study (e.g. necropsy).
Individual weights of animals were determined once during the acclimatisation period, before the exposure on test day 1, on test days 2, 4, 8 and 15. Changes in weight were calculated and recorded when survival exceeded one day. At the end of the test, all animals were weighed and sacrificed.
- Necropsy of survivors performed: yes
Necropsy of all animals was carried out and all gross pathological changes were recorded for each animal with particular attention to any changes in the respiratory tract.
Organ weights of the lungs were determined for all animals.

Statistics:
not applicable

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.06 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No animal died prematurely.
Clinical signs:
other: A 4-hour inhalation exposure to the test item at a concentration of 5.06 mg/L air revealed slight dyspnoea until 3 hours post exposure in all 3/3 male and 3/3 female animals, respectively. However, this effect is considered to be an overall clinical sign
Body weight:
All females appeared to be reduced in body weight gain at the end of the study period. The males appeared to be increased in body weight gain at the end of the study period
Gross pathology:
Necropsy revealed oedematous and marbled lungs in all animals.
Other findings:
Organ weights:
- males (mean): 2.10 ± 0.12 g
- females (mean): 1.93 ± 0.08 g

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
LC50 (rats; 4 hours) > 5.06 mg/L air (actual concentration)
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the inhalation route.