Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2018 to 23 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 433 “Acute Inhalation Toxicity: Fixed Concentration Procedure.”
Version / remarks:
Adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed concentration procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Off white powder
- Storage conditions: At ambient temperature (10 to 30 °C) protected from light
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test item prepared as follows:
Device used: Mechanical grinder
Number of bursts: Four
Burst time: 8 - 15 seconds
Method of sieving: Hand
Sieve used: 212 μm mesh

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 76 to 82 days
- Weight at study initiation: 320 to 341 g
- Housing: The animals were housed five per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood shavings (Lignocel 3/4) were used as bedding and were sterilised by autoclaving and changed at appropriate intervals each week. Cages, food hoppers and water bottles were changed at appropriate intervals.
- The cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary. The cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cage.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were allowed to acclimatize to the conditions described below for 12 days before treatment commenced.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 40 - 70 %
- Air changes: Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.4 µm
Geometric standard deviation (GSD):
1.99
Remark on MMAD/GSD:
The mean MMAD value was within the ideal range of 1 to 4 microns, indicating the generated test material aerosol was respirable to the rat.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure system consisted of a flow through nose only chamber and an aluminium alloy construction comprising a base unit, one animal exposure section with 20 exposure ports, and a top section. The dimensions of the chamber give an internal volume of 30 L. A curved aerosol conditioning pre-chamber was used on the exposure system, with an internal volume of 2.2 L. An elutriator, with an internal volume of 1.3 L, was connected to the aerosol generator. The elutriator connected to the pre-chamber by aerosol tubing.
- Exposure chamber volume: 33.6 L
- Method of holding animals in test chamber: Plastic nose-only restraint tube.
- Aerosol Generation: Solid Aerosol Generator: The SAG mechanism technique for the dispersion of dry dust and powders comprises two steps, the continuous supply of material to the disperser and the dispersal of the material as an aerosol. The method for metering powder to the disperser was to use a moving toothed belt. A regulated flow of compressed air conducted the aerosol to the inhalation chamber.
- Inlet Airflow: From in-house compressed air system (breathing quality). Flow: 28 L/minute
- Extract Airflow: Drawn by in-house vacuum system, filtered locally. Flow: 29 L/minute
- Temperature and humidity in air chamber: The mean chamber air temperature was 21.4 ± 0.35 °C and mean relative humidity was 52.2 ± 4.74 %.

TEST ATMOSPHERE
- Aerosol samples were collected as follows:
Sample type: Glass microfiber filter, held in an open face filter holder
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas metre
Sample frequency: 12 samples collected during exposure
Sample location: Animal exposure port
Sample analysis: Gravimetric
Aerosol samples were collected at irregular intervals due to additional samples being collected after adjustments made to operating conditions. A time-weighted average mean aerosol concentration was calculated.
- Particle Size Distribution was determined by cascade impaction – samples collected as follows:
Impactor type: Marple 290 Series Personal Cascade Impactor (Andersen Instruments, Smyrna, Georgia, USA)
Configuration: 298
Collection media: Stainless steel substrates and glass microfiber final stage filter
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: Two samples collected during exposure
Sample location: Animal exposure port
Sample analysis: Gravimetric, MMAD and GSD derived

DATA EVALUATION
- Determination of Time-Weighted Averages for Calculation of Aerosol Concentration of the test material: Samples of air were removed from the test chamber during exposure in order to determine the concentration of the test material in air. In the first instance, samples were obtained following equilibration and then at approximately 30-minute intervals. Additional samples were obtained as necessary to monitor the chamber concentration following adjustments to the exposure system. A time-weighted average was calculated from the individual data in order to prevent undue biasing of repeat samples in the overall mean.
Time-weighted average: sum of ‘weighted concentrations’ (sample concentrations weighted for time) calculated for each sampling occasion as follows:

‘Weighted concentration’ (mg/L) = (Concentration (mg/L) × ‘Time-weighting’ (min)) / Exposure duration (min)

Where the ‘time-weighting’ is the respective interval between adjustments to the exposure system or the midpoints between consecutive sampling occasions if no adjustment was made.

- Calculation of MMAD and GSD: MMAD and GSD - by linear regression of the probit of the cumulative percentage, by mass, of particles smaller than the ECD of each stage versus the logarithm of the ECD of each stage.

- Determination of Nominal Aerosol Calculation:
Nominal aerosol concentration (mg/L) = (Usage (g) × 1 000) / (Generation period (mins) × Airflow (L/minute))

- Determination of Chamber Air Changes:
Number of air changes per hour = (60 (minutes) × Airflow (L/minute)) / Chamber volume (L)

- Determination of T95 Equilibration Time:
Natural Logarithm (100 ÷ (100-95)) × ( Chamber volume (L) / Airflow (L/minute))
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Target: 5.0 mg/L
TWA Mean: 5.12 mg/L
No. of animals per sex per dose:
5 males
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Throughout the study, all cages were checked at least twice daily, once in the morning and again towards the end of the normal working day, for dead or moribund animals.
Clinical signs were recorded pre-exposure, at hourly intervals during exposure, immediately following exposure, 1 hour and 2 hours post-exposure and as late as possible in the working day.
During the observation period, the animals were observed once in the morning and once toward the end of the experimental day. On the day of study termination there was one observation (morning only).
Rats were weighed at least once during the week prior to exposure. The weight of each animal was recorded on Days 1 (prior to dosing), 2, 4, 8 and 15.
- Necropsy of survivors performed: Yes. At the end of the scheduled observation period, animals were killed by an overdose of pentobarbitone sodium followed by exsanguination.
All animals were subjected to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded. Tissues were discarded following necropsy.
Statistics:
In order to minimise the cumulative errors, which result from repeated rounding of numbers, some of the data in this report have been calculated continuously using unrounded numbers and only rounded for reporting. Consequently, any further calculation using these rounded numbers may include rounding errors in the last significant figure, possibly leading to small apparent discrepancies with other data in the report.

Days of pretreatment relate to study days, as follows:
Phase day P5
Day of study -8

Results and discussion

Effect levels
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
> 5.12 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no unscheduled deaths.
Clinical signs:
other: There were no clinical signs related to treatment.
Body weight:
Slight body weight losses were evident for all animals on Day 2. Body weight gains were observed on Day 4 and continued to increase for the remainder of the observation period for all animals.
Gross pathology:
The macroscopic examination performed after a single exposure and a 14 day observation period revealed no test material related lesions. No abnormalities were seen in any of the animals.
Other findings:
Chamber Atmosphere Conditions
- The mean achieved concentration value was 102 % of the target concentration of 5 mg/L. The MMAD was within the ideal range of 1-4 microns for an acute inhalation study, indicating that the aerosol was respirable to the rat.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the LC50 (4 hour) of the test material is in excess of 5.12 mg/L for male rats.
Executive summary:

The acute inhalation toxicity of the test material was investigated in accordance with the standardised guideline OECD 433, under GLP conditions.

One test group, consisting of five male RccHan™;WIST rats were exposed to the test material for a single 4-hour snout only exposure and observed for a period of fourteen days post exposure.

The mean achieved concentration value of the test material was 102 % of the target concentration of 5 mg/L. The MMAD was within the ideal range of 1-4 microns for an acute inhalation study, indicating that the aerosol was respirable to the rat.

There were no unscheduled deaths and no clinical signs related to treatment. Slight body weight losses were evident for all animals on Day 2. Body weight gains were observed on Day 4 and continued to increase for the remainder of the observation period for all animals. No macroscopic abnormalities were observed.

Under the conditions of this study, the LC50 (4 hour) of the test material is in > 5.12 mg/L for male rats.