Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a study on acute toxicity by the inhalation route is available
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 NOV 2021 to 09 DEC 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
September 2009
Deviations:
yes
Remarks:
Weight of re-used test item not noted. No impact on the overall study integrity or on the interpretation of the study results and conclusions.
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes
Specific details on test material used for the study:
- As this study required a lot of test item, both test item batches were used. Both batches are almost identical and therefore it was considered acceptable for this study.
- Before use, the test item was grinded with an automatic grinder (PM-100, Retsch, Ochten, The Netherlands) and passed through a 355 µm steel mesh sieve.
Species:
rat
Strain:
Wistar
Remarks:
Cri: WI (Han) (Wistar Han rat)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Age at the Initiation of Dosing: young adult animals, approximately 9 weeks old.
- Weight at the Initiation of Dosing: Males: 239 to 278 g - Females: 163 to 196 g.
- On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same exposure group together) in polycarbonate cages (Makrolon MW type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & S6hne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 22°C with an actual daily mean relative humidity of 42 to 59%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiâten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Municipal tap-water was freely available to each animal via water bottles, except during designated procedures. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.


Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Remarks:
The test item was administered once via the inhalation route on Day 1, by nose-only directed flow exposure for 4 hours.
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.5 µm
Geometric standard deviation (GSD):
2
Remark on MMAD/GSD:
The particle size distribution was characterized twice during the exposure period. The MMAD was 4.2 µm (GSD 1.8, sampling time 09:17) and 3.5 µm (GSD 2.0, sampling time 11:56). As the first measurement was higher than expected based on the results of the trail generations, a third measurement was performed after the exposure period for clarification. The MMAD was 2.6 µm (GSD 1.8, sampling time 14:64). Agglomeration of aerosol particles at this high concentration might have resulted in the MMAD value of the first measurement to fall outside the recommended range of 1 - 4 µm. The MMAD just exceeded this range and based on the clinical signs there was no evidence for test item deposition in the upper airways. In addition, the other MMAD values were below 4 µm and therefore it was considered not to have affected the study.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the design of the exposure chamber was based on the directed flow nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of animal sections with eight animal ports each. Each animal port had its own test atmosphere inlet and exhaust outlet.
- Method of holding animals in test chamber: the animals were placed in polycarbonate restraining tubes, which were connected to the exposure chamber. Before the animals were placed in the tubes, their eyes were treated with eye oitment (Ophtosan, AST Farma BV, Oudewater, The Netherlands) in order to protect them from possible adverse effects.
- Airflow: the number of animal sections and number of open inlets were adapted to the air flow in such a way that, at each animal port, the theoretical air flow was at least 1 L/min.
- System of generating dust: administering the test item to a stream of pressurized air using a combination of a spiral feeder (Hethon 30) and air mover (AIR-VAC, Milford, CT, USA) generated a dust. A magnetic vibrator was attached to the feeder to promote the feed. The dust was passed through a series of two cyclones, allowing larger particles to settle, before it entered the exposure chamber. The mean total airflow was 18 L/min.
- Treatment of exhaust air: from the exposure chamber, the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood in which the exposure chamber was placed.
- Temperature, humidity in air chamber: the temperature and relative humidity were measured with a humidity and temperature indicator (E+E Elektronik, Engerwitzdorf, Austria) and recorded after the animals were connected to the exposure chamber and at 30-minute intervals alter initiation of exposure. The probe was inserted in a tube mounted in one of the free animal ports of the exposure chamber. The temperature of the atmosphere during the exposure was between 20.5 and 21.0°C. The relative humidity was between 25 and 28%, which was considered appropriate for this relatively short 4 hours exposure duration.

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: a total of 23 representative samples were taken for determination of the actual concentration during exposure at 5 mg/L. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. Samples were drawn through a glass fiber filter (type APFC04700, Millipore, Billerica, MA, USA). Sample volumes were measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands). The collected amount of test item in the air sample was measured gravimetrically. Subsequently the time-weighted mean concentration with the standard deviation was calculated. The resulting time-weighted mean actual concentration was 5.2 ± 0.15 mg/L.The concentration was measured at time points (n=23) that were equally distributed over the exposure period, the results of which demonstrated that the item was sufficiently stable. The variation in concentration was caused by adjustments to the generation equipment. These fluctuations were considered not to have affected the study as the mean concentration was 5.2 mg/L and because the animals were not visually affected by the short-term exposure to the concentrations above the target concentration of 5 mg/L. The generation was interrupted on four occasions in order to remove test item deposits from the system. To compensate for these interruptions, the generation time was elongated by 20 minutes in order to achieve a 4-hour exposure. The actual exposure time was 232 minutes. This 8-minute decrease in time of exposure was judged not to have affected the study as this variance is considered acceptable for a 4-hour target time. By calculating the time-weighted mean concentration, effects of interruptions and variations were taken into account resulting in an actual reflection of the mean exposure concentration over time.

- Time needed for equilibrium of exposure concentration before animal exposure : Due to the small volume of the exposure chamber the equilibrium time was negligible.

TEST ATMOSPHERE
- Particle size distribution: the particle size distribution was characterized four times during each exposure period. The samples were drawn with a flow of 2 L/min from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (TE-290-GF. Tisch Environmental, Cleves, Ohio, USA) and a fiber glass back-up filter (SEC-290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test item collected were measured gravimetrically.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined based on OECD guidance document No 39. Graphs of the cumulative mass of test item collected (percentage of total collected) against the cut points of the impactor stages were drawn on log-normal paper. When drawing the graphs more weight was given to the cut points where the cumulative mass sampled was within the range of 5 to 95%. The Mass Median Aerodynamic Diameter (MMAD) was read from the graph and the geometric standard deviation (GSD) was calculated.
- Stability monitoring: it was considered that the opacity of the test atmosphere could not be reliably monitored by means of an aerosol monitoring system. An indication of stability of the test atmosphere was obtained from the concentration measurements equally distributed over time.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: the starting exposure level, 5 mg/L, was selected on the basis of the EC and UN classification guidelines and on the available test item data. It was expected not to cause mortality.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
The target concentration was 5 mg/L.
The time-weighted mean actual concentration was 5.2 ± 0.15 mg/L.
No. of animals per sex per dose:
One group of 3 males and 3 females was administered the dose level of 5 mg/L.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: all the animals were examined for reaction to exposure. The onset, intensity and duration of these signs was recorded (if appropriate). Animals were checked for clinical signs at least three times during exposure. Post exposure observations were performed at periodic intervals on the day of exposure (at least two times) and once daily thereafter. Particular attention was paid to the animals for the first hours after exposure. Animals were weighed individually on Day 1 (pre exposure), 2, 4, 8 and 15.
- Necropsy of survivors performed: yes.
- Clinical signs : animals were checked for mortality, behavioral signs of distress and effects on respiration.
- Other examinations performed: all animals were euthanized according to laboratories Standard Operating Procedures at the end of the observation period. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded. On completion of the necropsy, the animals' carcasses were disposed of and no tissues were retained.
Statistics:
No statistical analysis was performed (the method used was not intended to allow the calculation of a precise LC50 value).
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occurred.
Clinical signs:
irregular respiration
Remarks:
During exposure, increased and irregular breathing was seen in all animals. After exposure, increased and irregular breathing and hunched posture were observed up to 3h after Day 1 and erected fur were seen up to Day 3 in all animals.
Body weight:
Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.
Gross pathology:
No abnormalities were found at macroscopic postmortem examination of the animals.
Other findings:
None.
Interpretation of results:
GHS criteria not met
Conclusions:
The inhalation LC50, 4h value of the test substance in Wistar Han rats was established to exceed 5 mg/L. Therefore, according to this result, the test substance does not have to be classified according to GHS and CLP criteria.
Executive summary:

The study was carried out based on the OECD Guideline 436: Acute Inhalation Toxicity- Acute Toxic Class Method, September 2009.


The substance was administered as a dust by nose-only inhalation for 4 hours to one group of three male and three female Wistar Han rats at a target concentration of 5 mg/L. Mortality and clinical signs were observed daily during the 14-day post observation period and body weights were determined on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed after terminal sacrifice (Day 15).


The time-weighted mean actual concentration was 5.2 ± 0.15 mg/L.


The particle size distribution was characterized twice during the exposure period. The MMAD was 4.2 µm (GSD 1.8) and 3.5 µ.m (GSD 2.0).


No mortality occurred.


During exposure, increased and irregular breathing was seen in all animals. After exposure, increased and irregular breathing and hunched posture were observed up to 3 hours after exposure Day 1 and erected fur were seen up to Day 3 in all animals. Afterwards, no clinical signs were noted up to the end of the observation period.


Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity. No abnormalities were found at macroscopic postmortem examination of the animals.


The inhalation LC50-4h value of the test item in Wistar Han rats was established to exceed 5 mg/L.


According to these results, the test substance does not have to be classified according to GHS and CLP criteria.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion