Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
September 2009
Deviations:
yes
Remarks:
During characterization of the test atmosphere, the weight of the used test item was not correctly noted in the raw data, therefore no nominal concentration and generation efficiency could be determined.
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
white to brownish
Details on test material:
Batch 151222
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Before use the test item was grinded with an automatic grinder (ZM-100, Retsch, Ochten, The Netherlands).

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9-10 weeks old
- Weight at study initiation:
Males: 262 to 315 g.
Females: 178 to 223 g
- Fasting period before study: no
- Housing:
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 MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room(s) in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum):
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten 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.
- Water (e.g. ad libitum):
Municipal tap-water was freely available to each animal via water bottles.
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.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
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 20°C with an actual daily mean relative humidity of 50 to 65%. 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.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.2 - <= 4.5 µm
Geometric standard deviation (GSD):
>= 1.8 - <= 2.2
Remark on MMAD/GSD:
The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined at least twice during the exposure period. At 1 mg/L, the MMAD was 3.2 µm (gsd 1.9) and 3.3 µm (gsd 2.2). At 3.9 mg/L, the MMAD was 4.0 µm (gsd 1.9), 4.5 µm (gsd 1.8) and 4.1 µm (gsd 2.0). Agglomeration of aerosol particles at the high concentration might have resulted in the MMAD values to fall outside the recommended range of 1 - 4 µm. The MMAD only slightly exceeded this range and there was no evidence for item deposition in the upper airways. Since good distribution throughout the lung requires particles with an aerodynamic diameter between 1 and 5 μm, it can be assumed that sufficient deposition in the lower respiratory tract occurred during the exposure.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The design of the exposure chamber is based on the directed flow nose only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983).
- Exposure chamber volume: /
- Method of holding animals in test chamber: polycarbonate restraining tubes
- Source and rate of air: The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal. 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.
- Method of conditioning air: /
- System of generating particulates/aerosols: An aerosol was generated by administering the test item to a stream of pressurized air using a combination of a spiral feeder (Randcastle Extrusion Systems, Cedar Grove, NJ, USA) and air mover (AIR-VAC, Milford, CT, USA). Outside air was pressurized and passed through several filters before use. The aerosol was passed through a series of two cyclones (1 mg/L) or an elutriator and three cyclones (3.9 mg/L), allowing larger particles to settle, before it entered the exposure chamber.
From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
- Method of particle size determination: The particle size distribution was characterized at least twice 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. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined based on OECD guidance document No. 39.
- 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.
- Temperature, humidity, pressure 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 after 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 exposures was between 21.6 and 22.0oC. The relative humidity was between 27 and 36% which was considered appropriate for this relatively short 4 hours exposure duration.

TEST ATMOSPHERE
- Brief description of analytical method used: A total of 29 and 21 representative samples were taken for determination of the actual concentration during exposure at 1 mg/L and the technically maximum attainable concentration, respectively. 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.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): air
- Concentration of test material in vehicle (if applicable): 1 and 3.9 mg/L
- Justification of choice of vehicle: standard protocol
- Lot/batch no. (if required): /
- Purity: /

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: /
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): At 1 mg/L, the MMAD was 3.2 µm (gsd 1.9) and 3.3 µm (gsd 2.2). At 3.9 mg/L, the MMAD was 4.0 µm (gsd 1.9), 4.5 µm (gsd 1.8) and 4.1 µm (gsd 2.0).

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Standard starting concentration for aerosols (OECD 436 Appendix 3c) when no information is available on inhalation toxicity.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Remarks on duration:
To compensate for interruptions (maximum 3 minutes per treatment), the generation time was elongated by maximum 3 minutes in order to achieve an actual exposure time of 240 minutes.
Concentrations:
The study was performed following a stepwise exposure scenario (Annex 3c in OECD 436). Three animals of each sex were exposed to a test item target concentration of 1 mg/L. Based on the results, one additional group of three animals of each sex was exposed to the technically maximum attainable concentration of 3.9 mg/L.
No. of animals per sex per dose:
3 animals of both sexes per exposure group. Two exposure groups. Females were nulliparous and non-pregnant.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Animals were checked for mortality, behavioral signs of distress and effects on respiration 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.
Animals were weighed individually on Day 1 (pre-exposure), 2, 4 and 8 and 15.
- Necropsy of survivors performed: yes
All moribund animals and animals surviving to the end of the observation period were sacrificed by an intra-peritoneal injection of Euthasol® (at least 250 mg/kg). All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.
- Other examinations performed: mortality, behavioral signs of distress, effects on respiration and body weight
Statistics:
No statistical analysis will be performed (the method used is not intended to allow the calculation of a precise LC50 value).

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 3.9 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occurred.
Clinical signs:
other: At 1 mg/L, shallow breathing labored respiration and restless behavior were seen during exposure (not presented in the table). After exposure, lethargy, hunched posture, labored respiration, slow breathing and rales were seen for the animals. The animal
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 post mortem examination of the animals.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The inhalation LC50, 4h value of CH02906 in Wistar rats was established to exceed 3.9 mg/L.
Based on these results CH02906 does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

The objective of this study was to assess the acute inhalation toxicity of CH02906 in rats of both sexes in rats following a single 4 hour nose-only exposure to one or more defined concentrations. Animals were retained for a 14 day post-exposure observation period.

The study was carried out based on the guidelines described in:

·      Acute Inhalation Toxicity- Acute Toxic Class Method,September 2009.

CH02906 was administered as a dust by nose-only inhalation for 4 hours to two groups of three male and three female Wistar rats. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed after terminal sacrifice (Day 15).

 

At 1 mg/L, the time-weighted mean measured air concentration was 1.1 ± 0.02 mg/L.

At a target concentration of 5 mg/L, the time-weighted mean measured air concentration was 3.9 ± 0.09 mg/L. This concentration was found to be the technically maximum attainable concentration. The nominal concentration was 235 mg/L. The generation efficiency was 1.7%.

The air concentration was measured at several time points that were equally distributed over the exposure period, the results of which demonstrated that the item was sufficiently stable. Variation in measured air concentration was caused by adjustments to the generation equipment. By calculating the time-weighted mean measured air concentration, effects of these variations were taken into account resulting in a more accurate reflection of the mean exposure concentration over time.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined at least twice during the exposure period. At 1 mg/L, the MMAD was 3.2 µm (gsd 1.9) and 3.3 µm (gsd 2.2). At 3.9 mg/L, the MMAD was 4.0 µm (gsd 1.9), 4.5 µm (gsd 1.8) and 4.1 µm (gsd 2.0). Agglomeration of aerosol particles at the high concentration might have resulted in the MMAD values to fall outside the recommended range of 1 - 4 µm. The MMAD only slightly exceeded this range and there was no evidence for item deposition in the upper airways. Since good distribution throughout the lung requires particles with an aerodynamic diameter between 1 and 5μm, it can be assumed that sufficient deposition in the lower respiratory tract occurred during the exposure.  

No mortality occurred.

At 1 mg/L, shallow breathing labored respiration and restless behavior were seen during exposure (not presented in the table). After exposure, lethargy, hunched posture, labored respiration, slow breathing and rales were seen for the animals. The animals had recovered from the clinical signs between Days 3 and 6.

At 3.9 mg/L, slow breathing was seen during exposure (not presented in the table). After exposure, lethargy, hunched posture, labored respiration, slow breathing, rales, piloerection and a dull right eye were seen for the animals. The animals had recovered from the clinical signs by Day 3.

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 post mortem examination of the animals.

 

The inhalation LC50, 4hvalue of CH02906 in Wistar rats was established to exceed 3.9 mg/L.

Based on these results CH02906 does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).