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Diss Factsheets

Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 May 2022 to 9 June 2022
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
impurity 1
Reference substance name:
unknown impurities
unknown impurities
Test material form:
solid: particulate/powder

Test animals

Details on test animals or test system and environmental conditions:
- Source: Hylasco Biotechnology (India) Pvt. Ltd., Telangana - 500078.
- Females nulliparous and non-pregnant: yes.
- Rationale for use of males: No data are available to indicate that one sex is more susceptible than the other.
- Age at study initiation: The age at reception (29 April) was 6 weeks. Age at date of exposure (26 May) was 9 weeks.
- Weight at study initiation: Males: 253.4 to 257.2 g; Females: 245.5 to 248.9 g.
- Fasting: food and water were withheld during the exposure period.
- Housing: before and after exposures, animals were housed in groups of two/three per cage by sex in standard polysulfone cages (size: approximately L 425 x B 266 x H 185mm), with stainless steel top grill having facilities for pelletted food and drinking water. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and were changed along with the cage at least once a week. Steam sterilized corn cob was used as bedding and changed along with the cage at least once a week.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: After physical examination for good health and suitability for the experiment, the animals were acclimatized for six days before treatment. After grouping (prior to testing), i.e. on day 6 of acclimatization, the animals were acclimatized to the test apparatus (restrainers) for one hour, to lessen the stress caused by introduction to the new environment.

- Temperature (°C): 21.7-23.2°C. The maximum and minimum temperature in the experimental room were recorded once daily.
- Relative humidity (%): 61.7-66.2%. The relative humidity in the experimental room was recorded once daily and was calculated from dry and wet bulb temperature recordings.
- Air changes (per hr): 12.5 to 14.1 air changes/hour.
- Photoperiod (hrs dark/hrs light): 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Mass median aerodynamic diameter (MMAD):
2.31 µm
Geometric standard deviation (GSD):
Remark on MMAD/GSD:
Particle size of the aerosol generated in the chamber air was determined two times, during the first and fourth hour of the exposure period using any one port.
MMAD (µm) and GSD at the first hour were respectively: 2.31 µm and 1.71.
MMAD (µm) and GSD at the fourth hour of exposure were: 2.30 µm and 1.71.
Mean MMAD and GSD are reported in the relative sections.
The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) values for the test item for the G1 group were within the range recommended by OECD Guidance Document No. 39 on acute inhalation testing equal to MMAD of 1-4 µm and GSD of 1.5-3.0.
Details on inhalation exposure:
- Exposure apparatus: the animals were tested using nose only inhalation equipment (SMIT Fabricators). The equipment is designed and operated in slight positive pressure. The nose only, flow-past exposure chamber consists of two
chambers, the aerosol inlet chamber (Diameter 10 cm × Height 8 cm) and the aerosol exhaust chamber (Diameter 20 cm × Height 10 cm). The chambers are cylindrical in shape and placed one next to the other.
- Exposure chamber volume: 0.7 liters.
- Method of holding animals in test chamber: the rats were restrained in glass exposure restrainers (size approximately Length 23 cm, Diameter 7 cm). These restrainers make an airtight seal with the plenum tube through a rubber
gasket. Within the plenum is a central tube into which the aerosol enters at each port. The air flow carries the aerosol to the nose of the animals restrained in position at the open ends of the tubes.
- Source and rate of air: the airflow rate was monitored continuously and maintained at 8 LPM.
- Method of conditioning air: the compressed air is dehumidified, filtered and reduced by filter reducer assembly to the required pressure (2 kg/cm2) at the use point.
- System of generating particulates/aerosols: based on physico-chemical properties of the test item, a solid dust aerosol generation system (SMIT Fabricators) was used for technical pre-test and limit test study.
- Method of particle size determination: Seven Stage Cascade Impactor (SMIT Fabricators) was used to determine particle size distribution, Mass Median Aerodynamic Diameter (MMAD) and Geometric standard deviation (GSD).
- Treatment of exhaust air: exhausted air and rat exhaled air exits from the chamber outlet.
- Temperature, humidity, pressure in air chamber: the inhalation chamber monitor was used to measure oxygen content, carbon dioxide content, temperature and humidity in the inhalation chamber during exposure (Uniphos Envirotronic
Pvt. Ltd.).

- Samples taken from breathing zone: yes.
- Time needed for equilibrium of exposure concentration before animal exposure: 0.26 minutes.

- Composition of vehicle: 20.6-20.8% v/v oxygen; 0.03-0.04% v/v carbon dioxide.

Seven Stage Cascade Impactor (SMIT Fabricators) was used to determine particle size distribution, Mass Median Aerodynamic Diameter (MMAD) and Geometric standard deviation (GSD). Refer to data in this section (Administration/exposure) and to the attached full study report.

- Rationale for the selection of the starting concentration: as no information of the toxicity of the test compound was available, the concentration level at 5 mg/L (limit test) was used as the starting dose selected from Annex 3d of
OECD 436 guideline.
Analytical verification of test atmosphere concentrations:
The test item concentration at the animals’ breathing zone of the inhalation chamber was determined using gravimetric filter analysis method. Aerosol samples were drawn from one port at hourly intervals during the 4 hours exposure period.
Duration of exposure:
4 h
Nominal: 26.99 mg of test item/L of chamber air;

Measured mean concentration (± SD): 5.03 ± 0.03 mg of test item/L of chamber air.
No. of animals per sex per dose:
Three animals of each sex for limit test (5.03 mg/L air).
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: the rats were observed for pre-terminal deaths four times during day 0 (i.e. at hourly intervals during the exposure period), twice after release from the restrainers for clinical signs of
toxicity/pre-terminal deaths, and once daily during days from 1 to 14. Body weights were recorded once during the acclimatization period, on day 0 (pre-exposure), and days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes.
- Clinical signs: observation included body weight, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Other examinations performed: at the end of the observation period, all rats were euthanized and subjected to detailed necropsy by observing the respiratory tract and visceral organs.

Results and discussion

Effect levels
Key result
Dose descriptor:
Effect level:
> 5.03 mg/L air
Based on:
test mat.
Exp. duration:
4 h
No pre-terminal deaths were observed during the observation period.
Body weight:
All rats gained bodyweight throughout the experiment period.
Gross pathology:
No abnormalities were detected at necropsy in any of the exposed animals.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
The acute inhalation Median Lethal Concentration (LC50) after 4 hours exposure to the test item AP 729 is found to be >5.03 mg/L of chamber air in male and female Wistar rats.
The test item is therefore unclassified as per Annex 3d of the OECD 436 test guideline.
The test item is classified as category 5 according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Ninth Revised Edition (2021), as there was no mortality at 5.03 mg/L of chamber air in both male and female Wistar rats.
Executive summary:

To assess the toxic effects of AP 729, an acute inhalation toxicity study was conducted on Wistar rats according to Annex 3d of OECD Guideline 436, with a treatment group (G1, limit test). 

A technical pre-test was conducted without animals to check the feasibility of generating adequate test atmosphere (test item concentration, particle size distribution, Ocontent, CO2 content, temperature and relative humidity) after chamber equilibrium using a solid dust aerosol generation system. 

The inhalation toxicity study was performed in 3 male and 3 female Wistar rats by nose-only exposure to test item AP 729 as a dust aerosol generated at the same exposure conditions adopted during the technical pre-test.
The animals were housed in special rat restrainers and continuously exposed to the test item aerosol for 4 hours in an inhalation exposure chamber (nose-only, dynamic state). The post treatment observation period was 14 days.

The dust aerosol was sampled from the inhalation chamber to determine particle size distribution, Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD). Particle size of the aerosol generated in the chamber air was determined two times i.e., during the first and fourth hour of the exposure period using any one port. The MMAD and GSD were calculated using Seven Stage Cascade Impactor. The overall mean MMAD of 2.31 µm and GSD of 1.71 were observed for G1 group. 

The test item concentration at the animals’ breathing zone was also determined using gravimetric filter analysis method. The average actual concentration of AP 729 was 5.03 mg/L of chamber air. 

All animals were normal after the 4 hours exposure period. All rats gained bodyweight throughout the experiment period and no clinical signs were detected. There were no pre-terminal mortalities observed during the experimental period and no abnormalities were detected at necropsy at the end of the 14 days observation period. 

The acute inhalation (4 hours) Median Lethal Concentration (LC50) value of AP 729 is greater than 5.03 mg/L of chamber air in both male and female Wistar rats.