Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 October 2017 to 05 december 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes
Test type:
traditional method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PuyangOuya Chemical Technology Co., Ltd.; 2017073046
- Expiration date of the lot/batch: 31/Dec/2018
- Purity test date: 99.87% (w/w)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was aerosolized non-diluted and in its solid form utilized in the quantity of 110.85 g. The test item was ground and sieved (100# mesh sieve) in order to decrease the particle-size and to increase its respirability.

Test animals

Species:
rat
Strain:
other: Wistar Hannover
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ANILAB – Animais de laboratório Criação e Comércio Ltda., batches: 037/17 and 038/17
- Age at study initiation: 10 and 11 weeks of age on the day of exposure
- Weight at study initiation: 206 – 334g
- Housing: Polypropylene cages (41x34x19 cm) with autoclaved wood shavings and stainless steel mesh lids containing five rats of the same sex per cage were used in the experimental phase.
- Diet: Pelleted and autoclaved commercial diet for rats (Comercial name: Presence; Batch: 41EX170062037; Validity date: 24/Jan/2018) were available ad libitum.
- Water: The animals had free access to filtered water throughout the test, provided by SEMAE (Serviço Municipal de Água e Esgoto) in plastic bottles with capacity of 1000 mL.
- Acclimation period: 17 days (males) and 10 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5 °C to 24.0 °C
- Humidity: 43% to 70%.
- Air changes: 10 to 20 air changes per hour
- Photoperiod: A light system controller was adjusted to maintain a cycle of 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 2.53 - < 3.39 µm
Geometric standard deviation (GSD):
> 2.67 - < 3.01
Remark on MMAD/GSD:
The analysis of the aerodynamic particle-size distribution demonstrated that the Mass Median Aerodynamic Diameter (MMAD) was 2.53 μm and 3.39 μm (first and second samples, respectively) and the Geometric Standard Deviation (GSD) was 2.67 and 3.01 (first and second samples, respectively), indicating that at least 50% of the mass collected from the aerosol generated was within the respirable diameter (< 4 μm) (Table 4).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Preliminary test
A preliminary test without animals was conducted to define the appropriate conditions to reach a test atmosphere with adequate particle-size.

Limit test
From the results obtained in the preliminary test without animals, it was observed that the conditions established for the limit test would reproduce the maximum attainable concentration in the chamber atmosphere.

Exposure equipment
The chamber used was a Jaeger Inhalation Chamber, Mark II with a Wright Dust Feeder integrated system coupled to a cyclone, with a big scraper and previously calibrated to a 4.0 RPM rotation, with 4.25 cm of diameter and 3.5 cm in height. The air eliminated was treated with a filter absolute model F – 771 K03 Trox and filter of activated carbon F – 760 Trox. The chamber has a positive pressure. The source of air used came from a compressed air (21% of oxygen and 79% of nitrogen). The volume was approximately1.5 litres.

Exposure principle
Rats were exposed to the aerosolized test item in plexiglass nose-only tubes applying a direct-flow exposure principle. The total airflow (primary + secondary) in the chamber was 10 L/min. Considering that there are twelve exits in the chamber (10 to allocate the animals during the exposure and 2 to make the collects), the dynamic flow was of approximately 833 mL/min/animal.

Airflow and exposure conditions
The airflow rate was monitored continuously, through the use of an analogical flow meter and was recorded during the exposure. The total airflow did not show any variation (10 L/min.) (Table 1).

During the period of animals exposure to the test substance, four measurements of temperature and humidity, equally spaced in time, were performed. The temperature and relative humidity in the animal’s breathing zone were maintained in the range between 19 and 25ºC and 30 and 70%, respectively.

Exposure principle justification
Nose-only tubes were chosen to accommodate the rats’ size and to allow the rat’s tail to remain outside the tube, avoiding restrained-induced hyperthermia. This exposure principle is preferable compared to whole-body exposure because of scientific and technical reasons - rapid attainment of steady-state concentrations, lower consumption of test substance and minimization of uptake by non-inhalation routes.

Mean actual concentration
Three equally time-spaced air samples from the vicinity of the breathing zone were taken with an air sampling pump previously calibrated to a 1.2 L/min flow rate to collect the aerosol in filters (Millipore Field Monitor for contamination analysis, 0.8 μm micropore size) for four minutes. The filters were weighed before and after sampling on an analytical scale.

Aerodynamic particle-size distribution
Two equally time-spaced air samples were taken from the vicinity of the breathing zone using an air sampling pump previously calibrated to a 0.500 L/min flow rate, to collect the aerosol in a Seven Stage Cascade Impactor (In-Tox, Albuquerque, New Mexico, U.S.A.) for five minutes. Before and after sampling, the stages were weighed.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Mean actual concentration (mg/L) = 2.99 ± 0.120 (maximum attainable concentration)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: Animals were observed frequently during the exposure period. Immediately after the exposure period, the rats were removed from the chamber, their heads carefully cleaned of any material and transferred to the animal house facility. Following exposure, two clinical observations were made during the first 24 hours and then daily during a fourteen-day observation period. The clinical observations were recorded systematically and individually

- Frequency of observations and weighing: The body weight was measured on acclimatization, prior to exposure, on the day of exposure (day 0), the first, third, seventh and fourteenth days after exposure.

- Necropsy of survivors performed: Yes

- Other examinations performed: Microscopic examination of potential target organs was not considered necessary, due to the absence of compound-related clinical signs and macroscopic findings.
Statistics:
Only a limit test was performed, the four-hour inhalation LC50 was not calculated.

Results and discussion

Preliminary study:
A preliminary test without animals was conducted to define the appropriate conditions to reach a test atmosphere with adequate particle-size. The maximum attainable concentration was 2.99 ± 0.120 mg/L.
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 2.99 mg/L air (analytical)
Exp. duration:
4 h
Mortality:
No compound-related death was registered in this study.
Clinical signs:
other: No compound-related clinical sign was observed in this study. Individual data are displayed on Tables 5 and 6.
Body weight:
The mean body weight increased for both sexes in all post-exposure weighings, except in the first post-exposure weighing for the females (Table 3). All animals exceeded their initial body weight at the end of the observation period of 14 days. Individual body weights are displayed on Tables 9 and 10.
Gross pathology:
No compound-related macroscopic finding was registered in this study. Individual data are displayed on Tables 7 and 8.
Other findings:
- Histopathology: It was not considered necessary, due to the absence of compound-related clinical sign and macroscopic findings.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The combined (males and females) median lethal concentration in a 4-hour nose-only exposure period (4-h LC50) to Musk Ketone inhaled by Wistar Hannover rats was greater than 2.99 mg/L.
Executive summary:

In an acute inhalation toxicity study (16073.417.084.17), a group of young adult Wistar Hannover strain rats (5/sex) were exposed to a test atmosphere of musk ketone (99.87%) for 4 hours (nose only) at a mean actual concentration of 2.99 ± 0.120 mg/L (maximum attainable concentration). Animals were then observed for 14 days.

LC50 male/female = > 2.99 ± 0.120 mg/L

The MMAD was 2.53 -3.39 μm and GSD was 2.67 - 3.01, indicating that at least 50% of the mass collected from the aerosol generated was within the respirable diameter (< 4 μm).  No compound-related death was registered in this study. No compound-related clinical sign was observed in this study. The mean body weight increased for both sexes in all post-exposure weighing, except in the first post-exposure weighing for the females. All animals exceeded their initial body weight at the end of the observation period of 14 days. No compound-related macroscopic finding was registered in this study. Histopathology was not considered necessary, due to the absence of compound-related clinical sign and macroscopic findings.

This acute inhalation toxicity test in rats is acceptable and satisfies the guideline requirement for an OECD 403 study.