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Toxicological information

Acute Toxicity: inhalation

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
yes
Remarks:
Due to a technicians error six male animals were utilized for the first exposure instead of three males and three females.
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 193g to 350g
- Fasting period before study: no
- Housing: in groups of up to three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet (ad libitum): Harlan 20 14C Rodent Diet, Harlan Laboratories UK Ltd, Ox on, UK
- Water (ad libitum): drinking water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high).
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During each exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber '0' ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: Chamber air flow rates ranged from 50 to 60 L/min dependent on concentration, providing 100 to 120 air changes per hour.
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the SAG 410.
- System of generating particulates/aerosols: In order to facilitate aerosolisation and reduce particle size, the test item was ground using a small amount of diethyl ether in a Retsch Planetary Ball Mill (Retsch (UK) Ltd, Leeds, UK), the solvent was removed via evaporation prior to use. The absorption of the test item was not determined.
A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply.
- Treatment of exhaust air: The extract from the exposure chamber passed through a 'scrubber' trap and was connected with a high efficiency filter to a metered exhaust system.
- Temperature, humidity of air chamber: 19-20°C, 30-39%

TEST ATMOSPHERE
- Brief description of analytical method used: The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump. Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
An aerosol sample collected at an animal port of the inhalation chamber was analysed by UPLC/UV-MS for the verification of stability of EKKE after micronization. The chromatographic profile of the aerosol sample was identical to the EKKE used to generate the aerosol.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined three times during each exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): see table 1
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5.09 and 5.33 mg/L
No. of animals per sex per dose:
6 males and 3 females
Control animals:
no
Details on study design:
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any evidence of overt toxicity was recorded at each observation.

Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.

Necropsy
At the end of each fourteen day observation period, the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Statistics:
Not appropriate

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC0
Effect level:
>= 5.09 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: no mortality
Mortality:
No mortality was recorded.
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a
Body weight:
Group 1 - Five out of the six male animals exhibited body weight losses on the first day postexposure. Reasonable body weight gains were noted in all animals during the remainder of the recovery period.
Group 2 - One out of the three female animals exhibited no body weight body weight gain on Day 1 post-exposure. Two out of the three female animals exhibited no body weight gain from Days 1 to 3 post-exposure. Reasonable body weight development was noted in all animals during the remainder of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst the animals from Group 1. Dark patches on the lungs were noted in all animals at necropsy from Group 2.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No deaths occurred in groups of six or three rats (six males or three females) exposed to mean achieved atmosphere concentrations of 5.33 mg/L or 5.09 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of EKKE, in the RccHanTM: WIST strain rat, was > 5 mg/L.
Executive summary:

A study was performed to assess the acute inhalation toxicity of EKKE. The method used was compatible with the OECD Guidelines No. 436. Two groups of six male or three female Wistar rats were exposed to a dust atmosphere of the test item at mean achieved atmosphere concentrations of 5.33 and 5.09 mg/L with a Mean Mass Median Aerodynamic Diameter of 3.42 and 3.45 µm and a Geometric Standard Deviation of 2.07 and 2.82 µm, respectively. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period.

No deaths occurred in groups of six males or three females rats exposed to mean achieved atmosphere concentrations of 5.33 mg/L or 5.09 mg/L. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered to appear normal on Day 5 post-exposure. Five out of the six male animals exposed to 5.33 mg/L exhibited body weight losses on the first day post­exposure. Reasonable body weight gains were noted in all animals during the remainder of the recovery period. One out of the three female animals exposed to 5.09 mg/L exhibited no body weight body weight gain on Day 1 post-exposure. Two out of the three female animals exhibited no body weight gain from Days 1 to 3 post-exposure. Reasonable body weight development was noted in all animals during the remainder of the recovery period. No macroscopic abnormalities were detected amongst the animals from Group 1. Dark patches on the lungs were noted in all animals at necropsy from Group 2. The 4 hr LC0 of EKKE, in the Wistar rat, was higher than 5 mg/L. Therefore, EKKE should not be classified for the acute toxicity by inhalation exposure according to the CLP and GHS criteria.