Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March to 25 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 403 without any deviation.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 12-14 March 2014 / signed on 12 May 2014)
Test type:
traditional method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colorless liquid
- Storage condition of test material: Stored at 2-8 °C temperature and protected by Nitrogen.
Specific details on test material used for the study:
- Purity test date: 02 September 2014
- Storage condition of test material: Stored cold under nitrogen at approximately 4°C, in the dark

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™ : WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: 200-350 g
- Housing: Animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes.
- Diet: Food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 per hour
- Photoperiod: 12 h dark / 12 h light

N-LIFE DATES: 23 March to 25 June 2015

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.26 - <= 3.43 µm
Geometric standard deviation (GSD):
>= 2.06 - <= 2.26
Remark on MMAD/GSD:
Mean Mass Median Aerodynamic Diameter (MMAD) = 3.29, 3.26 and 3.43 μm at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively
Geometric Standard Deviation (GSD) = 2.18, 2.06 and 2.26 at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber.
- Exposure chamber volume: Cylindrical exposure chamber had a volume of approximately 30 L (dimensions: 28 cm diameter x 50 cm high).
- One day prior to the day of exposure each rat was acclimatized (for approximately 2 h) to a tapered polycarbonate restraining tube.
- Method of holding animals in test chamber: During the 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 ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: Chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).
- 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 and humidity in air chamber: Temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds.
, UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. Temperature and humidity in air chamber were 19-20 °C and 26-41%, respectively.
- Exposure chamber oxygen concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.
- Exposure Chamber Atmosphere Concentration: Prior to the start of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator between 18 and 20 °C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The non-volatile component of the batch used during the study was found to be approximately 8.62 % (n=2). Due to the very low levels of non-volatiles contained within this test item it was considered appropriate to use chemical analysis in order to determine test atmosphere concentrations for all exposure groups.
The test atmosphere was sampled nine times during each exposure period. The sampling procedure involved 2 to 3 liters of test atmosphere (dependent on concentration) being drawn through a glass impinger or a series of two glass impingers (dependent on concentration) containing Methanol (each made up to 80 mL). The samples were then submitted for chemical analysis.
The nominal chamber concentrations were calculated by dividing the mass of test item used by the total volume of air passed through the chamber during each exposure.
The nominal concentrations were 434 %, 366 % and 372 % of the actual mean achieved atmosphere concentration for Groups 1, 2 and 3 respectively and show that keeping the aerosol airborne was relatively straightforward for all dose groups.
- Pretreatment: Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied to achieve the required atmospheric conditions.
- Following an appropriate equilibration period three groups, each of five or ten rats (five males and/or five females), were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. Further concentrations were selected after consideration of the results of the previous exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test item was measured off-line by high performance gas chromatography (GC).
- Samples taken from breathing zone: Yes
- Particle size distribution: See table 7.2.2/2
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Mean Mass Median Aerodynamic Diameter (MMAD) = 3.29, 3.26 and 3.43 μm at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively; Geometric Standard Deviation (GSD) = 2.18, 2.06 and 2.26 at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Nominal concentration: 21.9, 11.9 and 17.2 mg/L
Mean achieved atmosphere concentration: 5.05, 3.25 and 4.62 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Duration of observation period following administration: 14 days
- Frequency of observations: 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 deaths or evidence of overt toxicity were recorded at each observation.
- Frequency of weighing: Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.
- Necropsy of survivors performed: Yes, at the end of the 14 day observation period all animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including those that died or were humanely killed during the study 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:
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) was calculated using validated data analysis software (R Environment, (The R Foundation for Statistical Computing, Vienna, Austria.)). This software utilized Log-Normal (Probit) regression models in order to calculate the LC50 values. LC50 values and 95% confidence limits were calculated for males and females separately.

Results and discussion

Preliminary study:
Not applicable
Effect levelsopen allclose all
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
5.23 mg/L air (analytical)
Based on:
test mat.
95% CL:
>= 4.45 - <= 6.01
Exp. duration:
4 h
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
4.84 mg/L air (analytical)
Based on:
test mat.
95% CL:
>= 4.29 - <= 5.38
Exp. duration:
4 h
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
5.18 mg/L air (analytical)
Based on:
test mat.
95% CL:
>= 3.98 - <= 6.38
Exp. duration:
4 h
Mortality:
At 5.05 mg/L, 1/5 male and 3/5 females died
At 3.25 mg/L, 0/5 female died (males not exposed)
At 4.62 mg/L, 2/5 females died (males not exposed)
Clinical signs:
other: Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection, and wet fur. There were frequent instances of labored respiration, occasional instances of noisy respiration, atax
Body weight:
Group 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were then noted in all surviving animals during the remainder of the recovery period.
Group 2 – All animals exhibited body weight losses on the first day post-exposure. With the exception of one animal, which showed no body weight gain during the final week of the recovery period, body weight gains were noted in all animals during the remainder of the recovery period.
Group 3 – All surviving animals exhibited body weight losses on Day 1 post-exposure and from Days 1 to 3 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.
Gross pathology:
With the exception of one male animal from Group 1 which exhibited dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods.
The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study: Lungs – dark patches; Small intestine – contained dark substance.
Due to the observations noted during the study and at necropsy it is considered that deaths noted during the study may have been mainly attributable to systemic toxicity.
Other findings:
None

Any other information on results incl. tables

Table 7.2.2/1: Exposure Chamber Concentration

The actual concentration of the test item was measured off-line by high performance gas chromatography (GC). The test atmospheres were sampled after theoretical chamber equilibration and then at approximately half-hourly intervals during each exposure period.

The concentration of the test item was shown to be stable and the mean values obtained were:

Group Number

 

Mean Achieved (mg/L)

 

Standard Deviation

 

Nominal (mg/L)

 

1

5.05

0.12

21.9

2

3.25

0.23

11.9

3

4.62

0.08

17.2

 

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

The theoretical chamber equilibration time (T99) was 3 minutes (Silver, 1946).

Table 7.2.2/2: Particle Size Distribution

Group Number

 

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (μm)

Inhalable Fraction

(% <4 μm)

Geometric Standard Deviation

1

5.05

3.29

60.0

2.18

2

3.25

3.26

61.2

2.06

3

4.62

3.43

57.6

2.26

 

Table 7.2.2/3: Mortality Data

Group Number

 

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

 

Male

 

Female

Total

1

5.05

1/5

3/5

4/10

2

3.25

NOT EXPOSED

0/5

0/5

3

4.62

NOT EXPOSED 

2/5

2/5

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The inhalation LC50 and 95% confidence limits of test item were calculated to be
All animals: 5.23 (4.45 – 6.01) mg/L; Males only: 5.18 (3.98 – 6.38) mg/L; Females only: 4.84 (4.29 – 5.38) mg/L
Under the test conditions, the test item is classified as Category 4 according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.
Executive summary:

In an acute inhalation toxicity study performed according to OECD Guideline 403 and in compliance with GLP, three groups of five or ten RccHan™ : WIST strain rats (five males and/or five females) were exposed to an aerosol atmosphere of the test item for 4 hours.

Nominal concentration: 21.9, 11.9 and 17.2 mg/L (corresponding to mean achieved atmosphere concentrations: 5.05, 3.25 and 4.62 mg/L, respectively)

Animals were observed for mortality, clinical signs and body weight for 14 days and at the end of the study the animals were sacrificed for macroscopic examination.

 

At 5.05 mg/L, 1/5 male and 3/5 females died;

At 3.25 mg/L, 0/5 female died (males not exposed);

At 4.62 mg/L, 2/5 females died (males not exposed).

 

Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection, and wet fur. There were frequent instances of labored respiration, occasional instances of noisy respiration, ataxia, coma and red/brown staining around the eyes and snout and isolated occurrences of exophthalmos, dehydration, pallor of the extremities, lethargy, ptosis and prostration. Surviving Group 1 animals appeared normal on Day 7 post-exposure. All Group 2 animals recovered to appear normal on Day 3 post-exposure and surviving animals from Group 3 recovered to appear normal on Day 6 post-exposure.

 

Group 1 (5.05 mg/L) – All surviving animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were then noted in all surviving animals during the remainder of the recovery period.

Group 2 (3.25 mg/L) – All animals exhibited body weight losses on the first day post-exposure. With the exception of one animal, which showed no body weight gain during the final week of the recovery period, body weight gains were noted in all animals during the remainder of the recovery period.

Group 3 (4.62 mg/L) – All surviving animals exhibited body weight losses on Day 1 post-exposure and from Days 1 to 3 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.

 

With the exception of one male animal from Group 1 which exhibited dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods. The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study: Lungs – dark patches; Small intestine – contained dark substance.

Due to the observations noted during the study and at necropsy it is considered that deaths noted during the study may have been mainly attributable to systemic toxicity.

 

The inhalation LC50 and 95% confidence limits of test item were calculated to be

All animals: 5.23 (4.45 – 6.01) mg/L; Males only: 5.18 (3.98 – 6.38) mg/L; Females only: 4.84 (4.29 – 5.38) mg/L

Under the test conditions, the test item is classified as Category 4 according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.