Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 06 November 2012 and 12 December 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Cis-hex-3-en-ol (LFA)
- Physical state: clear colourless liquid
- Analytical purity: 98.59 %
- Lot/batch No.: 1Z50650
- Expiration date of the lot/batch: 06 June 2015
- Storage condition of test material: room temperature, in the dark under nitrogen

Test animals

Species:
rat
Strain:
other: RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 200 to 350 g
- Fasting period before study:
- Housing: The animals were housed in groups of three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

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: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical exposure chamber
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatised (for approximately 2 h) to a tapered polycarbonate restraining tube. During the day of exposure, 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: The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. The extract from the exposure chamber passed through a 'scrubber' trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
- System of generating particulates/aerosols: the test item was aerosolised using a glass concentric jet nebuliser located at the top of the exposure chamber. The nebuliser was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply. 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 nebuliser.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined 3 times during the exposure period using a Marple Personal Cascade Impactor. This device consisted of 6 impactor stages (8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 µm cut points) with stainless steel collection substrates and a back up glass fibre filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 µm was calculated. The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50 % point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.
- Temperature, humidity in air chamber: 20 °C, 77- 82 %
- Oxygen concentration: the test chamber was generated to contain at least 19 % oxygen

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes, 5 times during the exposure period

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 2.67 µm/ 2.77
- Predicted amount less than 4 µm: 65.5 %

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Following an appropriate equilibration period a single group of 6 rats (3 males and 3 females) was exposed to an atmosphere of the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 99.8% of target and no deaths occurred, no further levels were required.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Target concentration of 5.0 mg/L
No. of animals per sex per dose:
6/sex/ dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 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, 1 h after termination of exposure and subsequently once daily for 14 d. Any evidence of overt toxicity was recorded at each observation; bodyweight, individual bodyweights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14
- Necropsy of survivors performed: yes, at the end of the 14 d observation period all 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

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.99 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no mortalities.
Clinical signs:
other: One day after exposure, all animals exhibited increased respiratory rate, hunched posture and pilo-erection. Animals recovered to appear normal from Days 5 to 7 post exposure.
Body weight:
All males and 2 female animals exhibited slight bodyweight losses on the first day post-exposure. Bodyweight gains were noted in all animals during the remainder of the recovery period, with the exception of 1 female animal which exhibited a bodyweight loss from Days 1 to 3 post-exposure.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.

Any other information on results incl. tables

Exposure chamber concentration

The test atmospheres were sampled after theoretical chamber equilibration and then at approximately hourly intervals during the exposure period. The mean values obtained were:

Atmosphere concentration

Mean achieved (mg/L)

Standard deviation

Nominal (mg/L)

4.99

0.20

9.33

 

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* (*the test atmosphere was generated for a total of 10 min prior to animal insertion to ensure test item concentration was being achieved).

Particle size distribution

The particle size analysis of the atmosphere drawn from the animals' breathing zone, was as follows:

Mean achieved atmosphere concentration (mg/L)

Mean mass median aerodynamic diameter (µm)

Inhalable fraction (%, 4 µm)

Geometric standard deviation

4.99

2.67

65.5

2.77

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was assessed for acute toxicity via the inhalation route, administered by nose only exposure to a mean acheived atmosphere concentration of 4.99 mg/L. The test substance was found to have an LC50 of >4.99 mg/L. Therefore, under the conditions of this study the test substance was not considered to be acutely toxic via the inhalation route.
Executive summary:

A study was performed to assess the acute inhalation toxicity of the test item. The method used was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals (2009) No. 436 "Acute Inhalation Toxicity - Acute Toxic Class Method".

A group of 6 RCCHan™: WIST strain rats (3 males and 3 females) was exposed to an aerosol atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a 14 -day observation period.

The mean achieved atmosphere concentration was as follows:

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

4.99

0.20

9.33

 

The characteristics of the achieved atmosphere were as follows:

Mean achieved atmosphere concentration (mg/L)

Mean mass median aerodynamic diameter (µm)

Inhalable fraction (%, 4 µm)

Geometric standard deviation

4.99

2.67

65.5

2.77

The mortality data were summarised as follows:

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

Male

Female

Total

4.99

0/3

0/3

0/6

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered to appear normal from Days 5 to 7 post-exposure.

All males and 2 female animals exhibited slight bodyweight losses on the first day post-exposure. Bodyweight gains were noted in all animals during the remainder of the recovery period, with the exception of 1 female animal which exhibited a bodyweight loss from Days 1 to 3 post-exposure.

No macroscopic abnormalities were detected amongst animals at necropsy.

No deaths occurred in a group of six rats exposed to a mean acheived atmosphere concentration of 4.99 mg/L for four hours.

It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of the test item was greater than 4.99 mg/L.