Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1st may to 16th May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study performed according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

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
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl dihydrojasmonate
- Substance type: pure active substance
- Physical state: colourless liquid
- Analytical purity: 99.63 %
- Isomers composition: ca 10% cis- and 90% trans- isomers
- Lot/batch No.: 73519
- Storage condition of test material: under Nitrogen at room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 200g to 350g.
- Housing: in groups of five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK) and provided with environmental emichment items: wooden chew blocks (B & K Universal Ltd, Grimston, Hull, UK) and cardboard "fun tunnels" (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum (excepted during exposure period) (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK)
- Water (e.g. ad libitum): ad libitum (excepted during exposure period)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
See Scheme of apparatus used in "Illustration"
- Exposure apparatus: The test material was aerosolised using a glass concentric jet nebuliser (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber. The nebuliser was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test material 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. 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. A schematic diagram of the dynamic (continuous flow) system employed is shown in "Illustration".
- Exposure chamber volume: approximately 30 litres (dimensions: 28 cm diameter x 50 cm high).
- Method of holding animals in test chamber: See "Illustration". 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.
- 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 (Schaefer Instruments Ltd, Oxon., UK). This device consisted of six impactor stages (9.8, 6.0, 3.5, 1.55, 0.93 and 0.52 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 material, 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 9.8, 6.0, 3.5, 1.55, 0.93 and 0.52 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 4µm (considered to be the respirable portion) was determined. All particle size distribution calculations were performed using a purpose designed computer programme (Chrom Series Data Server and Reg 2000 Graph Plotter).
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometerlhumidity meter (Comark Ltd, Welwyn Garden City, Hertfordshire, 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. The chamber was maintained under negative pressure.

TEST ATMOSPHERE
- Brief description of analytical method used: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowborough, East Sussex) located in a sampling port in the animals breathing zone during each exposure period. The test atmosphere was generated to contain at least 19% oxygen. The actual chamber test material concentration was measured at regular intervals during the exposure period. The gravimetric method used employed glass fibre filters (Gelman type A/E 25 mm) 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 material. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration. The nominal chamber concentration was calculated by dividing the mass of test material used by the total volume of air passed through the chamber.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): at least 19% oxygen

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: See Executive summary and "Graph showing particle size distribution" in "Attached background material"
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.36 um/1.91 um
Analytical verification of test atmosphere concentrations:
yes
Remarks:
See "Any other information on material and methods incl. tables"
Duration of exposure:
4 h
Remarks on duration:
none
Concentrations:
5 mg/l air (male + female)
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 and weighing: Observations: 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 fourteen days. Wheighing: Prior to treatment on the day of exposure, on day 7 and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
Not applicable

Results and discussion

Preliminary study:
Not applicable
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.93 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: 95% CL not applicable
Mortality:
None
Clinical signs:
other: During exposure, wet fur was noted in all animals and there were instances of increased respiratory rate. On removal from the chamber, animals showed increased respiratory rate, hunched posture, pilo-erection and wet fur and there was an isolated instance
Body weight:
Normal bodyweight development was noted. during the study. Two females showed reduced bodyweight gain but such variations are not uncommon in female rats of this strain or age.
Gross pathology:
With the exception of a single instance of dark foci on the lungs, no abnormalities were detected at necropsy.
Other findings:
None

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Executive summary:

Introduction. A study was performed to assess the acute inhalation toxicity of the test material.

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 403 "Acute Inhalation Toxicity" referenced as Method B2 in Commission Directive 92/69/EEC "Acute Toxicity - Inhalation" (which constitutes Annex V of Council Directive 67/548/EEC).

 

Methods. A group of ten Sprague-DawleyCrl:CD® (SD) IGS BR strain rats (five males and five females) was exposed to an aerosol atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period.

 

Results. The mean achieved atmosphere concentration was as follows:

Atmosphere concentration

Mean achieved (mg/l)

Standard deviation

Nominal (mg/l)

4.93

0.50

51.0

 

The characteristics of the achieved atmosphere were as follows:

Mean Achieved Atmosphere Concentration (mg/l)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable fraction

(% < 4µm)

Geometric Standard Deviation

4.93

3.36

60.6

1.91

 

The mortality data were summarized as follows:

Mean Achieved Atmosphere Concentration (mg/l)

Deaths

Male

Female

Total

4.93

0/5

0/5

0/10

 

Clinical Observations. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. There were instances of red/brown staining around the snout and an isolated instance of noisy respiration. Animals recovered steadily to appear normal from Days 3 - 6.

Bodyweight. Normal bodyweight development was noted during the study.

Necropsy. With the exception of a single instance of dark foci on the lungs, no abnormalities were detected at necropsy.

 

Conclusion. No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 4.93 mg/l. It was therefore considered that the acute inhalation median lethal concentration (LC50) of MethylDihydrojasmonate, in the Sprague-DawleyCrl:CD® (SD) IGS BR strain rat, was much greater than 4.93 mg/l (LC50 > 4.93 mg/l air) as no death occured at this concentration.