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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January 1990 to 15 February 1990
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
study report
Report date:
Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
equivalent or similar to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
according to guideline
EPA OPP 81-3 (Acute inhalation toxicity)
GLP compliance:
Test type:
traditional method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-2-(4-chloro-2-methylphenoxy)propionic acid
EC Number:
EC Name:
(R)-2-(4-chloro-2-methylphenoxy)propionic acid
Cas Number:
Molecular formula:
(R)-2-(4-chloro-2-methylphenoxy)propionic acid
Test material form:
solid: flakes
Brown coloured hard flakes
Details on test material:
- Storage conditions: Room temperature protected from light.
Specific details on test material used for the study:
- Storage condition of test material: The sub-sample of test material used during this study was transferred to an amber glass screw top container after milling.

Test animals

other: CD strain (remote Sprague-Dawley origin)
Details on test animals or test system and environmental conditions:
- Age at study initiation: On arrival the rats were about 6 to 11 weeks old. At the time of dosing the animals were about seven to 12 weeks old.
- Weight at study initiation: On arrival the male rats were within the bodyweight range of 182 to 200 g and the females 191 to 212 g. At the time of dosing the male rats were within the bodyweight range 259 to 289 g and females from 243 to 254 g.
- Fasting period before study: Animals were not fed during the exposure procedure.
- Housing: The rats were housed five of one sex per cage. Each cage consisted of a high-density polypropylene body measuring 56 x 38 x 18 cm with a stainless-steel lid and floor. The cages were suspended above absorbent crepe paper which was changed daily.
- Diet: A commercially available complete pelleted rodent diet was fed ad libitum. This was an expanded diet which contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Drinking water was taken from the public supply, controlled by the East Anglian Water Company, Lowestoft, Suffolk, England and offered ad libitum to each cage in polyethylene bottles with sipper tubes.
- Acclimation period: 8 days
- Microbiological status when known: The animals had been bred under barriered conditions and travelled from the supplier to the animal-holding room in sealed boxes with filter protected air-vents.

- Temperature: Target value 21° (18 to 25 °C)
- Humidity: Target value 55 % RH (40 to 70 % RH)
- Air changes: The room was kept at positive pressure with respect to the outside and had its own supply of filtered, fresh air which was passed to atmosphere and not re-circulated. Ventilation equipment was designed to provide approximately 15 air changes per hour.
- Photoperiod: Electric time switches regulated a lighting cycle of 12 hours of artificial light per day.

From: 21 January 1990
To: 15 February 1990

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
snout only
Mass median aerodynamic diameter (MMAD):
6.31 µm
Geometric standard deviation (GSD):
>= 2.39 - <= 10.23
Remark on MMAD/GSD:
The small percentage of inhalable particles was considered to be associated with the difficulties encountered during milling of the waxy, low melting point test material. The percentage of the atmosphere that was less than 4 μm EAD was 37 %.
Details on inhalation exposure:
- Exposure apparatus: The exposure chamber consisted of 30 cm diameter aluminium alloy cylinder comprised of modules assembled to give a volume of ca. 60 litres. The cylinder incorporated three animal exposure sections each having 20 exposure ports (ADG Instruments Ltd, Codicote, Hitchin, Herts, England). The exposure chamber and generation apparatus were positioned in a large cabinet equipped with an extract fan exhausting to atmosphere through a collection filter.
- Exposure chamber volume: ca. 60 litres
- Method of holding animals in test chamber: Each rat was placed in an individual polycarbonate restraining tube so that only the snout protruded. The restraining tubes were attached to the chamber so that only the snout projected into the chamber.
- Source and rate of air (airflow): A dust feed mechanism (Wright, 1950, 1963) mounted into the top of the chamber, was used to produce the test atmosphere. The mechanism was designed to produce and maintain an atmosphere containing dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The concentration of dust suspended in the airstream could be varied by changing the gear ratio of the generator mechanism. Dry, oil free compressed air was passed through the mechanism to give a flow rate of 25 L/min.
- System of generating particulates/aerosols: It was not possible to generate atmospheres containing the test material as supplied. Mechanical milling by ultracentrifugal mill and by crossbeater mill resulted in the formation of a waxy solid on the milling screens. As a consequence of this characteristic of the test material, hand milling with a pestle and mortar was performed. Small quantities of the test material were ground gently with a pestle and mortar, the minimum effective pressure was applied to minimise the tendency of the test material to form a solid waxy film in the mortar. Waxy residues lining the pestle and mortar were scraped off and reground with a small quantity of fresh test material. The milled test material formed a solid cake during storage; therefore, the prepared material was brushed through a 150 μm test sieve before use to break up aggregates and ensure thorough mixing.
A Wright Dust Feed Mechanism powder canister was packed with previously milled test material to a pressure of 40 kg/cm^2 using a hydraulic press. Even density of packing was achieved by packing the canister in stages. The canister was weighed before use. A dust feed mechanism was positioned on top of the chamber and the gear ratio set to give the maximum practicable canister advance rate.
The powder canister of the Wright Dust Feed Mechanism was advanced until a trace of suspended dust could be seen in the chamber air. The generator gearing was then engaged and the generator motor switched on. After a 5.5 minute equilibration period, the theoretical time required for the concentration of aerosol to reach 90 % of the final value under the conditions of exposure employed (Silver and Arsenal, 1946), the exposure was timed for four hours. After four hours the generator was switched off and the rats were removed from the restraining tubes for examination.
- Method of particle size determination: Cascade Impactor
- Treatment of exhaust air: Exhaust air was drawn from the base of the chamber at a rate of 25 L/min and vented to atmosphere after first passing through an absolute filter.
- Temperature, humidity, pressure in air chamber: The chamber and generator air supplies were switched on and balanced to give a zero-pressure difference between the chamber and extract cabinet air. Wet and dry bulb thermometers located in the animal breathing zone were used to measure the temperature and humidity within the chamber. Readings were monitored frequently and recorded at 30-minute intervals throughout the exposure period. The mean chamber temperature and relative humidity during exposure were 22 °C and 53 % (RH), respectively.
- Respirable fraction determination: The fraction of the test atmosphere considered to be inhalable to the laboratory rat was calculated from the combined mass of test material collected on stages three to seven of Sierra Marple cascade impactor samples as a percentage of the total material collected by the device i.e. all particles with an Equivalent Aerodynamic Diameter of 6.0 μm or less.

- Brief description of analytical method and equipment used: The total aerosol concentration was determined on five occasions during the four-hour exposure. The atmosphere samples were withdrawn through a previously weighed Whatman GF/A glass fibre filter held in an open face filter holder. Samples were collected at an accurately calibrated flow rate of approximately four litres per minute. The filter and collected material were weighed to determine the atmosphere concentration of the test material.
- Time needed for equilibrium of exposure concentration before animal exposure: 5.5 minutes

- Composition of vehicle: Dry, oil free compressed air.

- Particle size distribution: The particle size distribution of the chamber atmosphere was measured once during each hour of the exposure period. The aerosol was characterised by drawing a continuous atmosphere sample at two litres per minute through a cascade impactor (Sierra Marple Model 296. Sierra Instruments Incorporated, Carmel Valley, California, U.S.A.) which was located in a spare animal exposure port.
At a sampling rate of two litres per minute the collection characteristics of the Sierra Marple Model 296 cascade impactor are:
Stage 1: Particles and droplets larger than 9.8 μm Equivalent Aerodynamic Diameter (EAD) and droplets.
Stage 2: Particles and droplets between 6.0 and 9.8 μm EAD
Stage 3: Particles and droplets between 3.5 and 6.0 μm EAD
Stage 4: Particles and droplets between 1.55 and 3.5 μm EAD
Stage 5: Particles and droplets between 0.93 and 1.55 μm EAD
Stage 6: Particles and droplets between 0.52 and 0.93 μm EAD
Stage 7: (Filter) Particles and droplets less than 0.52 μm EAD
The values of chamber concentration reported were determined by gravimetric analysis of five samples of chamber air taken during each exposure. Atmosphere concentrations are reported in terms of mg of test material per litre of chamber air.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 6.31 µm ± 3.92
The Mass Median Equivalent Aerodynamic Diameter (MMEAD) and geometric standard deviation of the test material present in the exposure atmosphere was calculated by linear regression of the mean cumulative percentage of the material collected on each Sierra Marple Cascade Impactor Stage (on a probability scale) against the logarithm of the manufacturers published stage cut points (μm) for the device.
The achieved chamber concentration was 0.87 mg/L (SD 0.07, 11.3 % generation efficiency). The relatively poor efficiency of atmosphere generation achieved during the exposure was considered to be associated with the waxy nature of the milled test material.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
One nominal concentration of 7.67 mg/L (actual 0.87 mg/L)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed immediately before exposure, 15 and 30 minutes after the start of exposure and at 30 minute intervals for the remainder of the exposure period. Following return to their cages, the animals were observed at 30 minute intervals during the first three hours after exposure. Subsequently, they were examined twice daily until completion of 14 days of observation. The type, time of onset and duration of reactions to treatment were recorded. Each rat was weighed daily from the day of delivery until the end of the observation period.
- Necropsy of survivors performed: Yes. Rats that survived the observation period were placed under deep sodium pentobarbitone anaesthesia by intraperitoneal injection and killed by rapid exsanguination. All rats killed on completion of the observation period were subjected to necropsy with a minimum of delay.
- Macroscopic pathology: All animals were subjected to a detailed necropsy. The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices. The cranial cap was lifted and the brain dissected free of the meninges. The frontal and nasal elements were removed and any abnormality of the nasal passages, meninges, brain and pituitary recorded. The ventral abdominal skin was reflected to allow observation of the subcutaneous structures, in particular mammary glands and superficial lymph nodes. The abdominal viscera were examined in situ.
The entire gastro-intestinal tract was re-examined after removal. The stomach and caecum were opened along the greater curvature and rinsed in isotonic saline. After weighing the liver and kidneys were sectioned at intervals and the cut surfaces examined. The thorax was opened and the viscera examined before removal. The lungs were removed, dissected clear of surrounding tissue and weighed. Pleural surfaces were examined before inflation with 3 to 6 mL of buffered 4 % formaldehyde saline, injected slowly via the trachea. The trachea was then ligated.
- Organ weight: The organs specified were dissected free of adjacent fat and other contiguous tissue and the weights recorded: Lungs (with bronchi), liver and kidneys. Samples of the following tissues, together with macroscopic abnormalities, were preserved in buffered 4 % formaldehyde saline against possible future histological processing and microscopic examination: Larynx, lungs (with bronchi), liver and kidneys. Kidney weights wereseparately recorded for left and right sides. The recordings were sumed for presentation and before calculation of individual relative organ weights as a percentage of bodyweight.
Group mean values were calculated from individual values unless otherwise specified. Standard deviations were calculated where appropriate using the sample statistic.
Group mean and standard deviation are presented to the same level of accuracy as the individual values.
Standard deviations are not presented where the number of individual values is less than three.

Results and discussion

Preliminary study:
An additional range-finding test was performed using a single group of two male and two female rats exposed to a nominal concentration of 2.85 mg/L. The results of this exposure, which are not reported in detail, indicated that the main study should be performed at the maximum practical chamber concentration.
Effect levels
Key result
Dose descriptor:
Effect level:
> 0.87 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
There were no deaths as a result of exposure.
Clinical signs:
Brown staining around the snout and wet fur were observed during the exposure period. These signs were considered consistent with excessive salivation.
Body weight:
Bodyweight was unaffected by treatment. The observed weight gain was in the expected range for rats of this age and strain.
Gross pathology:
There were no significant findings at necropsy. The lung, liver and kidney weights for all animals were within the expected ranges for rats of this age and strain.
Other findings:
Test material around the snout was recorded for most animals at the end of the exposure period. A reduction in respiratory rate was seen in some animals. The signs observed were considered to be consistent with exposure to a slightly irritant particulate atmosphere.
During the three hours immediately following the exposure, test material around the snout, brown staining around the snout and wet fur were recorded for most animals. Brown staining around the snout persisted after the exposure for up to three days in male rats and up to four days in females. Subsequently, all rats were normal in behaviour and appearance.

Any other information on results incl. tables

Particle Size Distribution

The mean proportion by weight of particles of inhalable size (< 6 μm Equivalent Aerodynamic Diameter) derived from the cascade impactor samples was:

Percent < 6 μm E.A.D. ± SD: 42.6 ± 9.2

Percent < 4 μm E.A.D.: 36.9

Mass Median E.A.D. ± geometric SD: 6.31 ± 3.92 μm

The small percentage of inhalable particles was considered to be associated with the difficulties encountered during milling of the waxy, low melting point, test material.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Under the conditions of the study, there were no deaths following the exposure for four hours of five male and five female rats to the maximum chamber concentration that could be generated using the methods described in this report.
The median lethal chamber concentration for four hours' exposure (LC50 4-hour) is therefore greater than 0.87 mg per litre of air.
Executive summary:

The acute inhalation toxicity of the test material was assessed according to EPA APP 81-3 and equivalent to OECD Test Guideline 403 and in compliance with GLP by exposing a single group of five male and five female rats, for a continuous period of four hours, to an atmosphere containing the maximum practicable concentration of the test material.

The acute toxicity was investigated by exposing a single group of five male and five female albino rats to an atmosphere containing the maximum practicable concentration of test material. The test group was subjected to a single four-hour, continuous snout-only exposure. Signs of reaction to treatment were recorded during a subsequent 14-day observation period. The animals were sacrificed at the end of the observation period and were subjected to a detailed necropsy.

The nominal concentration of the test material was 7.67 mg/L, with the actual concentration being 0.87 mg/L. The relatively poor efficiency of atmosphere generation and the small percentage of inhalable particles (< 6.0 μm EAD) achieved during the exposure were considered to be associated with the difficulties encountered during milling of the waxy, low melting point test material. The Mass Median EAD of the atmosphere generated was 6.31 μm with a geometric standard deviation of 3.92 μm. The percentage of the atmosphere that was less than 4 μm EAD was 37 %.

There were no deaths during the study. Reduced respiratory rate, test material around the snout, brown staining around the snout and wet fur were recorded during the exposure period. Signs evident immediately following the exposure were test material around the snout, brown staining around the snout and wet fur; brown staining around the snout persisted for up to four days in some rats. Subsequently, all animals were normal in appearance and behaviour. Bodyweight gain was unaffected by treatment and there were no macroscopic findings that were attributed to treatment. Examination of the absolute and bodyweight-relative organ weights did not reveal any changes that were attributed to treatment.

Under the conditions of this study, there were no deaths following the exposure for four hours of five male and five female rats to the maximum chamber concentration that could be generated using the methods described in this report.

The median lethal chamber concentration for four hours' exposure (LC50 4-hour) is therefore greater than 0.87 mg per litre of air.