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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
Study period:
1988-02-20 - 1988-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 81-3 (Acute inhalation toxicity)
Deviations:
no
GLP compliance:
yes
Remarks:
Federal Register, Vol 48, No 230, 1983
Test type:
traditional method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation:fine black powder
- storage conditions: at ambient temperature in the dark in closed container
The test substance was stored in the dark at ambient temperature in the company dispensary at the Elphinstone Research Centre when not in use.


Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) Limited, Mansion, Kent, England (one batch (16 males and 16 females was received at IRI on 2 February 1988 whilst the second batch (16 males and 16 females) was received on 4 February 1988).Five males and 5 females of the first batch received and 15 males and 15 females of the second batch received were used on the study.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-7 weeks old (arrival)
- Weight at study initiation: approximately 110-135 g (arrival)
- Fasting period before study: None. No water or food during the 4 h exposure period.
- Housing: semi-barrier maintained animal room. The animals were housed in Room C16 of the Rodent Toxicology Complex, at the Elphinstone Research Centre. Rats were housed 5 males or 5 females per cage in suspended polypropylene cages with detachable stainless steel tops and bottoms. All cages were suspended over trays containing absorbent paper
- Diet (e.g. ad libitum): ad libitum. Rat and Mouse (modified) No. 1 Diet SQC Expanded, supplied by Soecial Diet Services limited
- Water (e.g. ad libitum): ad libitum. Each cage was supplied with a polypropylene water bottle (capacity 500 ml) with a rubber washer and melamine cap
- Hygiene: Cages were changed weekly and cage tray papers were changed as required (at approximately 3 day intervals), water bottles were cleaned weekly. Each afternoon, when other work in the room was finished, floors were swept then washed with a disinfectant solution. Once each week walls, benches and racking within the animal room were washed with a disinfectant solution.
Disinfectant used was either 1% Tego from T.H. Goldschmidt and Company Limited, or 3% Hycogen from Hy-co Products (Scotland) Limited.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C + 2°C (Temperature extremes recorded were 16°C-24°C)
- Humidity (%): ca 55% (humidity extremes were 30%-54%)
- Photoperiod (hrs dark / hrs light): A 12 h light-dark cycle was controlled by a time switch, light hours being 0700-1900 h.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
other: snout only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.1 - <= 2.9 µm
Remark on MMAD/GSD:
The test material was tested as a particulate aerosol in its original form. The particle size distribution of the dispersed material inside the exposure chamber was estimated twice during the exposure period using a Marple (model 296) Caccade Impactor. The device was positioned on the chamber using a vacuum pump, at a constant rate of 2 litres/min for a recorded time period. The impaction substrates (for each stage) were weighed before and after sampling and the weight increase on each substrate determined to be the mass of particles in the size range of that impactor stage. From the results obtained, the total weight of particles on all stages and filter was summed and the percent particle mass in each size range calculated. The data may be presented graphically as a cumulative size distribution, showing the percent of particle mass smaller than the aerodynamic particle diameter, and the respirable mass fraction determined from the particle size distribution.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test material was tested as a particulate aerosol in its original form. The test atmospheres were generated by means of a Wright dust feeder (Wright, B.M., 1950, J. Sci. Instr., 27, 12). In this electrically operated device, a scraper blade removed the powder at a steady rate from a prepacked canister of test material. The loose powder was removed from the canister by a stream of clean, dry compressed air.
After passing through a jet the air containing the finely dispersed dust passed directly into the exposure chamber.
The aluminium exposure chamber was cylindrical in cross section and had a volume of approximately 41.5 litres. An extract duct from the chamber was connected by way of a high efficiency filter to a metered vacuum system. The exposure chamber was mounted inside an extract cabinet for the protection of operators and the environment.
Compressed air was supplied by means of 2 Broomwade compressors (Type CAR 31) fitted with automatic pressure control switches. These supplied filtered, conditioned, oil-free compressed air for subsequent dilution of the test atmosphere.
The canister of the Wright dust feeder was packed in stages to ensure an even density. The canister was positioned on the Wright dust feeder which was located at the top of the exposure chamber. The exit nozzle of the dust feeder was passed through a rubber diaphragm into the chamber space and the exposure started by switching on the dust feeder and engaging the scraper blade with the powder.
The exposure system was truly dynamic, incorporating a single pass of the freshly generated material. The particles dispersed throughout the chamber and exited through the base to a filtered vacuum line. Chamber air flow rates were monitored continuously and the values recorded at 30 min intervals.
The inhalation exposures were conducted in a room adjacent to the animal holding room. Each animal was removed from its cage and examined for general health status. The ear number was checked, the animal weighed and then loaded into a tapered, polycarbonate restraint tube which fitted onto the exposure chamber and sealed by means of a push fit through a rubber 'o' ring. All animals were exposed on a single tier eliminating exposure variations. Only the animals' snouts were exposed to the test atmosphere. The animals were not allowed access to food or water during the 4 h exposure period.
During exposure the animals were observed at regular intervals for signs of any adverse reactions to treatment. On completion of the 4 h exposure period, the animals were removed from the chamber, unloaded from the restraint tube, returned to their cages in the animal holding room and observed for clinical signs.
The study protocol stated that only 3 groups would be exposed to a range of concentrations of the test material. However, due to the 100% mortality observed following the first 2 concentrations tested, an additional group was included in the study to give a total of 4 groups each comprising 5 male and 5 female rats. The animals were exposed to a range of concentrations of the test material via the inhalation route by snout only exposure for a single continuous 4 h period. The exposures were conducted over the period 20 February -1 March 1988.
The first concentration tested was 1.01 mg/litre, the remaining exposure concentrations were selected from the mortality pattern recorded following this initial concentration.
During the exposure periods the temperature within the exposure chamber was measured by a mercury thermometer located at the animals' breathing zone whilst the relative humidity was monitored using wet/dry bulb mercury thermometers. The chamber temperature and relative humidity were measured at 30 min intervals throughout the exposure periods and ranged from 17°C-22°C and 13%-62% respectively for temperature and humidity. The humidity readings at the higher chamber concentrations tended to be extremely low due to the accumulation of test material on the wet bulb thermometer.

TEST ATMOSPHERE
Chamber concentrations were measured at regular intervals during the exposure periods. The gravimetric method used employed pressed glass fibre filters (Whatman GF7B) placed in a filter holder. The conical input side of the holder was positioned and temporarily sealed in a port in the exposure chamber at the animals' breathing zone. Chamber air was drawn through the filter at a measured rate of 1.0 litre/min using a vacuum pump. The air flow during each sample was controlled by acritical orifice and timed for a suitable period. Each filter was weighed before and after sampling in order to calculate by difference the weight of collected material. The chamber concentration was estimated by further calculation using the sample air volume.
The nominal chamber concentration was estimated using the following equation:

Nominal concentration (mg/litre) = Weight of material used (mg) / Total air flow through chamber /(litres)
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
1.01 (not estimated), 0.52 (3.72), 0.15 (0.83) and 0.31(2.10) mg/L measured (nominal)
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All the rats were weighed immediately before dosing and on Days 2, 3, 4, 7, 10 and 14 post exposure.
- Necropsy of survivors performed: yes (Any gross lesions observed were recorded in descriptive terms, including location(s), size (mm), colour and number. The respiratory tract was subjected to detailed macroscopic examination for signs of irritancy or local toxicity. All organs were examined in situ)
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: All the rats were observed for clinical signs at approximately hourly intervals throughout the exposure period, for the first 1-2 h post dosing and thereafter at least once daily during the subsequent 14 day observation period. The onset, intensity and duration of any signs observed were recorded. Lungs: Body Weight Ratio:
The lungs of each animal were removed and weighed to allow calculation of lung: body weight ratio.
Statistics:
not reported

Results and discussion

Preliminary study:
not performed
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
0.281 mg/L air
Based on:
test mat.
Remarks:
aerosol
95% CL:
>= 0.207 - <= 0.361
Exp. duration:
4 h
Mortality:
The mortality pattern observed as a result of exposure to the test material was as follows:
Group       Concentration (mg/L)       Mortality
Males        Females     Total %
3              0.15                                  0/5            0/5                0
4              0.31                                  3/5            3/5                60
2              0.52                                  5/5            5/5                100
1              1.01                                  5/5            5/5                100
All mortalities occurred overnight following exposure, or on Day 1 post exposure.
Clinical signs:
other: Marked respiratory depression was observed during exposure for the animals in Groups 1 and 2, exposed to 1.01 or 0.52 mg/L respectively. On return to their cages (following completion of exposure) all animals showed heavy deposition of the test material
Body weight:
All animals which survived exposure to the test material showed a marked body weight loss following exposure. During the subsequent 14 day observation period the animals showed a gradual body weight recovery. The overall body weight gains over the 14 day post exposure observation period were lower then normal.
Gross pathology:
Gross post mortem examinations of the animals that died following exposure to the test material revealed extensive haemorrhage in the lungs and resultant pulmonary oedema.
Other findings:
Lung:Body Weight Ratio:
Lung:body weight ratios for those animals that died as a result of exposure to the test material were markedly increased. In addition, the ratios observed for the surviving animals in Group 3 (exposed to 0.31 mg/L) were marginally increased.

Any other information on results incl. tables

Chamber Atmospheric Conditions

Particle Size Distribution

Estimation of the particle size distribution revealed that the percentage of particles <3.5 µm was 67.0%, 74.2%, 81.9% and 85.1% by weight respectively for Groups 1, 2, 3 and 4.

The mass mean diameter of the aerosol particles generated for Groups 1, 2, 3 and 4 was determined to be 2.9 µm, 2.7 µm, 2.2 µm and 2.1 µm respectively.

Applicant's summary and conclusion

Interpretation of results:
other: Category 2 based on EU GHS criteria
Conclusions:
The LC50 value of the test material was calculated to be 0.281 mg/L. The results obtained indicated that an exposure concentration as low as 0.15 mg/L produced toxic effects.
Executive summary:

This study was undertaken to investigate the acute inhalation toxicity of the test material and to determine the median lethal concentration (LC50) in rats following a single 4 hour snout only exposure. The study was conducted in compliance with EPA Guideline Section 81-3 and was in compliance with Good Laboratory Practice Standards (FED. Reg. Vol 48, No. 230, 1983).

Four groups of Sprague Dawley rats consisting each of 5 males and 5 females were exposed to 0.15, 0.31 0.52 and 1.01 mg/L concentration of the test material. No mortality was observed at 0.15 mg/L whereas at concentrations of 0.52 and 1.01 mg/L all animals died. In the exposure group at 0.31 mg/L 3 males and 3 females died.

Marked respiratory depression was observed during exposure at all concentrations. All or some of the following signs were recorded for all groups: hunched/subdued appearance, piloerection, laboured respiration, hypokinesia and ataxia following exposure. The signs showed a dose related response; being more severe in the animals exposed to 0.52 or 1.01 mg/L. Surviving animals showed a body weight loss following exposure. A gradual body weight recovery was recorded over the 14 day post exposure observation period. Gross pathological examination of the animals that died following exposure to the test material revealed extensive haemorrhage in the lungs and resultant pulmonary oedema. Lung:body weight ratios were markedly increased for the premature decents.

The LC50 of the test material was calculated to be 0.281 mg/L with 95% confidence limits of 0.207-0.361 mg/L.