Pullulanase

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 25 - July 9, 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pullulanase

EC Number:

232-983-9

EC Name:

Pullulanase

Cas Number:

9075-68-7

Molecular formula:

not available

IUPAC Name:

Pullulanase

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Pullulanase SP 247, Toxbatch PPY 1323
- Substance type: UVCB
- Physical state: Powder
- Lot/batch No.: PPY 1323
- Expiration date of the lot/batch: At least stable until 1992-08-05
- Stability under test conditions: The test material is stable for at least 24 hours at room temperature
- Storage condition of test material: 4 degrees of Celcius

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River UK Ltd, Kent UK.
- Housing: Five animals per cage, the sexes being kept separately
- Weight at time of dosing: Between 155-167 g (females), 195-202 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Temperature (°C): 18-24°C
- Humidity: 45-70 %

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: An exposure chamber connected to 2 Wright dust feeders (Wright, B. M., 1950, J. Sci. Instr., 27, 12.) and two Broomwade compressors (type CAR 31) to supply compressed air.
- Exposure chamber volume: 41.5 L
- Method of holding animals in test chamber: Nose only
- Source and rate of air: Air was supplied in a volume of 18.0 L/min to prepare and disperse the dust.
- Method of conditioning air: The dust feeders were positioned at the base of the chamber. An extract duct at the top of the chamber was connected by way of a high efficiency filter to a metered vacuum system. The test compound particles were dispersed evenly throughout the chamber and exited through a series of small holes connected to a filtered vacuum line operating at a flow rate of approximately 2.0 L/min greater that the input flow rate (to reduce risk of contamination of the environment). The exposure chamber provided a single pass of the freshly generated dust.
- System of generating particulates/aerosols: The test atmosphere was produced by 2 Wright dust feeders. A scraper blade remoced powder from a prepacked canister at a steady rate. The loose powder was removed from the canister by a stream of clean, dry, compressed air. The dust passed through a jet and was impinged on a baffled plate to break up any aggregates. The air containing the finely dispersed dust was passed directly into the exposure chamber.
- Method of particle size determination: The particle size was estimated twice during the exposure period by using an Andersen Mini Sampler (2000 Inc., Salt Lake City, Utah, USA). The device consists of 4 metal impaction plates and a back-up glass fiber filter housed within an anodized aluminium can. The sampler was positioned and temporarily sealed in a port in the exposure chamber in the animal’s breathing zone. Chamber air was drawn through the sampler at a rate of 1.4 L/min using an Andersen 2000 vacuum pump for a recorded time period. Each impaction plate together with the back-up filter was weighed before and after sampling and the weight of material collected on each stage calculated by difference. From the results obtained the weight distribution of 5 size ranges of particles was calculated.
- Treatment of exhaust air: An extract duct at the top of the chamber was connected by way of a high efficiency filter to a metered vacuum system. The particles dispersed evenly throughout the chamber, and were exited through a series of small holes connected to a filtered vacuum line operation at a flow rate of approximately 2. 0 L/min greater than the input flow rate and therefore reducing risk of contamination of the environment.
- Temperature, pressure in air chamber: 21oC ± 2oC, normal pressure of the atmosphere

TEST ATMOSPHERE
- Brief description of analytical method used: The chamber concentration was estimated gravimetrically during the exposure periods at regular intervals. The gravimetric method used employed pressed glass fiber filters placed in a filter holder. The conical input side of the holder was positioned and temporarily sealed in a port in the exposure chamber in the animal’s breathing zone. Chamber air was drawn through the filter at a measured rate of 2.5 L/min using a vacuum pump. Seven air samples (volume between 15-37.5 L) during the 4 hour exposure period were taken. The collected material was weighed to determine the concentration of test material in the exposure chamber.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: 25.71% respirable (< 4.7 um)
- MMAD (Mass mean aerodynamic diameter) / GSD (Geometric st. dev.): MMAD=15.0 µm, GSD=4.1 µm

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

2.14 mg/L

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: During exposure, immediately after and frequently for the first 4 hours post exposure, and at least twice daily during the subsequent 14 day post exposure period. Weighing: Immediately before dosing and on days 2, 3, 4, 7, 10 and 14 post exposure.
- Necropsy of survivors performed: yes

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.14 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality.

Clinical signs:

other: All animals showed initial struggling with increased urination and defecation during the early part of the exposure period. Test animals showed a slight reduction in respiratory rate during exposure. In addition a clear nasal secretion and red nasal encru

Body weight:

No effects on body weight gain were observed.

Gross pathology:

No abnormalities.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Pullulanase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 2.14 mg/L. Thus it was concluded that the LC50 for pullulanase is in excess of 2.14 mg/L.

Executive summary:

In accordance with OECD guideline No. 403, a Limit Test was performed with two groups, one control group and one test group. Both groups consisted of 5 females and 5 males rats.

The animals were exposed by snout only exposure for 4 hours to air containing dust of Pullulanase, batch PPY1323, at a concentration of 2.14 mg/L.

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 4.7 um) was 25.71%. The animals were observed during exposure, immediately after and frequently for the first 4 hours post exposure, and at least twice daily during the subsequent 14 day post exposure period. They were weighed immediately before dosing and on days 2, 3, 4, 7, 10 and 14 post-exposure. After the observation period, the animals were sacrificed and examined pathologically.

The clinical signs observed during exposure of the test material were confined to a slightly depressed respiratory rate, a clear nasal secretion after 30 minutes exposure and red nasal encrustation after 3 hours. No animals died during exposure or during the observation period, and the pathological examination revealed no abnormalities.

In conclusion, the LC50 (4 h) for pullulanase in Sprague-Dawley rats was not demonstrated other than an indicuation of the value being in excess of 2.14 mg/L.