Cellulase

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 28, 1991 to June 12, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Constituent 2

Reference substance name:

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

Molecular formula:

Not available. See remarks.

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. See remarks.

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. See remarks.

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. See remarks.

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Lot/batch No.: PPC 3425
- Expiration date of the lot/batch: At least stable until January 2001

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
- Weight at time of dosing: 187-224 g (females), 283- 315 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Rat Modified No 1 Diet expanded from Special Diets Service Ltd., Essex, ad libitum, except during the 4 hours exposure
- Water: Tap water ad libitum, except during the 4 hours exposure
- Acclimation period: 18 days
- Temperature (°C): 18-22°C
- Humidity : 34-65 %

IN-LIFE DATES: From: May 28, 1991 To: June 12, 1991

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG Instruments Ltd., England
- Exposure chamber volume: 41.5 L
- Method of holding animals in test chamber: Snout only
- Source and rate of air: Clean dried air was provided by the aerosol generator at a flow rate of 21 L/min. The volume of air was measured continuously using flowmeters and was recorded at 30 min intervals.
- Method of conditioning air: Filtered, oil-free compressed air for the production of the test atmosphere was supplied by Hydrovane compressors.
- System of generating particulates/aerosols: A rotating brush aerosol generator ( RBG - 1000, Palas, Germany) scraped the powdered test material from a canister to micronize it and a stream of air ducted the test material to the exposure chamber.
- Method of particle size determination: Particle size was estimated twice during exposure using a Marple Cascade Impactor (Anderson Samplers Inc., Model 296) capable of fractionating the aerosol into the size range 0.25 -> 10 µm. Samples were taken from the breathing zone. The material collected on the impaction stages of the sampler was weighed before and after sampling to determine the particle size distribution in the atmosphere.
- Temperature, humidity, pressure in air chamber: 23-26°C, 24-67% humidity, normal pressure of the atmosphere.

TEST ATMOSPHERE
- Brief description of analytical method used: 16 air samples were taken during exposure. Chamber air was drawn at a measured rate of 1 L/min using a vacuum pump, with 2 liters of air sampled. The gravimetric method used employed pressed glass fiber filters (Whatman GF/B) placed in a filter holder. The collected material was weighed before and after sampling to determine the concentration of test material in the exposure chamber in relation to the air flow in liters.

- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: 89% respirable (< 10 um)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

4.86 mg/L

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 for clinical signs of effect: During exposure, immediately after the exposure (ca 1-2 hrs) and subsequently at least every day in the 14-day observation period. Body weights: Just before dosing and on days 2, 3, 4, 7, 10 and 14.
- Necropsy of survivors performed: Yes
- Other examinations performed: Organ weights: The lungs of each animal were weighed and the lung:body weight ratio determined. Histopathology was performed on the lungs of all animals.

Statistics:

Not performed.

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.86 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Effect levels 2

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.72 mg/L air

Based on:

other: enzyme concentrate dry matter

Exp. duration:

4 h

Mortality:

No mortality.

Clinical signs:

other: No clinical signs. Immediately following exposure, the rats exhibited a wet and unkempt appearance due to the method of restraint but this cleared within 2 hrs after the end of exposure and all animals were normal the following day.

Body weight:

All body weights and body weight gains were normal throughout the study.

Gross pathology:

No abnormalities.

Other findings:

Lung:body weight ratios were within normal limits for all animals.
Histological examination: Slightly increased number of mucous cells in the bronchial epithelium of 2 animals, one male and one female. Otherwise, the changes seen were all compatible with the ususal background level seen in Sprague-Dawley rats of this age at the present contract research laboratory.

Any other information on results incl. tables

Table 1. Cellulase: Acute inhalation toxicity study in rats, particle size distribution

 

Group/ Conc. (mg/L)	Sample	Stage	Particle size range (um)	Amount collected (g)	% of total	% respirable (<0.9 um)	% respirable (< 6um)	% respirable (< 10um)
1 (4.86)	1	1 2 3 4 5 6 Filter back up	>10 6-10 3.5-6 2.0-3.5 0.9-2.0 0.5-0.9 0.25-0.5	0.27 0.91 0.51 0.57 0.07 0.03 -	11.4 38.6 21.6 24.2 3.0 1.3 -	1.3	50.1	88.7
	2	1 2 3 4 5 6 Filter back up	>10 6-10 3.5-6 2.0-3.5 0.9-2.0 0.5-0.9 0.25-0.5	1.31 1.45 0.74 0.72 0.20 0.14 -	28.7 31.8 16.2 15.8 4.4 3.1 -	3.1	39.5	71.3
Mean						2.2	44.8	80.0

 

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 10um) was 80% (44.8% of the particles were < 6 um).

 

Applicant's summary and conclusion

Interpretation of results:

other: Data insufficient for classification since the highest dose < 5 mg active enzyme protein/L.

Conclusions:

Cellulase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 4.86 mg/L (corresponding to 4.72 mg enzyme concentrate dry matter/L). The LC50 for Cellulase is in excess of 4.72 mg enzyme concentrate dry matter/L.

Executive summary:

In accordance with OECD guideline No. 403, a Limit Test was performed with one group of rats consisting of 5 females and 5 males.

The animals were exposed by snout only exposure for 4 hours to air containing aerosolised freeze-dried powder of Cellulase, batch PPC 3425, at a concentration of 4.86 mg/L (equivalent to 4.72 mg enzyme concentrate dry matter/L).

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 10um) was 80% (44.8% of the particles were < 6 um). The animals were observed during exposure, for two hours after the exposure and subsequently every day in the 14-day observation period. After the observation period, the animals were sacrificed and examined pathologically.

Immediately following exposure, the rats exhibited a wet and unkempt appearance due to the method of restraint but this cleared within 2 h after the end of exposure. Body weight and body weight gain were unaffected by exposure. During the rest of the observation period, all rats were normal in appearance and behaviour. No animals died during the observation period, and the pathological examination revealed no abnormalities related to the exposure.

In conclusion, Cellulase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 4.86 mg/L (corresponding to 4.72 mg enzyme concentrate dry matter/L). The LC50 for Cellulase is in excess of 4.72 mg enzyme concentrate dry matter/L.