Cellulase

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April 2011 - 31 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Cellulase is also used in animal feed. Thus, in vivo skin irritation study were required to assess worker safety according to the provisions of Regulation EC 429/2008. Also, the findings from these studies were used for product classification and labelling in the US (OSHA, HSC).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

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 4

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 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:

other: Fermentation liquid

Details on test material:

Substance type: UVCB
Physical state: fermentation liquid (brown)
Stability under test conditions: the undiluted test material and dilutions in water (50 and 25%) for at least 5 hours at room temperature. The undilutedtest material is stable at least 7 days at 4 degrees Celsius.
Storage conditions of test material: approximately minus 20 degrees Celsius in the dark

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Two New Zealand White strain rabbits were supplied by Harlan Laboratories U.K. Ltd., Loughborough, UK. At the start of the study the animals weighed 2.31 or 2.35 kg and were twelve to twenty weeks old.
The animals were individually housed in suspended cages with free access to drinking water and food.
The temperature and relative humidity were set to achieve limits of 17 to 23 C and 30 to 70% respectively.
The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

Test system

Preparation of test site:

clipped

Vehicle:

not specified

Controls:

no

Amount / concentration applied:

0.5 ml of test item on three sites on the back of the rabbit

Duration of treatment / exposure:

One patch of removed at each of three time points: 3 minutes, 1 hour and 4 hours after application

Observation period:

Test sites were examined for evidence of primary irritation approximately 1, 24, 48 and 72 hours later.

Number of animals:

Two

Details on study design:

One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit. A quantity of 0.5 ml of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.

One patch was removed at each of three time points: 3 min, 1 hour and 4 hours after application. Any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water.

After consideration of the skin reactions produced in the first animal, an additional animal was treated with 0.5 ml of test item. One patch was applied to the back of the rabbit and was allowed to remain in contact with the skin for a period of four hours.

Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to a scale.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Results 2

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

4

Results 3

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 5

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 6

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: sum of 24 and 72 hours reading

Score:

0.3

Max. score:

1

Reversibility:

fully reversible

Irritant / corrosive response data:

No evidence of skin irritation was observed at any time follwoing 3 minute or 1 hour exposures. Slight erythema was noted at one treated site at the 24 and 48 hour observation points following the 4 hour exposure period. This effect was fully reversed by 72 hours. The substance is not classified as a skin irritant under GHS and CLP criteria.

Other effects:

No corrosive effects were noted.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Cellulase is classified as "Non Irritant".

Executive summary:

The objective of this study was to assess the local irritant effect of Cellulase when applied topically as a single dose. The study was conducted according to the method recommended in the OECD Guideline No 404, “Acute Dermal Irritation/Corrosion” and Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No. 440/2008. The study complied with all GLP standards. A sequential approach was used. In the initial test, the back of one New Zealand rabbit was divided into four test sites. Cellulase (0.5 ml which is equivalent to 82.91 mg enzyme concentrate dry matter/kg bw or 43.21 mg active enzyme protein/kg bw) was applied (semi-occlusive) to three sites whereas the fourth site served as control. One patch was removed at each of the three time points, 3 minutes, 1 hour and 4 hours after application. After consideration of the skin reaction in the initial test, a confirmatory test was conducted with one rabbit. One patch is applied and removed after 4 hours of exposure.  Immediately after removal of the patches and at 1, 24, 48 and 72 hours later, all animals were examined for signs of erythema and edema and the responses scored. No deaths or overt signs of toxicity were observed. No effects on feed consumption and body weight were recorded. At the 3-minute and 1-hour exposure periods, no evidence of skin irritation was noted. At the 4-hour exposure period, very slight erythema was noted in one animal at the 24 and 48-hour observation periods with a maximum score of 1. All findings were fully reversible at the 72-hour observation period. No edema was observed in any of the observation period. Cellulase shall be classified as “Non Irritant”.