Subtilisin

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion, other

Remarks:

Skin irritation, in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 12-27, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report Date:

1995

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. certificate)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Type:

Constituent

Type:

Constituent

Type:

Constituent

Test material form:

liquid

Details on test material:

- Substance type: UVCB
- Physical state: Liquid
- Lot/batch No.: PPA 4852
- Expiration date of the lot/batch: No specific expiration date. At least 10 years or as long as enzyme activity is preserved
- Stability under test conditions: Solutions in water are stable for at least 24 hours at room temperature or 4 degrees Celcius
- Storage condition of test material: minus 18 degrees of C

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Bred by Charles River, UK
- Weight at study initiation: between 2.45 - 2.65 kg
- Age at study initiation: Young adults, approx. 3 months
- Housing: Individually, in animal room with control of temperature (15-23C) and humidity (40-70%RH)
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Automatic, ad libitum
- Acclimation period: 7 days
- Photoperiod (hrs dark / hrs light): 12 hrs/12hrs

IN-LIFE DATES: From: 1994-10-12 To: 1994-10-27

Test system

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

not required

Amount / concentration applied:

0.5 mL of the test material diluted to retail activity (445 mg test material made up to 1000 mg with water), corresponding to a solution with 4.5% active enzyme protein

Duration of treatment / exposure:

4 hours

Observation period:

16 days

Number of animals:

3

Details on study design:

The test compound Subtilisin was assessed by semiocclusive application of 0.5 mL of the test material formulation to the closely-clipped dorsa of three New Zealand White rabbits for 4 hrs.
The study was conducted to comply with the following guidelines:
- Organisation for Economic Co-operation and Development (OECD) test guideline 404.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: Mean of 24, 48, 72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: Very slight exfoliation was evident until termination of the study (day16) in the present animal.

Results 2

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: Mean 24, 48, 72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Results 3

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: Mean 24, 48, 72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Results 4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: Mean 24, 48, 72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Results 5

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: Mean 24, 48, 72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: Exfoliation was seen until day 13 in this animal, all other effects were cleared at day 10.

Results 6

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: Mean 24, 48, 72 h

Score:

2.3

Max. score:

3

Reversibility:

fully reversible

Irritant / corrosive response data:

Very slight or well defined erythema and very slight to moderate oedema were observed at the test sites of two animals during the first 72 hrs after bandage removal. One of these animals also showed a dry abrasion over the majority of the test site during this time. One week after treatment, very slight erythema and exfoliation were evident in both animals. The exfoliation persisted until day 13 in one animal and to termination of the study in the other. No dermal response was observed at the tests site of the third animal at any time during the observation period.
The mean values for erythema and oedema recorded 24, 48 and 72 hours after treatment did not exceed the limit values considered to indicate a significant inflammatory response.

Applicant's summary and conclusion

Interpretation of results:

Category 3 (mild irritant) based on GHS criteria

Conclusions:

According to EC-standards (as published in the Official Journal of the European Communities, L 110 A, 4 May 1993), the test material is not irritating to skin. Concentration of solution applied was 4.5% expressed as active enzyme protein.

Executive summary:

An in vivo skin irritation test was conducted to assess the potential of the test substance, Subtilisin, batch PPA 4852, to cause skin irritation. The OECD test guideline 404 was followed. 0.5 mL of the test material formulation was applied under semiocclusive conditions to the closely-clipped dorsa of three New Zealand White rabbits for 4 hrs. The study was conducted in accordance with GLP. The study concluded that the test substance, Subtilisin, was non-irritant to the skin.

However due to the very slight exfoliation still seen in one animal at the termination of the study, Subtilisin should be regarded as slightly irritant to skin.