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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April-May, 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
Test procedure was according to Draize (1959) for the evaluation of household products (under the U.S. Hazardous Substances Labelling Act) at the time of testing. The study was performed before GLP was implemented but was performed according to state of the art at that time.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1977
Report Date:
1977

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US Federal Register, § 191.11, 17 Sept. 1964
Deviations:
not specified
Principles of method if other than guideline:
Test procedure was according to the US Federal Register, § 191.11, 17 Sept. 1964:
The primary irritation to the skin was measured by a patch-test technique on the abraded and intact skin of the albino rabbit, clipped free of hair. Six animals were used.
Under a square patch of two layers of surgical gauze measuring 1 inch by 1 inch, i.e. 6.4 cm2, 0.5 mL of the test substance was applied to the skin. The patches were secured in place by adhesive tape and the entire trunk of the animal then wrapped with an impervious material. This material aids in maintaining the test patches in position and simulates an occlusive bandage. After 24 hours of exposure, the patches were removed and the resulting reactions were evaluated after a scoring system identical to the OECD grading. Reading of the reactions were performed 24 and 72 hours after exposure started.
GLP compliance:
no
Remarks:
Before GLP but according to the same principles

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: between 2.5 - 3.5 kg
- Age at study initiation: Young adults
- Housing: Individually, in animal room without control of temperature and humidity
- Diet (e.g. ad libitum): Standard diet, ad libitum
- Water (e.g. ad libitum): Automatic, ad libitum
- Acclimation period: minimum 7 days
- Temperature (°C): 14-18C
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs

Test system

Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
other: water respectively Borate buffer, pH 9.1
Controls:
not required
Amount / concentration applied:
0.5 mL of the test material, prepared as a 25% w/v solution in water (pH 7) and 25% w/v in borate buffer, pH 9.1, respectively. Concentration of solution applied was 3.4% expressed as active enzyme protein.
Duration of treatment / exposure:
24 hours
Observation period:
72 hours
Number of animals:
2 x 6
Details on study design:
The test compound Subtilisin was assessed by semiocclusive application of 0.5 mL of the test material formulation in water or borate buffer to the closely-clipped dorsa of six New Zealand White rabbits per type of formulation for 24 hrs. Intact and abraded skin was evaluated.
Test procedure was according to the US Federal Register, § 191.11, 17 Sept. 1964

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
edema score
Basis:
animal #3
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #4
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
1.5
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
edema score
Basis:
animal #4
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
erythema score
Basis:
animal #5
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
1.5
Max. score:
4
Reversibility:
other: Not concluded within the 72 hr study period
Irritation parameter:
edema score
Basis:
animal #5
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #6
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
1.5
Max. score:
4
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
edema score
Basis:
animal #6
Time point:
other: Mean of 24, 72 h (intact skin, only)
Score:
0
Max. score:
4
Irritant / corrosive response data:
Both formulations of Subtilisin (batch PFP 312/20) i.e. buffered and unbuffered solutions, were indistinguishable by effect. Each provoked a mild to moderate erythematous response in the entire group of animals at 24 hrs, with a single animal displaying a barely perceptible oedema. At 72 hrs, the reactions had subsided and only the occasional animal displayed a barely perceptible erythema. Only the unbuffered reactions are displayed above as they are almost identical to the buffered reactions (results can be seen in table 2 at page 8 in the report).
The mean values for erythema and oedema recorded 24 and 72 hours (after the 24 hr exposure started) did not exceed the limit values considered to indicate a significant inflammatory response.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
The test material was mildly irritating to skin under the conditions of the study. According to EU (CLP 2008) this test material should not be classified as irritant. (Criteria used for interpretation of results: EU)

CLP: not classified
Executive summary:

An in vivo skin irritation test was conducted to assess the potential of the test substance, Subtilisin, batch FPF 312/20, to cause skin irritation. The procedure of the US Federal Register, § 191.11, 17 Sept. 1964, was followed. 0.5 mL of the test material formulation was applied under semiocclusive conditions to the closely-clipped dorsa of 12 New Zealand White rabbits for 24 hrs. The study was conducted before GLP was implemented but the principles was the same and state of the art was followed. The study concluded that the test substance, Subtilisin, was a mild irritant to the skin. Concentration of solution applied was 3.4% expressed as active enzyme protein.