Subtilisin

Endpoint summary

Description of key information

Due to the catalytic activity, Subtilisin has the potential to cause slight skin and eye irritation. The hazard category and statements are as follows: ‘Causes skin irritation, cat 2 (H315)’ and ‘Causes serious eye damage, cat. 1 (H318)’.

For historical reasons, the eye irritation potential of subtilisin needs some explanation: When Subtilisin induces eye irritation, the effects are highly reversible. As said above, the current harmonized classification of subtilisin in EU for eye irritation is ‘serious eye damage, eye damage, cat. 1 (H318)’. This classification was established back in 1995, where the involved industrial enzyme producers agreed with the Authorities that subtilisin should be re-classified from Xi; R36 (= irritating for eyes) to Xi; R41 (= risk of serious damage to eyes) and this has been valid since. The background was results from one study, using only one single rabbit that was killed after the 1-hour reading. More recent data indicate, however, that R41/H318 is an over-classification of subtilisin. The same batch of subtilisin was re-tested in vivo in 1998 with an extended observation beyond the 1-hour reading. The conclusion was that subtilisin should be classified Xi; R36 (= irritating for eyes). Further investigations of the eye irritation potential of representative subtilisins showed that these test materials are mild to moderate eye irritants. The in vivo studies typically show a quick onset of predominantly conjunctival reactions but also a quick clearance of the effects. Subtilisin has the potential to cause superficial effects in the eye but there is no depth of injury, confirmed by histological investigation of the corneas from various ex vivo investigations. The eye irritation potential of subtilisin has been discussed in details in the attached document in the endpoint summary of the section on Irritation/Corrosion.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2019 - 6 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

640/2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit supplied by EpiSkin Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After 15 minutes, each tissue was rinsed with sterile Dulbeccos Phosphate Buffered
Saline (DPBS) to remove residual test substance. Each insert was then transferred to a well containing 2 ml maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT
and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in
air.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

78.4

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the test substance with a mean tissue viability of 78.4%, was predicted as a non irritant to the skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 78.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The criteria required for acceptance of results in the test were satisfied.

It was concluded that the test substance with a mean tissue viability of 78.4%, was predicted as a non irritant to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Sept. 23-24, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Test procedure was a GLP study performed similar to the principles of OECD guideline 431 (2010)

Qualifier:

equivalent or similar to guideline

Guideline:

other: Similar to the principles of OECD Test No. 431, 2010: In Vitro Skin Corrosion: Human Skin Model Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Type of coverage:

other: Direct application to the EpiDermTM

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

25 mg was applied to the individual reconstituted skin membranes and wetted with 50 uL milliQ water

Duration of treatment / exposure:

3 min and 1 hour

Details on study design:

The test compound Subtilisin and two other enzyme samples were assessed by being applied topically to the EpiDermTM Skin Model for 3 minutes and 60 minutes. Subsequently, the effect on the tissue viability was determined, based on the reduction of MTT to a purple formazan precipitate by mitochondrial dehydrogenase activity. The test compound was tested as a neat product. Positive control substance was 8M KOH.
The study was conducted to comply with the following guidelines:
- Organisation for Economic Co-operation and Development (OECD). New test guideline 431. OECD Guideline for the testing of chemicals. Draft proposal for a new guideline 431. In vitro skin corrosion: human skin model test (2002).
- Commission directive 2000/33/EC. Official Journal of the European Communities L136/90 – 107. Annex I, B.40. Skin Corrosion.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

79.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

80.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Formazan production in EpiDerm^TMskin membranes and negative and positive controls for 3 minutes and 1 hour:

                                                                

3-minute exposure
Test group	Test compound	A540(% of control)
NC	Negative control (demineralised water)	100.0
A	Subtilisin (PPA 1619)	79.1
PC	Positive control (8 M KOH)	10.2
1-hour exposure
Test group	Test compound	A540(% of control)
NC	Negative control (demineralised water)	100.0
A	Subtilisin (PPA 1619)	80.8
PC	Positive control (8 M KOH)	8.2

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro EpiDermTM Skin Model (EPI-200), the test compound Subtilisin was predicted as non-corrosive.

Executive summary:

An in vitro EpiDerm^TM Skin irritation test was conducted to assess the potential of the test substance, Subtilisin (batch PPA 1619), to cause skin corrosion. The EpiDerm^TM test has been accepted as a replacement to the in vivo rabbit skin irritation test, by the European Centre for the Validation of Alternative Methods (ECVAM), and a draft proposal for a new OECD test guideline (TG 431) was followed. The study was conducted in accordance with GLP.

It was concluded that the test substance, subtilisin, was predicted as a non-corrosive to the skin.

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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

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 12.5% enzyme concentrate dry matter.

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.

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.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: Mean 24, 48, 72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: Mean 24, 48, 72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: Mean 24, 48, 72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

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.

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.

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 12.5% expressed as enzyme concentrate dry matter.

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.

Reference 4

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: see 'Remark'

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.

Qualifier:

according to guideline

Guideline:

other:

Deviations:

no

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

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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

IN-LIFE DATES: April 1977

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.

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

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: Mean of 24, 72 h (intact skin, only)

Score:

1

Max. score:

2

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:

0

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: Mean of 24, 72 h (intact skin, only)

Score:

1

Max. score:

2

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:

0

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: Mean of 24, 72 h (intact skin, only)

Score:

1

Max. score:

2

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:

0

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

other: Mean of 24, 72 h (intact skin, only)

Score:

1.5

Max. score:

3

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:

1

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:

2

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:

0

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

other: Mean of 24, 72 h (intact skin, only)

Score:

1.5

Max. score:

3

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:

0

Irritant / corrosive response data:

The two 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 edema. 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 edema recorded 24 and 72 hours (after the 24 hrs exposure started) did not exceed the limit values considered to indicate a significant inflammatory response.

Interpretation of results:

GHS criteria not met

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.

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.

Reference 5

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March 6-17, 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure was a human volunteer study according to Kligman and Wooding (1967). The study was performed before GLP or GCP was implemented but was performed according to state of the art at that time.

Qualifier:

according to guideline

Guideline:

other:

Deviations:

no

Principles of method if other than guideline:

Test procedure was according to Kligman AM and Wooding WM : A method for the measurement and evaluation of irritants of human skin, Journal of Investigative Dermatology, 1967; 49 : 78-94:
The cumulative irritation to the skin was measured by a patch-test technique on intact skin of the volar forearms of 10 subjects.
Under a square patch measuring 1 inch by 1 inch, 0.5 mL of the test substance was applied to the skin. The patches were secured in place by occlusive tape. Twenty-four hours after patch application, the test site was clinically evaluated and a new patch applied. The test was repeated for 10 days.

GLP compliance:

no

Remarks:

Before GLP or GCP but according to the same principles

Species:

human

Strain:

not specified

Details on test animals or test system and environmental conditions:

The study included 4 females and 6 males, age 18 - 45 yrs.
IN-LIFE DATES: from March 6 1978 to March 17, 1978

Type of coverage:

occlusive

Preparation of test site:

other: no preparation needed

Vehicle:

water

Amount / concentration applied:

0.5 mL of the test material, prepared as a 1% w/v solution in water.

Duration of treatment / exposure:

24 hours x 10

Observation period:

every 24 hours

Details on study design:

The cumulative irritation to the skin was measured by a patch-test technique on intact skin of the volar forearms of 10 subjects.
Under a square patch measuring 1 inch by 1 inch, 0.5 mL of a 1% solution in water of the test substance Subtilisin was applied to the skin. The patches were secured in place by occlusive tape. 24 hours after each patch application, the test site was clinically evaluated and a new patch applied. The test was repeated for 10 days.

Irritation parameter:

other:

Basis:

other:

Time point:

other: day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10

Score:

0

Max. score:

0

Remarks on result:

other: No human primary skin irritation at all

Irritant / corrosive response data:

Subtilisin (batch PFP 312/20) as a 1% solution in water did not give rise to any instances of primary irritation at all.

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the present test material (1% solution in water) would not present a risk of primary skin irritation for humans under normal use conditions.

Executive summary:

An in vivo human skin irritation test was conducted to assess the potential of the test substance, Subtilisin (batch FPF 312/20), to cause cumulative skin irritation. The procedure of Kligman AM and Wooding WM: A method for the measurement and evaluation of irritants of human skin, Journal of Investigative Dermatology, 1967; 49: 78-94, was followed. Under a square patch measuring 1 inch by 1 inch, 0.5 mL of the test substance was applied to the intact skin of the volar forearms of 10 subjects. The patches were secured in place by occlusive tape. 24 hours after each patch application, the test site was clinically evaluated and a new patch applied. The test was repeated for 10 days with daily readings. The study was conducted before GLP or GCP was implemented but the state of the art of the time of the study was followed. The study concluded that the present test material (1% subtilisin solution in water) did not present a risk of primary skin irritation for humans under normal use conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 24-27 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Centre Lagro, France
- Age at study initiation: young adults
- Weight at study initiation: 2008-2210 g
- Housing: induvidually in stainless steel cages with perforated floor
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23.9 C (target 17 - 23 C)
- Humidity (%): 30-70 %
- Air changes (per hr): ca 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark cycle

IN-LIFE DATES: From: 24 June 2002 To: 27 June 2002

Vehicle:

other: Tested neat

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): Undiluted test material

Observation period (in vivo):

The reactions were judged 1, 24, 48, 72 hours and at 7 days after treatment

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No washing

SCORING SYSTEM: According to OECD 405

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.7

Max. score:

1

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: Small haemorrhage on the nictitating membrane at 1h reading, cleared at 24h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: Small haemorrhage on the nictitating membrane at 1h reading, cleared at 24h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hrs

Remarks on result:

other: Discharge score 1 at 1 h, fully cleared at 24h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: Small haemorrhage on the nictitating membrane at 1h reading, cleared at 24h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 hrs

Remarks on result:

other: Discharge score 2 at 1 h, fully cleared at 24h

Irritant / corrosive response data:

At the 1 hour reading, Subtilisin generally caused slight or moderate redness and slight or moderate swelling of the conjunctivae and slight or moderate ocular discharge in the rabbits. In addition, all rabbits showed a small haemorrhage on the nictitating membrane. After 24 hours, slight to moderate redness and swelling was observed in all treated rabbits. After 48 hours only slight redness was observed and 72 hours after treatment, all eye effects had cleared completely.

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Conclusions:

According to EC-standards (as published in the Official Journal of the European Communities, L 110 A, volume 36, 4 May 1993), the test material is not irritating to eyes. However, due to the small haemorrhage on the nictitating membrane, the test material is regarded as a slight eye irritant.

Executive summary:

The study was carried out according to OECD guideline no. 405. Three rabbits were treated as follows: One rabbit was treated first and two more the following day. An amount of 0.1 ml of the undiluted test substance was instilled in the conjunctival cul-de-sac of one of the eyes of the rabbit. After administration, the upper and the lower eye lid were carefully closed and subsequently held together for at least one second before releasing to prevent loss of material. The other eye remained untreated and served as a control. The reactions of the eyes were judged at 1 h, 24 h, 48 h and 72 h after treatment.

At the 1 hour reading, Subtilisin generally caused slight or moderate redness and slight or moderate swelling of the conjunctivae and slight or moderate ocular discharge in the rabbits. In addition, all rabbits showed a small haemorrhage on the nictitating membrane. After 24 hours, slight to moderate redness and swelling was observed in all treated rabbits, while the haemorrhage on the nictitating membrane was no longer present. After 48 hours only slight redness was observed and 72 hours after treatment, all eye effects had cleared completely. According to EC-standards (as published in the Official Journal of the European Communities, L 110 A, volume 36, 4 May 1993), Subtilisin batch PPA 6865 is not irritating to eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study was performed according to GLP and the procedures were according to the later OECD guideline 438 (adopted 2009).

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 438 (adopted 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

other: Test material was applied neat

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The chicken eye cornea was treated with 0.03 mL
- Concentration (if solution): undiluted test sample

Duration of treatment / exposure:

The exposure period was 10 seconds

Observation period (in vivo):

The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment.

Details on study design:

Subtilisin was examined for potential irritation/corrosive properties in an ex vivo bioassay, the Chicken Enucleated Eye Test (CEET). Three main parameters were measured to disclose possible adverse effects, the corneal swelling, corneal opacity and fluorescein retention of damaged epithelial cells.
The study was carried out at the contract lab. TNO. Three enucleated chicken eyes per sample and one control per test run were selected for testing. At time t = 0, immediately after a zero reference measurement, the following procedure was applied for each test eye: the eye cornea was treated with 0.03 ml of the undiluted test sample. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 ml isotonic saline of ambient temperature. The control eye was treated with saline only. The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment. Fluorescein retention was only evaluated at ca 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
In addition, the eyes were collected for histopathological examination of the cornea.

Irritation parameter:

corneal swelling 

Value:

1

Irritation parameter:

cornea opacity score

Value:

0

Irritation parameter:

fluorescein retention score

Value:

0

Other effects / acceptance of results:

After treatment, the corneal thickness of the test eyes generally remained unchanged or only showed a very slight increase. None of the three eyes showed any corneal opacity or fluorescein retention. Microscopic examinations of the treated corneas did not reveal any additional information on possible adverse effects.

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the results obtained by slit-lamp examination and according to the EC classification system of the Chicken Enucleated Eye Test, it was concluded that the tested Subtilisin batch was not irritating to eyes.

Executive summary:

Subtilisin was examined for potential irritation/corrosive properties in an ex vivo bioassay, the Chicken Enucleated Eye Test.

Three main parameters were measured to disclose possible adverse effects, the corneal swelling, corneal opacity and fluorescein retention of damaged epithelial cells.

Three enucleated chicken eyes per sample and one control were selected for testing. The individual eye cornea was treated with 0.03 ml of the undiluted test sample. After an exposure period of 10 seconds, the corneal surface was rinsed with 20 ml isotonic saline. The control eye was treated with saline only. The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were carried out with the slit-lamp microscope.

In addition, the eyes were collected for histopathological examination of the cornea.

The results showed that after treatment, the corneal thickness of the test eyes generally remained unchanged or only showed a very slight increase. None of the three eyes showed any corneal opacity or fluorescein retention. Microscopic examinations of the treated corneas did not reveal any additional information on possible adverse effects.

According to the EC classification system of the Chicken Enucleated Eye Test, it was concluded that the tested Subtilisin batch was not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation potential of subtilisin has been tested in vitro and in vivo in rabbits and in the case of skin irritation, also in humans:

- In vitro EpiDerm™ Skin Model (EPI-200), the test compound Subtilisin was predicted as non-corrosive.

- In vivo skin irritation studies in rabbits: Two studies conclude Subtilisin to be slightly irritant to skin.

- An occluded in vivo human skin irritation test with daily 24-hour applications for 10 days and readings every day was performed. No primary skin irritation was seen.

The eye irritation potential of subtilisin has been tested in vitro and in vivo in rabbits:

- In in vitro enucleated chicken eye test, the test compound Subtilisin was predicted as non-corrosive.

- In in vivo eye irritation studies in rabbits, Subtilisin was in spite of highly reversible effects concluded to be slightly irritant due to a small hemorrhage on the nictitating membrane.

For an explanation why the listed classification of subtilisin in section 2.2 CLP classification is ‘serious eye damage, cat. 1 (H318)’, please read ‘Short description of key information’ above. 

The overall conclusion is that subtilisin due to the catalytic activity has the potential to cause slight skin and eye irritation. 

Effects on skin irritation/corrosion: slightly irritating

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

The skin and eye irritation potential of subtilisin has been tested in many studies (in vitro, in vivo in animals and in vivo skin irritation in humans) since the introduction of these products more than 40 years back. Representative studies have been selected for the present dossier and the overall conclusion is that subtilisin due to the catalytic activity has the potential to cause slight skin and eye irritation.

The eye irritation potential of subtilisin has been discussed in detail in the attached document.

For an explanation on, why the listed classification of subtilisin in section 2.2 CLP classification is ‘serious eye damage, cat. 1 (H318)’, please read ‘Short description of key information’ above.