Registration Dossier

Administrative data

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

Referenceopen allclose all

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:
other: Similar to the principles of OECD Test No. 431, 2010: In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
yes
Remarks:
Fresh MTT solution was prepared - no negative influence on the study outcome.
GLP compliance:
yes (incl. certificate)
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 EpiDermTMskin 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 EpiDermTMSkin irritation test was conducted to assess the potential of the test substance, Subtilisin (batch PPA 1619), to cause skin corrosion, by means of an in vitro test. The EpiDermTMtest 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.

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:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
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.

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 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.

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:
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 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. 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

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:
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.
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. Concentration of solution applied was 3.4% expressed as active enzyme protein.

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:
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:
other: White
Details on test animals 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. Concentration of solution applied was 0.14% expressed as active enzyme protein.
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. Twenty-four 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 is concluded that it is unlikely that the present test material (1% solution in water) would 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. Twenty-four hours after each patch application, the test site was clinically evaluated and a new patch applied. The test was repeated for 10 days with reading every day. 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 it is unlikely that the present test material (1% subtilisin solution in water, 0.14% expressed as active enzyme protein) would 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

Referenceopen allclose all

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:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
yes
Remarks:
the temperature exceeded the limit slightly - not judged to have any influence on the outcome.
GLP compliance:
yes (incl. certificate)
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 and 72 hours 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:
conjunctivae score
Basis:
animal #1
Time point:
other: mean 24-72h
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:
other: mean 24-72h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean 24-72h
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:
other: mean 24-72h
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:
other: mean 24-72h
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:
other: mean 24-72h
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.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting 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:
other: OECD 438 (adopted 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
fluorescein retention score
Value:
0
Irritant / corrosive response data:
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 testedin vitroandin vivoin rabbits and in the case of skin irritation, also in humans:

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

-In vivoskin irritation studies in rabbits: Two studies conclude Subtilisin to be slightly irritant to skin. Concentration of the enzyme in the formulation applied in the two studies was 4.5% respectively 3.4%, expressed as active enzyme protein.

- 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 haemorrhage on the nictitating membrane. Concentration of solution applied was 5.8% expressed as active enzyme protein.

According to REACH, the substance identification of enzymes is based on ”dry matter” of the enzyme concentrate, i.e. the following components: The active enzyme protein, other proteins, peptides and amino acids, carbohydrates, lipids and inorganic salts. However, when the focus is on skin and eye irritation, the active enzyme protein is the important component, characterized by the catalytic activity. The concentration of the solutions applied in the irritation studies is therefore expressed as active enzyme protein, typically as % w/w.

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. 

Justification for selection of skin irritation / corrosion endpoint:
Study is old but performed in human volunteers.

Justification for selection of eye irritation endpoint:
Key study selected as most relevant study of highest concern.

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 vivoanimal andin vivoskin 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 details in the attached document.

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.