Subtilisin

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Subtilisin

EC Number:

232-752-2

EC Name:

Subtilisin

Cas Number:

9014-01-1

Molecular formula:

Not available.

IUPAC Name:

Subtilisin

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

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 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates 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:

liquid

Details on test material:

- Lot/batch No.: PPA 6865
- Expiration date of the lot/batch: May 14 2004

Test animals / tissue source

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

Test system

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

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 6

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results 7

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

Results 8

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

Results 9

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

Results 10

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

Results 11

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

Results 12

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.

Applicant's summary and conclusion

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.