Proteinase, Aspergillus acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

One animal used in previous eye irritation study as control. Deviation did not affect the outcome of the study as a 17-day washout period allowed prior to inclusion on this study. Also, similar reactions observed in all treated animals during the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

One animal used in previous eye irritation study as control. Deviation did not affect the outcome of the study as a 17-day washout period allowed prior to inclusion on this study. Also, similar reactions observed in all treated animals during the study.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Aspergillopepsin I (EC no. 232-796-2, CAS no. 9025-49-4, EC name: Proteinase, Aspergillus acid, Enzyme Class no. 3.4.23.18)

Molecular formula:

Not applicable, see remarks

IUPAC Name:

Active enzyme protein of Aspergillopepsin I (EC no. 232-796-2, CAS no. 9025-49-4, EC name: Proteinase, Aspergillus acid, Enzyme Class no. 3.4.23.18)

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. See remarks.

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.: AFP501K1

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, D-88353 Kisslegg, Germany
- Age at study initiation: not specified
- Weight at study initiation: 2.7 - 3.8 kg
- Housing: The animals were kept individually in PPO-plast cages (Noryl®, floor area: 4200 cm2) with perforated floor. The tray under the cage was cleaned 2 times a week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 46 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

Single application

Observation period (in vivo):

1, 24, 48, and 72 hours

Number of animals or in vitro replicates:

3 females

Details on study design:

REMOVAL OF TEST SUBSTANCE: No

TOOL USED TO ASSESS SCORE: Fluorescein stain was used after the 24-hour observation.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: mean of 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

iris score

Basis:

animal: mean of 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

chemosis score

Basis:

animal: mean of 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

conjunctivae score

Basis:

animal: mean of 3 animals

Time point:

24/48/72 h

Score:

0.1

Max. score:

3

Reversibility:

fully reversible

Irritant / corrosive response data:

At the examination 1 hour after exposure, conjunctiva appeared red (some blood vessels were definitely hyperaemic – equal to score 1) in the initial animal. This reaction persisted the following day (24 hours after exposure). However, it was considered ethically acceptable to proceed with 2 additional animals as only very mild reactions had been observed in the initial animal. Similar scoring of conjunctival redness was observed in the 2 additional animals at the examination 1 hour after exposure. At all other examinations, all animals appeared normal.

Other effects:

No clinical signs were observed during the daily observations.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Only slight signs of irritation were observed at 1 and 24 hours in one rabbit. All eyes were normal by 48 hours.

Executive summary:

The eye irritant effect of the test substance was investigated according to the method OECD Guideline 405. Three female albino rabbits were used in the study. Initially, one animal was exposed to 0.1 mL of the test substance in the left eye. The right eye remained untreated and served as control. Both eyes of all animals were examined and the changes were graded according to a numerical scale 1, 24, 48, and 72 hours after treatment.

 

No clinical signs were observed in the animals during the daily observations. At the examination 1 hour after exposure, conjunctiva appeared red (some blood vessels were definitely hyperaemic – equal to score 1) in the initial animals. The reaction persisted the following day (24 hours after exposure). However, it was considered ethically acceptable to proceed with two additional animals as only very mild reactions had been observed in the initial animal. Similar scoring of conjunctival redness was observed in the two additional animals at the examination 1 hour after exposure. At all examinations, all animals appeared normal. Mean values, based on the results from the 24, 48, and 72 hour readings were calculated. The mean values were 0.0, 0.0, 0.0, and 0.1 for cornea opacity, iris lesion, chemosis of conjunctiva, and redness of conjunctiva, respectively.