Licheninase

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

In vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2016 - 11 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted on 26 July 2013.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Licheninase

EC Number:

819-930-0

Cas Number:

37288-51-0

Molecular formula:

Not applicable, please see remarks

IUPAC Name:

Licheninase

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.: PPB41588
- Expiration date of the lot/batch: 05-07-2026
- Storage condition of test material: < -10⁰C

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age at study initiation: 7 weeks old
- Weight at study initiation: 1.5-2.5 kg
- Housing: The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The chicken eye cornea was treated with 30 µL.
- Concentration (if solution): undiluted test sample, 6.5% TOS.

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.

Number of animals or in vitro replicates:

3 eyes for the positive control and the test enzyme, and one eye for the negative control.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES:
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES: 1 eye for the negative control and 3 eyes for the negative and positive control, respectively.

NEGATIVE CONTROL USED: Physiological saline.

POSITIVE CONTROL USED: Benzalkonium Chloride (BAC) 5%

APPLICATION DOSE AND EXPOSURE TIME: undiluted test sample, 6.5% TOS for a treatment time of 30, 75, 120, 180 and 240 minutes.

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

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline.
- Indicate any deviation from test procedure in the Guideline: No deviation

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland
- Damage to epithelium based on fluorescein retention: Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland
- Swelling: Corneal thickness was measured using the Haag-Streit slit-lamp microscope, set at 0.095 mm.
- Macroscopic morphological damage to the surface: Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland
- Others (e.g, histopathology): The microscopic slides were not subjected to histopathological examination.

SCORING SYSTEM:
- Mean corneal swelling (%): 1%
- Mean maximum opacity score: 0.0
- Mean fluorescein retention score at 30 minutes post-treatment: 0.0

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No opacity or fluorescein retention were observed. Microscopic examination of the corneas did not reveal any abnormalities.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test has already been established in labs initially performing the ICE test, and does not need to be demonstrated again as mentioned in OECD guidelines.

ACCEPTANCE OF RESULTS:
The test was considered acceptable if the concurrent negative or vehicle/solvent and the concurrent positive controls are identified as UN-GHS Non-Classified and UN-GHS Category 1, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the results obtained by slit-lamp examination and applying the classification criteria of the Isolated Chicken Eye (ICE) test, it was concluded that the tested licheninase batch was not irritating to eyes.

Executive summary:

Licheninase was examined for potential irritation/corrosive properties in an ex vivo bioassay, the Isolated Chicken Eye (ICE) 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 30 µL of the undiluted test material. After an exposure period of 10 seconds, the corneal surface was rinsed with 20 mL isotonic saline. The control eye was treated with physiological saline only. The eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were carried out with a slit-lamp microscope. In addition, the eyes were collected for histopathological examination of the cornea.

Licheninase, batch PPB41588 only caused very slight swelling of the cornea (mean of 1%). No opacity or fluorescein retention were observed. Microscopic examination of the corneas did not reveal any abnormalities. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal

any abnormalities. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas revealed severe erosion and very slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis.

Applying the classification criteria of the ICE test, the following irritation classifications can be assigned:

Licheninase, batch PPB41588:

- NC: “Not Classified” (UN-GHS and EU-CLP classifications).