Licheninase

Endpoint summary

Description of key information

The mean viability of the skin membranes was 98 ± 9% compared to the negative control group. Based on the results obtained in the present study, the test substance licheninase, batch PPB41588, was classified as non-irritant.

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.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2016 – 23 August 2016

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:

Adopted 28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate issued by Health Care Inspectorate of the Ministry of Health, The Netherlands.

Test system:

human skin model

Remarks:

EpiDerm™ reconstructed skin membranes

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

other: PBS

Details on test system:

SKIN DISC PREPARATION
- Procedure used: EpiDerm™ reconstructed skin membranes. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2).
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC and 5% CO2
- Temperature of post-treatment incubation (if applicable): Same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: each skin model was removed from the well, rinsed with PBS to remove the study substance (and mesh), blotted dry and transferred to a 6-well plate containing medium.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Yes, but not specified.
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
The in vitro skin irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
Mean tissue viability (% of negative control): ≤ 50 %, classification and labelling required, irritant or corrosive.
Mean tissue viability > 50 %, Non-irritant (No Category).

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate tissues each for test substance, negative control Phosphate Buffered Saline and positive control 5% Sodium Dodecyl Sulphate.
For test enzyme licheninase, one replicate was excluded due to a notably insufficient extraction of the MTT.

Type of coverage:

open

Preparation of test site:

other: Not necessary since it is a three-dimensional human skin model.

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): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (6.5% TOS)

Duration of treatment / exposure:

60 minutes

Details on study design:

TEST SITE
- Area of exposure: 0.64 cm2
- % coverage: 100%
- Type of wrap if used: Nylon mesh

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With PBS
- Time after start of exposure: Exposure time 60 min, post-exposure time approx. 42 h.

SCORING SYSTEM:
- Method of calculation: OD measurement with a spectrophotometer

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

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:
- Direct-MTT reduction: No reduction was observed.
- Colour interference with MTT: None observed.

The experiment was considered valid if:
- the OD of the viable negative control was ≥ 0.8 and ≤ 2.8.
- skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control.

The test group was considered valid if the standard deviation (SD) calculated from individual tissue viability percentages of the three replicates was ≤ 18%.
- Range of historical values if different from the ones specified in the test guideline: Although the data are not within the historical range (on the edge), they are within the criteria. Since the acceptance criteria were met, the experiment was considered valid.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of the skin membranes was 98 ± 9% compared to the negative control group. Based on the results obtained in the present study, the test substance licheninase, batch PPB41588, was classified as non-irritant.

Executive summary:

This study was performed to determine the in vitro skin irritation potential of licheninase, BatchPPB41588, using EpiDerm™ reconstructed skin membranes.

The skin membranes were topically exposed to the test substance for 60 min. Viability of the epidermal cells was assessed using the MTT test at ca. 42 h post-exposure. Negative control (PBS) and positive control (5% SDS) were run in parallel. The general principle for the detection of viability via the MTT test is the conversion of the yellow tetrazolium salt (MTT) to the blue/purple coloured product formazan by mitochondrial enzymes. The formation of formazan was measured using a spectrophotometer.

The acceptance criteria of the negative and positive control were met and therefore the study was considered valid.

The mean viability of the skin membranes was 98 ± 9% compared to the negative control group. One replicate was excluded due to a notably insufficient extraction of the MTT.

Based on the results obtained in the present study, the test substance licheninase, batch PPB41588, was classified as non-irritant (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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.

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin and eye irritation potential of licheninase has been tested according to OECD guidelines and in compliance with GLP. No skin or eye irritation was observed at any doses during the studies. The conclusion was that licheninase is neither a skin nor an eye irritant. 

Justification for classification or non-classification

The skin and eye irritation potential of licheninase has been tested according to OECD guidelines and in compliance with GLP. The conclusion was that licheninase did not exert any skin or eye irritation and therfore, licheninase should not be classifed.