Arabinofuranosidase, α-l-

Endpoint summary

Description of key information

- This study was performed to determine the in vitro skin irritation potential of arabinofuranosidase, batch PPH40331 using EpiDerm™ reconstructed skin membranes. 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 104 ± 5 % compared to the negative control group. Based on the results obtained in the present study, the test substance was classified as non-irritant (UN GHS No Category).

- Arabinofuranosidase, batch PPH40331 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. it caused no significant corneal effects consisting of very slight corneal swelling (3%), no corneal opacity (mean score of 0.0) and no fluorescein retention (mean score of 0.0). Microscopic examination did not reveal any abnormalities.

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

Arabinofuranosidase, batch PPH40331:

- “Not Classified” (UN-GHS classification);

- “Not Classified” (EU-CLP classification).

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:

18-01-2016 to 01-03-2016

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)

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:

unchanged (no vehicle)

Details on test system:

SKIN 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 (10.7% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (10.7% TOS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (10.7% TOS)

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

about 43 hours.

Number of replicates:

Triplicate tissues each for test substance, negative control Phosphate Buffered Saline and positive control 5% Sodium Dodecyl Sulphate.

Type of coverage:

open

Preparation of test site:

other: Not necessary since they are reconstructed skin membranes

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 (10.7% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (10.7% TOS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (10.7% 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. 43 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: At the end of the incubation period of the test substance with a MTT solution, the MTT solution had turned blue/purple, indicating that the test substance was considered to have the potential to reduce MTT and consequently interfere with the MTT test. Therefore, frozen controls were included in the test.
- Colour interference with MTT: None observed.

The experiment was considered valid if
- the OD of the 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%.

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed to determine the in vitro skin irritation potential of arabinofuranosidase, batch PPH40331 using EpiDerm™ reconstructed skin membranes. 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 104 ± 5 % compared to the negative control group. Based on the results obtained in the present study, the test substance was classified as non-irritant (UN GHS No Category).

Executive summary:

This study was performed to determine the in vitro skin irritation potential of arabinofuranosidase, batch PPH40331 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. 43 h post exposure. Negative and positive controls 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 104 ± 5 % compared to the negative control group.

Based on the results obtained in the present study, the test substance 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:

13-01-2016 to 07-03-2016

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, 10.7% 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 positive control, respectively.

NEGATIVE CONTROL USED: Physiological saline 0.9%.

POSITIVE CONTROL USED: Benzalkonium Chloride (BAC) 5%

APPLICATION DOSE AND EXPOSURE TIME: Undiluted test sample, 10.7% TOS for a treatment time of 30, 75, 120, 180 and 240 minutes.

OBSERVATION PERIOD: The eyes were examined at 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): There were no histopathological findings.

SCORING SYSTEM:
- Mean corneal swelling (%): 3%
- 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

Run / experiment:

1

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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:

Arabinofuranosidase, batch PPH40331 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. it caused no significant corneal effects consisting of very slight corneal swelling (3%), no corneal opacity (mean score of 0.0) and no fluorescein retention (mean score of 0.0). Microscopic examination did not reveal any abnormalities.
Applying the classification criteria of the ICE, the following irritation classifications can be assigned:
Arabinofuranosidase, batch PPH40331:
- “Not Classified” (UN-GHS classification);
- “Not Classified” (EU-CLP classification).

Executive summary:

Arabinofuranosidase, batch PPH40331 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (BAC 5%). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 μL neat test material for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed. Arabinofuranosidase, batch PPH40331 caused no significant corneal effects consisting of very slight corneal swelling (3%), no corneal opacity (mean score of 0.0) and no fluorescein retention (mean score of 0.0). Microscopic examination did not reveal any abnormalities.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. Microscopic examination did not reveal any abnormalities.

The positive control eyes showed severe corneal effects and demonstrated the ICE test as valid to detect severe eye irritants. Microscopic examination revealed moderate or severe erosion, very slight, slight or moderate necrosis and slight vacuolation (one cornea; mid and low region) of the epithelium, and the epithelium partly detached from the basement membrane (one cornea).

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

Arabinofuranosidase, batch PPH40331:

- “Not Classified” (UN-GHS classification);

- “Not Classified” (EU-CLP classification).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Arabinofuranosidase is classified as a non-irritant.

Categories Display