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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-11-2016 to 10-01-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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

Constituent 1
Reference substance name:
Nuclease, deoxyribo-
EC Number:
232-667-0
EC Name:
Nuclease, deoxyribo-
Cas Number:
9003-98-9
Molecular formula:
n.a.
IUPAC Name:
Nuclease, deoxyribo-
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
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPW42035
- Expiration date of the lot/batch: No specific expiry date. At least 10 years or as long as enzyme activity is preserved.
- Storage condition of test material: Refrigerated in the dark.

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, 7.2% 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: Isotonic saline.

POSITIVE CONTROL USED: Benzalkonium Chloride (BAC) 5%

APPLICATION DOSE AND EXPOSURE TIME: undiluted test sample, 7.2% 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 (%): 2%
- 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
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 starting 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 ICE, it was concluded that the tested deoxyribonuclease batch was not irritating to eyes.
Executive summary:

Deoxyribonuclease, batch PPW42035 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 for 10 seconds followed by a 20 mL saline rinse. The following 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.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Deoxyribonuclease, batch PPW42035 only caused very slight swelling of the cornea (mean of 2%). No opacity or fluorescein retention were observed. Microscopic examination of the corneas did not reveal any abnormalities.

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

Deoxyribonuclease, batch PPW42035:

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

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