Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August - 10 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
09 October 2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No 1152/2010
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A - test item applied undiluted.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A - test item applied undiluted.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A - test item applied as supplied.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS:

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): Cattle were ≥ 9 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: Same day testing.
- indication of any existing defects or lesions in ocular tissue samples: Only cornea free of defects were used in the test.
- Indication of any antibiotics used: HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) during storage and shipping of isolated eyes.

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): 0.75 mL
- Concentration (if solution): N/A - test item applied undiluted.

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A - vehicle not used.
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
10 mins ± 30 seconds
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
120 mins - for second opacity measurement (defined as t130).
A further 90 mins - for permeability determination.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O- ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0). At the end of the incubation period, the basal opacity was determined (t0).

QUALITY CHECK OF THE ISOLATED CORNEAS : Only corneae with a value of the basal opacity < 7 were used.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes (n=3), Saline (0.9% NaCl in deionised water)

SOLVENT CONTROL USED (if applicable) : N/A

POSITIVE CONTROL USED : Yes (n=3), 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME : The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes (± 30 seconds).

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: For opacity measurements - After the test item or control items, respectively, were rinsed off from the application side with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium is free of test item the corneas are given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).

For permeability determination - Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least 3 times.
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: To determine light transmission through tthe cornea, an opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was used.
- Corneal permeability: The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490).
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean IVIS (n=3)
Value:
0.04
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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, values of 65 studies with liquid test items sharing 37 sets of controls, performed between January 2016 and July 2018 reported.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Tthe negative control responses resulted in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: The positive control indicated an IVIS that falls within two standard deviations of the current historical mean (updated every three months).
- Range of historical values if different from the ones specified in the test guideline: N/A

Any other information on results incl. tables

Table 1       IVIS scores

 

Test Group

Rep.

Opacity value

(t130– t0)

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Negative control

1

0.00

0.056

0.84

0.97

2

0.00

0.062

0.93

3

0.00

0.075

1.13

Mean

0.00

0.064

-

Positive control

1

89.00

0.697

99.45

102.35

2

88.00

0.611

97.16

3

101.00

0630

110.45

Test item

1

0.00

-0.002

-0.04

0.04

2

0.00

-0.002

-0.04

3

0.00

0.013

0.19

*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the current study and under the experimental conditions reported, the test item does not meet the GHS classification criteria.
Executive summary:

OECD 437 (2018) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using the test item in accordance with OECD Guideline 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared. Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 30 min incubation in the vertical position with sodium fluorescein. Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490).

The quality criteria required for acceptance of the results were met.  The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)).

The mean corrected opacity reading and permeability readings for the test item were 0.00 and 0.003, resulting in an In Vitro Irritation Score (IVIS) of 0.04.

According to the current study and under the conditions of the experimental conditions reported, the test item does not meet the criteria categorisation in accordance with UN GHS and EU CLP Regulation (EC) No. 1272/2008