Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
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:
adopted in Oct 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted in Feb 2017
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany

Test material

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH, Birkenfeld, Germany
- Characteristics of donor animals: The cattles were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 h.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes.
- Indication of any existing defects or lesions in ocular tissue samples: After the arrival of the corneas, they were examined and only corneas which were free from damages were used.
- Indication of any antibiotics used: 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL)

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL

NEGATIVE CONTROL
- Amount(s) applied: 750 µL

POSITIVE CONTROL
- Amount(s) applied: 750 µL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed complete Minimum Essential Medium (MEM, 32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1 °C.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 2 h

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Thorough rinsing with complete MEM with phenol red and final rinsing with complete MEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD492). The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
0.44
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
With the negative control (Hank’s Balanced Salt Solution) neither an increase of opacity nor permeability of the corneas could be observed (mean IVIS = 1.30).
The positive control (dimethylformamide) was tested undiluted and showed clear opacity and distinctive permeability of the corneas (mean IVIS = 110.48) corresponding to a classification as serious eye damaging (CLP/GHS (Cat 1)).
Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.44 (threshold for serious eye damage: IVIS > 55).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The values of mean opacity (0.63) and mean permeability (0.01) were within the range of historical data of the test facility (range of opacity: -1.17 – 2.63; range of permeability: -0.01 – 0.06).
- Acceptance criteria met for positive control: IVIS of the positive control was 110.48 and thus within the range of historical data of the test facility (IVIS: 36.23 - 150.41).

Any other information on results incl. tables

Table 1: Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter Negative Control Test Item Positive Control
  1. Replicate 2. Replicate 3. Replicate 1. Replicate 2. Replicate 3. Replicate 1. Replicate 2. Replicate 3. Replicate
1. Measurement 0.061 0.050 0.036 0.055 0.046 0.066 1.367 0.630 0.852
2. Measurement 0.061 0.048 0.031 0.054 0.044 0.066 1.411 0.647 0.881
3. Measurement 0.057 0.051 0.036 0.055 0.044 0.066 1.390 0.648 0.871
1. Measurement – blank 0.0243 0.0133 -0.0007 0.0183 0.0093 0.0293 13.303 0.5933 0.8153
2. Measurement – blank 0.0243 0.0113 -0.0057 0.0173 0.0073 0.0293 13.743 0.6103 0.8443
3. Measurement – blank 0.0203 0.0143 -0.0007 0.0183 0.0073 0.0293 13.533 0.6113 0.8343
Mean of each replicate 0.0230 0.0130 -0.0023 0.0180 0.0080 0.0293 13.527 0.6050 0.8313
Mean of the 3 replicates 0.0112 -- --
Corrected -- -- -- 0.0068 -0.0032 0.0181 13.414 0.5938 0.8201
Corrected mean of the 3 replicates -- 0.0072 0.9184

Table 2: IVIS Values

Test Group IVIS Mean IVIS Relative Standard Deviation IVIS
Negative Control 1.52 1.30 14.60%
1.17
1.21
Test Item 0.26 0.44 115.04%
1.01
0.04
Positive Control 133.11 110.48 21.85%
113.27
85.07

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of this test, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.44 in the in vitro Bovine Corneal Opacity and Permeability Test. Therefore, the test substance does not require classification for eye irritation or serious eye damage.
CLP: not classified