Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-14 (experimental phase)
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 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:
OECD Guideline for Testing of Chemicals 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 (July, 2013).
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: B. 47. Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (Official Journal of the European Union, L 324, 9.12.2010).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature

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
- Characteristics of donor animals (e.g. age, sex, weight): at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Excess tissue was removed from the excised eyes. The 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 until 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
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin)

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): undiluted
Duration of treatment / exposure:
The incubation time lasted ten minutes.
Duration of post- treatment incubation (in vitro):
further two hours
Number of animals or in vitro replicates:
3 cornea for each test item, negative and positive control
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES
Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

NEGATIVE CONTROL USED
0.75 mL Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
0.75 mL 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, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted ten minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment.
- POST-EXPOSURE INCUBATION: Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). In the second step of the assay, permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0).
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Corneal permeability: 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.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. --> yes
Criteria for Determination of a Valid Test
The test will be acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result 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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value
Value:
2.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Not categorized
Irritation parameter:
in vitro irritation score
Run / experiment:
cornea 1
Value:
2.22
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
cornea 2
Value:
3.21
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
cornea 3
Value:
3.07
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Testing was performed via a GLP OECD 437 guideline study on the registered substance itself. It is hence sufficiently reliable to assess the eye irritating potential of the test item. The present in vitro method is recommended in a bottom up approach. The present in vitro method allows the identification of corrosive chemical substances and mixtures or of substances which do not require classification. The test item 3,5-Diethyltoluene-2,4-diisocyanate was tested undiluted. Relative to the negative control, the test item 3,5-Diethyltoluene-2,4-diisocyanate did not cause a relevant increase of the corneal opacity or permeability. The calculated mean IVIS was 2.83 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.
This test may hence serve as a stand-alone test, no further testing is required, and the test item does not need to be classified as eye irritant.
Executive summary:

This in vitro study was performed to assess the corneal damage potential of 3,5-Diethyltoluene-2,4-diisocyanate by means of the BCOP assay using fresh bovine corneae in a OECD 437 study under GLP.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability ofthe corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

 

Relative to the negative control, the test item 3,5-Diethyltoluene-2,4-diisocyanate did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 2.83. According to OECD 437 the test item is not categorized (GHS).