[(2,4-dibromophenoxy)methyl]oxirane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02. February - 31. May 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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(2,4-dibromophenoxy)methyl]oxirane

EC Number:

243-606-2

EC Name:

[(2,4-dibromophenoxy)methyl]oxirane

Cas Number:

20217-01-0

Molecular formula:

C9H8Br2O2

IUPAC Name:

2-[(2,4-dibromophenoxy)methyl]oxirane

impurity 1

Reference substance name:

2,4-dibromophenol

EC Number:

210-436-5

EC Name:

2,4-dibromophenol

Cas Number:

615-58-7

Molecular formula:

C6H4Br2O

IUPAC Name:

2,4-dibromophenol

impurity 2

Reference substance name:

[(p-bromophenoxy)methyl]oxirane

EC Number:

218-656-3

EC Name:

[(p-bromophenoxy)methyl]oxirane

Cas Number:

2212-06-8

Molecular formula:

C9H9BrO2

IUPAC Name:

2-[(4-bromophenoxy)methyl]oxirane

impurity 3

Reference substance name:

[(2,4,6-tribromophenoxy)methyl]oxirane

EC Number:

226-140-4

EC Name:

[(2,4,6-tribromophenoxy)methyl]oxirane

Cas Number:

5296-40-2

Molecular formula:

C9H7Br3O2

IUPAC Name:

2-[(2,4,6-tribromophenoxy)methyl]oxirane

Test material form:

liquid

Details on test material:

sample used for testing,
no further details

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
CG272
- Expiration date of the lot/batch: 11. July 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
direct applicabion
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: direct application

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Specification
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been
cultured to form a stratified squamous epithelium similar to that found in the human cornea.
It consists of highly organized basal cells. These cells are not transformed or transfected
with genes to induce an extended life span. The EpiOcularTM tissues are cultured in
specially prepared cell culture inserts with a porous membrane through which nutrients
can pass to the cells. The tissue surface is 0.6 cm2.

Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories,
Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 21. Feb. 2017
Batch no.: 23767

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

1h

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

2

Details on study design:

according to Guideline

EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft
Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with
rounded values may lead to slightly different results.
Calculation
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100 % viability
To calculate the relative absorbance, the following equation was used:
% viability = [OD corrected of test item / OD corrected of mean negative control] 100%

Assessment of Eye Irritation
% Viability Assessment GHS classification
> 60 % Non eye irritant No GHS category
for eye irritation
≤ 60 % Eye irritant GHS category 1 or 2

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

1

Value:

>= 85.8 - <= 89.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity criteria and results are stated in the following table:
Criterion Demanded Found
OD of negative control > 0.8 and < 2.5 1.7
% viability of positive control < 50% of negative control 24.2%
Variation within replicates ≤ 20% 2.3% (negative control)
1.9% (positive control)
3.5% (test item)
Values for negative control and for positive control were within the range of historical data
of the test facility

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, DENACOL EX-147 is considered as
non-eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

One valid experiment was performed.

The test item DENACOL EX-147 was applied to a three-dimensional human cornea tissue

model in duplicate for an exposure time of 28 minutes.

50 μL of the liquid test item was applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of

the tissues was evaluated by addition of MTT, which can be reduced to formazan. The

formazan production was evaluated by measuring the optical density (OD) of the resulting

solution.

Demineralised water was used as negative control and methyl acetate was used as positive

control.

The controls showed the following results: After treatment with the negative control, the

absorbance values (OD was 1.7) were within the required acceptability criterion of mean

OD > 0.8 and < 2.5. The positive control showed clear eye irritating effects, the relative

absorbance value was reduced to 24.2 % (< 50%).

Variation within tissue replicates was acceptable (≤ 20%).

After treatment with the test item, the mean value of tissue viability was 87.6 %.

This value is well above the threshold for eye irritation potential (≤ 60%).

Under the conditions of the test, DENACOL EX-147 is considered as

non-eye irritant in the EpiOcularTM Eye Irritation Test.