[(2,4-dibromophenoxy)methyl]oxirane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03. February - 18 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU B.40-BIS

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Cell source:

other: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

Details on animal used as source of test system:

no animals used as source of test system

Justification for test system used:

according to Guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have
been cultured to form a multi-layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers arranged in patterns analogous to
those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures
inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SCT
Day of delivery: 14. Feb. 2017
Batch: 23393

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not specified
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1°C and 5.0 ±0.5%
CO2, the inserts were removed from the plates using sterile forceps. The inserts were
thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective
holding plate, using the wells containing assay medium. After transfer of all inserts,
they were immediately moved to the wells containing MTT medium, blotting the bottom
with cellulose tissue again before setting the insert into the MTT well. The tissues were
incubated with MTT medium for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
After this time, the MTT medium was aspirated and replaced by DPBS. This was then aspirated,
too, and replaced several times. At last, each insert was thoroughly dried and set
into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted,
taking care to reach the upper rim of the insert. The plate was then covered with Parafilm®
and shaken on an orbital shaker for 2 hours at room temperature.
Then, the inserts were pierced with an injection needle, taking care that all colour was extracted.
The inserts were then discarded and the content of each well was thoroughly
mixed in order to achieve homogenisation.
From each well, three replicates with 200 μL solution (each) were pipetted into a 96-wellplate
which was read in a plate spectrophotometer at 570 nm.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
In the following table, the means of the negative controls and positive controls of all performed
experiments up to 19. Jan. 2017 are stated and compared with the values which
were found in this study.
Historical Data
Parameter Optical Density Optical Density % Tissue % Tissue
Negative Control Negative Control Viability Viability
Positive Control Positive Control
Time 3 min. 1 h 3 min. 1 h
Mean 1.966 1.911 25.1 % 12.2 %
Standard 0.280 0.224 7 % 3.9 %
Deviation
Range 1.197 - 3.077 1.377 - 2.571 9.6 - 57.3 % 4.1 - 24.2 %

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no direct interference detected in pre-tests

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 min and 1 h

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

>= 113.8 - <= 115.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corrosivity of the Test Item
The relative absorbance value was increased to 115.8% after 3 minutes treatment. This
value is above the threshold for corrosivity (50%). After 1 h treatment, the relative absorbance
value was increased to 113.8%, lying above the threshold for corrosivity (15%).
Therefore, the test item is considered as not corrosive to skin.
Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical
density was 1.7 (3 minutes) resp. 1.7 (1 hour).
The positive control showed clear corrosive effects. The criterion for the viability of the
1 h experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The
viability was 8.8 %.
Values for negative control and for positive control were within the range of historical data
of the test facility.
Therefore, the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

DENACOL EX-147 is considered as non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Executive summary:

One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with DENACOL EX-147 for

3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to

match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability

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

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

solution.

After treatment with the negative control, the absorbance values were within the required

acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing

the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.7 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals.

The relative absorbance value was reduced to 8.8 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the relative absorbance value was increased

to 115.8 %. This value is above the threshold for corrosion potential (50%). After 1 hour

treatment, relative absorbance value was increased to 113.8 %. This value, too, is above

the threshold for corrosion potential (15%). In the guideline, values greater or equal to the

threshold are considered as “non-corrosive to skin”.

Therefore, DENACOL EX-147 is considered as

non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.