[(2,4-dibromophenoxy)methyl]oxirane

Endpoint summary

Description of key information

two in vitro Guideline studies performed for skin irritation/corrosion.

A conclusion can be drawn only by combination of both study results.

One in vitro Guideline study for eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EU B.40-BIS

GLP compliance:

yes (incl. QA statement)

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 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

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.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18. April - 14. August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

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 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: The test system is a commercially available EpiDermTM-Kit, procured by MatTek.

Details on animal used as source of test system:

no animal used as sources

Justification for test system used:

according to Guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
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 representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on spe-cially prepared cell culture inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Designation of the kit: EPI-212-SIT
Day of delivery: 25. Apr. 2017
Batch no.: 25809

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 a total incubation time of 42 hours and 30 minutes, a 24-well-plate was prepared with 300 μL freshly prepared MTT-medium in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incuba-tor 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 buffer. 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 shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, 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 thor-oughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Historical Data
Parameter Negative Control (OD) Positive Control (% OD compared to Negative Control)
Substance DPBS buffer Sodium Dodecyl Sulphate
Solution 5%
Mean 1.861 4.9%
Standard deviation 0.329 3.3%
Range 0.476 - 2.471 1.8 - 17.1%

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

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 or irritant to skin if viability is <= 50% of negative control
- The test substance is considered to be non-corrosive to skin if if viability is > 50% of negative control
- 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:

30 µL

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

23.5 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

>= 17.6 - <= 19.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Skin Irritation Potential of the Test Item
The relative absorbance value was reduced to 18.6% after the treatment. This value is below the threshold for skin irritation (50%). Therefore, the test item is considered as irri-tant to skin.

Validity and Acceptability
Validity criteria and results are stated in the following table:
Criterion Demanded Found
OD of negative control ≥ 0.8 and ≤ 2.8 1.7
% tissue viability of positive control SDS <= 20% of negative control 3.8%
SD of mean viability of the tissue replicates (%) ≤ 18% 1.0% (negative control)
0.2% (positive control)
0.9% (test item)
All validity criteria were met.
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.

Interpretation of results:

study cannot be used for classification

Conclusions:

DENACOL EX-147 is considered as irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.
The OECD guideline 439 addresses the human health endpoint skin irritation. Therefore further testing on skin corrosion potential with another suitable in vitro study (e.g. OECD 431) may be required.

Executive summary:

Three tissues of the human skin model EpiDermTM were treated with DENACOL EX-147 for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.7. The positive control showed clear irritating effects. Relative absorbance was reduced to 3.8% (required: <= 20%).

Variation within the tissue replicates was acceptable (required: ≤ 18%).

After the treatment with the test item, the relative absorbance value was reduced to 18.6%. This value is below the threshold for skin irritation potential (50%).

Therefore, DENACOL EX-147 is considered as irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

The OECD guideline 439 addresses the human health endpoint skin irritation. Therefore further testing on skin corrosion potential with another suitable in vitro study (e.g. OECD 431) may be required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available information for skin irritation/corrosion is conclusive and sufficient for classification as skin irritant cat. 2.

The avaiable information for eye irritation is conclusive but not sufficient for classification.