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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2011 to 16 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 431 guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 431 In Vitro Skin Corrosion: Human Skin Model Test and Method B.40 BIS of Commission Regulation EC) No 440/2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Molecular formula (if other than submission substance): C12H12Cl2O2
- Molecular weight (if other than submission substance): 259.1
- Physical state: Brown powder
- Storage condition of test material: <30°C

Test animals

Species:
other: EpiDerm tissues (three-dimentional human skin model
Details on test animals and environmental conditions:
No test animals were used.
EpiDerm tissues were supplied by MatTek Corporation, Ashland, Massachusetts, USA and stored refrigerated. Tissues were transferred to 6-well plates with the assay medium 90 minutes before starting the assay.

Test system

Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 mg
- Concentration (if solution): Undiluted test substance
Duration of treatment / exposure:
Contact time of 3 minutes or 1 hour
Observation period:
Incubated at 37°C for 3 hours
Number of animals:
No animals were used on this test.
Four EpiDerm tissues were used per test substance, negative and positive control
Details on study design:
APPLICATION OF TEST AND CONTROL MATERIALS:
Immediately prior to treatment being initiated the assay medium was replaced.
The test was performed on a total of four tissues per test article, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. Approximately 40 mg solid was placed onto the tissue and moistened with 25 µL water to ensure good contact with the tissue surface. Further tissues were treated with 40 µL distilled water (negative control) and with 40 µL 8N potassium hydroxide (positive control).
After the 3-minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material.

CELL VIABILITY MEASUREMENTS:
The assay medium was replaced with 300 µL of 1 mg/mL MTT-medium and tissues were incubated for 3 hours at 37°C. After incubation, the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight, without shaking.
The optical density of the extracted formazan was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
99
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. Reversibility: no data Not measured. (migrated information)
Irritation / corrosion parameter:
other:
Value:
140
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hour. Reversibility: no data Not measured. (migrated information)
Other effects / acceptance of results:
The OD values obtained for each test sample were used to calculate the percentage viability relative to the negative control, which is arbitrarily set at 100%. The prediction of corrosivity associated with the EpiDermTM model is:
1. The test substance is considered to be corrosive to skin if the viability after a three minute exposure is less than 50%, or if the viability after three minutes exposure is greater than or equal to 50% and the viability after one hour is less than 15%.
2. The test substance is considered to be non-corrosive to skin if the viability after a three minute exposure is greater than or equal to 50% and the viability after a one hour exposure is greater than or equal to 15%.
For the three minute treatment, no significant differences in tissue viability were observed following test article treatment relative to the negative control. The relative survival of tissues treated with the test article was 99% when compared to the negative control.
For the one hour treatment, no significant differences in tissue viability were observed following test article treatment relative to the negative control. The relative survival of tissues treated with the test article was 140% when compared to the negative control.
No other test substance related effects were observed.

Any other information on results incl. tables

Skin Corrosion Assay; Cell Viability Measurements

        3 minute treatment

Substance

Tissue replicate

OD570

Mean

Tissue mean

% difference between Rep’s

% Relative survival

Aliquot 1

Aliquot 2

Aliquot 3

Negative control

A

1.985

1.958

1.738

1.894

1.797

-11.3

100

B

1.693

1.716

1.695

1.701

Test article

A

1.766

1.778

1.774

1.773

1.771

-0.2

99

B

1.776

1.762

1.768

1.769

Positive control

A

0.340

0.341

0.341

0.340

0.340

0.5

19

B

0.337

0.343

0.337

0.339

 

        1 hour treatment

Substance

Tissue replicate

OD570

Mean

Tissue mean

% difference between Rep’s

% Relative survival

Aliquot 1

Aliquot 2

Aliquot 3

Negative control

A

1.732

1.725

1.697

1.718

1.641

-9.8

100

B

1.469

1.608

1.615

1.564

Test article

A

2.365

2.408

2.338

2.370

2.306

-5.8

140

B

2.212

2.257

2.254

2.241

Positive control

A

0.235

0.244

0.243

0.241

0.259

-15.0

16

B

0.273

0.279

0.279

0.277

 

Applicant's summary and conclusion

Interpretation of results:
other: Not corrosive
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
The test substance was considered not to be corrosive to the in vitro skin testing model, EpiDerm.
Executive summary:

The result indicates that the substance should be non-corrosive to skin. Without further in vivo data the result has to be considered as inconclusive regarding classification.