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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 12, 2017 - December 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-4-(trifluoromethyl)benzoic acid
EC Number:
700-368-9
Cas Number:
328-90-5
Molecular formula:
C8H5F3O3
IUPAC Name:
2-hydroxy-4-(trifluoromethyl)benzoic acid
Test material form:
solid: particulate/powder
Details on test material:
Beige to brownish solid

In vitro test system

Test system:
human skin model
Remarks:
SkinEthicTM RHE
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
The human skin model, epidermis has been validated for corrosion testing and it can be use for skin corrosion test accordiing the OECD guideline 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): 17-RHE-127
- Delivery date: 12/12/2017
- Date of initiation of testing: 12/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1 °C for 60 minute exposure and room temperature for 3 minute exposure.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times in constant soft stream of 1 mL DPBS
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 uL of 1.00 mg/mL
- Incubation time: 180 min.
- Spectrophotometer: absorbance microplate reader
- Wavelength: 570 nm
- Filter: no

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Specification O.D. >0.7, Results: O.D.=1.1
- Barrier function: Using TRITON X-100 1%. Specification: 4.0 h<=ET50>=10.0 h. Results:4.5 h
- Morphology: Specification: number of cell layers >=4. Results: 6 cell layers (absence of significant histological abnormalities)
- Contamination: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Freeze killed tissues (for positive control)-Non-specific MTT reduction calculation (NSMTT)
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were prepared by incubating the viable tissues at -20 ± 5°C for 48 hours.
- N. of replicates: 2
- Method of calculation used: The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the positive minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same, calculated relative to the negative control run concurrently (%NSMTT). NSMTT was < 0% relative to the negative control, hence true MTT metabolic conversion of treated tissue was not determined.

- Live tissues (negative control and test item): Non-specific Color (NSC)
- N. of replicates : 2
- Method of calculation used: The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solutions minus the percent non specific color obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT.For positive control test item, %NSC was between 0 to 0.1 % relative to the negative control, hence TOD and relative viability calculation was not determined.

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

PREDICTION MODEL / DECISION CRITERIA
If the viability after 3 minutes exposure is strictly less than 50% or greater or equal to 50% and the viability after 1 hour exposure is strictly less than 15 %, test item has to be classified as CORROSIVE.

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 %, test item has to be classified as NON-CORROSIVE.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg ± 3 mg of test item/0.5 cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2 of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2 of 8N KOH
Duration of treatment / exposure:
2 exposure periods: 30 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours (incubation in MTT solution)
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
ca. 99.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
ca. 80.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: For adapted positive control treated tissues, NSMTT was < 0% relative to the negative control, therefore true MTT metabolic conversion of treated tissue is not determined.
- Colour interference with MTT: For adapted controls to correct colour interference due to test item, treated tissues were exposed for period of 3 minute at room temperature and 60 minutes at 37±1 °C and 5±1% CO2 in a 95% humidified incubator. %NSC was between 0 to 0.1% relative to the negative control, hence TOD and relative viability calculation were not determined.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes. It was performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Criteria: 0.8>=OD<=3.0. Results:2.271-2.404. Criteria met.
- Acceptance criteria met for positive control: Cell viability should be less than 15% according the guideline. The results: Cell viability =5.31%. Criteria met.
- Acceptance criteria met for variability between replicate measurements: The viability should not exceed 30% between tissue replicates. Viability was less than 1.46%.Criteria met.

Any other information on results incl. tables

Pre-Tests

Color Interference Test

Treatment

Optical Density (nm)

Interaction

Negative Control (Isopropanol)

0.043

No

0.049

4-Trifluoromethylsalicylic Acid (ATFMS)

0.257

Yes

0.261

Direct MTT Reduction Test

Treatment

Interaction

Negative Control (Maintenance medium)

No

4-Trifluoromethylsalicylic Acid (ATFMS)

No

Mean percent viability of the 4-trifluoromethylsalicylic acid (ATFMS)

Treatment

Viability

3 Minutes Exposure

60 Minutes Exposure

Negative control

(Sterile distilled water)

100%

100%

4-Trifluoromethylsalicylic Acid (ATFMS)

99.3%

80.7%

Positive control

(8N KOH)

-

5.31%

Individual results (positive and negative control)

Treatment

Exposure

Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean O.D. of Replicate tissues

% Viability/

Tissue

Mean % Viability

S.D. of % Viability

% C.V. of % Viability

Corrosivity Class

Negative Control

(Sterile Distilled water)

3 Minutes

1

2.352

2.309

2.294

2.328

100

100

NA

NA

NA

2.331

2.288

2.328

2.285

2

2.411

2.368

2.353

2.434

2.391

2.343

2.3

3

2.329

2.286

2.336

2.433

2.39

2.374

2.331

60 Minutes

1

2.408

2.365

2.306

2.324

100

100

NA

NA

2.324

2.281

2.314

2.271

2

2.335

2.292

2.299

2.332

2.289

2.360

2.317

3

2.447

2.404

2.368

2.342

2.299

2.444

2.401

Positive Control

(8N KOH)

60 Minutes

1

0.172

0.129

0.125

0.123

5.38

5.31

0.07

1.32

Corrosive

0.165

0.122

0.166

0.123

2

0.172

0.129

0.123

5.29

0.167

0.124

0.160

0.117

3

0.150

0.107

0.122

5.25

0.179

0.136

0.167

0.124

Individual results (test item)

Treatment

Exposure

Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean O.D. of Replicate tissues

% Viability/

Tissue

Mean % Viability

S.D. of % Viability

% C.V. of % Viability

Corrosivity Class

4-Trifluoromethylsalicylic acid (ATFMS)

3 Minutes

1

2.453

2.411

2.343

2.312

100.6

99.3

1.15

1.16

Non-corrosive

2.356

2.314

2.347

2.305

2

2.332

2.29

2.290

98.4

2.345

2.303

2.318

2.276

3

2.324

2.282

2.303

98.9

2.351

2.309

2.36

2.318

60 Minutes

1

1.923

1.881

1.908

1.877

82.1

80.7

1.18

1.46

1.98

1.938

1.948

1.906

2

1.899

1.857

1.862

80.1

1.895

1.853

1.918

1.876

3

1.882

1.84

1.86

80

1.901

1.859

1.922

1.88

Adapted Control forNon-Specific MTT Reduction (NSMTT)

Treatment

Exposure Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean OD of Replicate tissues

% NSMTT

Mean % NSMTT

TODTT

% Relative Viability

Negative Control

(Untreated killed tissues)

60 minutes

1

0.17

0.128

0.127

0.129

NA

NA

NA

NA

0.17

0.128

0.168

0.126

2

0.175

0.133

0.13

0.168

0.126

0.172

0.13

Positive Control

(Positive control treated killed tissues)

60 minutes

1

0.046

0.004

0.005

0.005

-5.3

-5.4

NA

NA

0.047

0.005

0.047

0.005

2

0.046

0.004

0.004

-5.4

0.045

0.003

0.046

0.004

Adapted Control for Non-Specific Color (NSC)

Treatment

Exposure Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean OD of Replicate tissues

% NSC

Mean % NSC

TODCT

% Relative Viability

Negative Control

 

3 minutes

1

0.043

0.001

0.001

0.001

NA

NA

NA

NA

0.044

0.002

0.042

0

2

0.043

0.001

0.000

0.041

-0.001

0.042

0

Negative Control

 

60 minutes

1

0.042

0

0.000

0.001

NA

NA

NA

NA

0.043

0.001

0.041

-0.001

2

0.043

0.001

0.001

0.042

0

0.043

0.001

4-Trifluoromethylsalicylic acid (ATFMS)

3 minutes

1

0.046

0.004

0.003

0.003

0.1

0.1

NA

NA

0.045

0.003

0.045

0.003

2

0.045

0.003

0.002

0.0

0.044

0.002

0.044

0.002

4-Trifluoromethylsalicylic acid (ATFMS)

60 minutes

1

0.045

0.003

0.002

0.002

0.0

0.0

NA

NA

0.043

0.001

0.043

0.001

2

0.043

0.001

0.001

0.0

0.044

0.002

0.043

0.001

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under experimental conditions, the test item was concluded to be non-corrosive in IN VITRO skin corrosion test using reconstructed human epidermins (RHE) tissues.
Executive summary:

An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis SkinEthic RHE model, according to OECD TG 431 (GLP study). The tissues were exposed to test item and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37±1 °C and 5±1% CO2. Since potassium hydroxide is direct MTT reducer, adapted control with two freeze killed tissues were used and treated with 8N KOH for exposure of 60 minutes. To evaluate the non-specific OD due to the residual test item staining, adapted control with two live tissues were used for test item and negative control for exposure of 3 minutes and 60 minutes. Percent relative viability in the tissues treated with the test item was 99.3% at 3 minute exposure period and 80.7% at 60 minute exposure period. Significant reduction in percent cell viability was not observed at the either 3 minute or 60 minute exposure period in the treated tissues when compared with the concurrent negative control. Differences between the viability of treated tissues was ≤ 1.46% i.e. %CV. OD values for negative control replicates were between 2.271 -2.404 and positive control showed 5.31 % cell viability. All criteria were valid. From the results of this study, the test item is concluded as non-corrosive using reconstructed human epidermins (RHE) tissues.

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