2-hydroxy-4-(trifluoromethyl)benzoic acid

Skin irritation / corrosion

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

Test material information

Constituent 1

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

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 99.3

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

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.