2-hydroxy-4-(trifluoromethyl)benzoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 25, 2018 - May 2, 2018

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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

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

Details on the study design:

TEST SYSTEM
The parental spontaneously immortalized human keratinocyte cell line (HaCaT) was developed through long-term culture of normal human adult skin keratinocytes at reduced calcium concentration and elevated temperature. The genetically modified HaCaT cell line (KeratinoSensTM) obtained from Givaudan Schweiz AG, was used in this study. KeratinoSensTM cell line contains a stable insertion of the luciferase gene under the transcriptional control of a constitutive SV40 promoter fused with an antioxidant response element (ARE) of the human AKR1C2 gene. KeratinoSens culture was maintained at the Mutagenicity section, Jai Research Foundation. The batch of the cell line was tested for mycoplasma contamination before using the cells in the experiment. Cultures free from any contamination were used in the study. KeratinoSensTM cell line passage number 21 (Experiment-1 and 2) and 24 (Experiment-3) were used in the study. Cell line with lot number JRF/HaCaT/2016/01 was used in the study.

CULTURE MEDIUM
The medium for culturing cells consists of D-MEM containing Glutamax supplemented with 9.1% fetal bovine serum and 500 μg/mL geneticin. The medium for exposure of cells with test item consists of D-MEM supplemented with 1% foetal bovine serum (495 mL D-MEM + 5 mL FBS).

CONTROLS
Concurrent positive and negative controls were included in each set of experiment.
Positive control: Trans cinnamaldehyde (stock concentrations from 0.4 to 6.4 mM were prepared in DMSO, which were further diluted to achieve final concentration of positive control ranging from 4 to 64 µM).
Negative control: DMSO

CULTURE VESSELS
Disposable tissue culture flasks of 75 cm2 culture (Nunc during main study experiment) area with vented neck were used to culture the cell line and the treatment was performed in 96-well white assay plates (for Luciferase assay) in triplicate and one 96-well transparent plates (for cytotoxicity assay).

PREPARATION OF CELL CULTURES
Cells were maintained in D-MEM containing Glutamax supplemented with 9.1 % fetal bovine serum and 500 μg/mL geneticin. After cells attained 80-90% confluency, they were washed twice with DPBS containing 0.05% of EDTA. To each flask 2-3 mL of 0.05% Trypsin-EDTA was added and flasks were incubated at 37 ± 1 oC (usually 5–10 minutes). After cells were detached completely, they were resuspended in DMEM with 9.1% FBS without geneticin. Cell count was taken and cell density was adjusted to 8 x 10E4 cells/mL. 125 µL of this culture (containing approx. 10,000 cells) was then dispensed in each well of 96 well plates, leaving one cell empty (to assess background values). Plates were incubated for 24 hours in 5 ± 1 % CO2 at 37 ± 1 ºC. Test item stock solution concentrations were between 19.53125 and 40000 µg/mL.

TEST ITEM EXPOSURE PROCEDURE
After 24 ± 2 hours of incubation, medium from all the 4 plates was aspirated and discarded. It was replaced with 150 µL of DMEM containing 1% FBS without geneticin. The 25 fold dilution of the 100X master plate was performed into a fresh plate (10 µL test solution + 240 µL of DMEM with 1% FBS without geneticin). Resulting 4X plate was further distributed to assay plates: 50 µL of this stock solutions were added 3 white assay plates and one cytotoxicity plate already containing 150 µL of culture medium. Final test concentrations used for the exposure were 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 µg for both the experiments. Plates were then covered with foil or petri-seal and incubated for 46 hours in 5 ± 1 % CO2 at 37 ± 1 °C.

LUCIFERASE ACTIVITY:
After 48 ± 2 hours of incubation, the medium from 96 well white assay plates was aspirated and discarded. Cells were washed once with 150 µL of DPBS. After washing, 20 µL of passive lysis buffer was added in each well. Cells were then incubated for 30 ± 10 minutes at 37 °C. After incubation, the plates with cell lysate were placed in luminometer. 50 µL of 1X Luciferase substrate was added and luciferase activity was recorded for 2 seconds.

CYTOTOXICITY EVALUATION:
Master mix was prepared by combining 27 µL of MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin. After 48 ± 2 hours of incubation, the medium from 96 well transparent plate was replaced with 227 µL above prepared master mix. The plate was covered with foil and incubated for 4 hours. After incubation, the medium was removed and 200 μL of a 10% SDS solution was added to each well. The plate was sealed and covered with a foil and placed in the incubator for overnight incubation. Following incubation the absorption at 570 nm was measured.

For the test item three experiments were conducted and out of these two valid experiments (experiment 1 and 3) were considered for final evaluation. Reason for conducting the repeating the experiments was due to failure of negative control (i.e., variation in 3*6 solvent control well was higher than 20%) to meet the acceptance criteria (experiment 2).

CALCULATIONS:
The following parameters were calculated in the KeratinoSensTM test method:
1. The maximal fold gene-induction (Imax) value observed at any concentration of the test item and positive control.
2. The EC1.5 value representing the concentration for which a gene induction above the 1.5-fold threshold (i.e., 50% enhanced gene activity).
3. The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Mean (run 1 and 3)

Parameter:

other: Imax

Value:

2.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Mean (run 1 and 3)

Parameter:

other: EC1.5 (µg/mL)

Value:

8.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: Mean (run 1 and 3)

Parameter:

other: IC50 (µg/mL)

Value:

306.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency of the facility in performing KeratinosensTM Assay was demonstrated by performing validation study (JRF Study N° 628-1-06-15806) .

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The coefficient of variation observed for the negative control during experiment 1 and 3 were 8.59% and 12.24%, respectively, which was below 20%. Since variation between the replicates was less than 20%, results of this run were considered as valid.
- Acceptance criteria met for positive control:
The gene induction for positive control was found to be >1.5 at concentrations of 32 µM and 64 µM in both the repetitions. The E.C1.5 value for positive control was found to be 35.84 µM and 12.79 µM in experiment 1 and 3, respectively. The average gene induction for positive control at 64 µM was found to be 1.93 and 6.31 for experiment 1 and 3, respectively. Clear dose response with increasing gene induction at increasing dose levels was observed in experiment 1 and experiment 3.
- Range of historical values if different from the ones specified in the test guideline:
Positive contro: Mean EC1.5 = 22.77 (SD ± 5.26) (Min 16.61 - Max. 32.12)
95% Control Limits: 12.24 - 33.30

Any other information on results incl. tables

The EC1.5 mean value was 8.80 µg/mL, which was less than 200 µg/mL. The value of maximum induction (Imax) was 1.76 and 1.68 at the tested concentration of 25 and 50 µg/mL in experiment 1 and 1.68, 1.86, 2.30, 2.39 and 1.94 at the tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively in experiment 3, which was higher than 1.5 fold. The cellular viability was 96.90 and 77.59 % at the test concentration of 25 and 50 µg/Ml in experiment 1 and 96.63, 95.70, 97.22, 89.27 and 87.52 % at the tested concentration of tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively, in experiment 3 with induction of luciferase activity above 1.5 fold. Observed dose response for luciferase induction was also statistically significant.

For the test item three experiments were conducted and out of these two valid experiments (experiment 1 and 3) were considered for final evaluation. Reason for conducting the repeating the experiments was due to failure of negative controls (i.e., variation in 3*6 solvent control well was higher than 20%) to meet the acceptance criteria (experiment 2).

Results of the present study indicate that, test item met all the evaluation criteria to conclude as sensitisers in KeratinoSens assay. Negative and positive controls met the acceptance criteria for the controls and were correctly identified as non-sensitiser and sensitiser, respectively. This showed the suitability of test system and procedures used in the test facility.

TEST ITEM:

Imax

Rep1	Rep3	Average
1.76*	2.39*	2.07

 

EC1.5

Rep1	Rep3	GeoMean
14.54µg/mL	5.32µg/mL	8.80µg/mL

 

IC50

Rep1	Rep3	GeoMean
316.31µg/mL	297.58µg/mL	306.80µg/mL

Keys: * = Significant induction was observed, Rep = Replicate/Experiment.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

From the results of this study, under the specified experimental conditions, test item was concluded as sensitisers in KeratinoSens assay.

Executive summary:

An In Vitro Skin Sensitization Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test was performed according to OECD Guideline 442D (GLP study). Test item was found to be soluble at 40 mg/mL in DMSO. Test item was tested in two independent experiments. KeratinosensTM (HaCaT) cells were exposed to test item between test concentrations of 400 µg to 0.1953125 µg and with positive control between 4 to 64 µM for 48 ± 2 hours in 5 + 1% CO2 at 37 ± 1 o C.  After incubation cells were analyzed for luciferase activity. Cell viability of the concurrently treated cells was also evaluated using MTT test with separate set of plate. Imax and EC1.5 values were calculated based on luciferase activity i.e., luminescence measured (reading of three plates) while IC50 was calculated based on results of cytotoxicity (OD values, reading of one plate). For positive control trans cinnamldehyde, EC1.5 value was found to be 21.41 µM, when run concurrently. The IC50 value for test item was found to be 306.80 µg. The EC1.5 mean values for test item was 8.80 µg/mL, which was less than 200 µg/mL. The value of maximum induction (Imax) for for test item was 1.76 and 1.68 at the tested concentration of 25 and 50 µg/mL in experiment 1 and 1.68, 1.86, 2.30, 2.39 and 1.94 at the tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively in experiment 3, which was higher than 1.5 fold. The cellular viability was 96.90 and 77.59 % at the test concentration of 25 and 50 µg in experiment 1 and 96.63, 95.70, 97.22, 89.27 and 87.52 % at the tested concentration of tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively, in experiment 3 with induction of luciferase activity above 1.5 fold. Observed dose response for luciferase induction was also statistically significant. All criteria for a valid study were met as described in the study plan. From the results of this study, under the specified experimental conditions, test item was concluded as sensitiser in KeratinoSens assay.