4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2019 to 9 December 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

EC Number:

850-929-8

Cas Number:

1584-79-8

Molecular formula:

C15H13F3N2O3S

IUPAC Name:

4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

Test material form:

solid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

16.09 mg of the cysteine peptide was weighed, and 30.4 mL of 100 mM phosphate buffer (pH 7.5) was added to prepare 0.667 mM cysteine peptide standard stock solution. The 0.667 mM standard stock solutions were diluted with acetonitrile to prepare 0.534 mM standard solution. The 0.534 mM standard solution was serially diluted with the solution of phosphate buffer/acetonitrile (8/2 v/v) to prepare 0.267, 0.134, 0.0667, 0.0334 and 0.0167 mM standard solution.

16.96 mg of the lysine peptide was weighed, and 31.0 mL of 100 mM ammonium acetate buffer (pH 10.2) was added to prepare 0.667 mM lysine peptide standard stock solution. The 0.667 mM standard stock solution was diluted with acetonitrile to prepare 0.534 mM standard solution. The 0.534 mM standard solution was serially diluted with the solution of ammonium acetate buffer/acetonitrile (8/2 v/v) to prepare 0.267, 0.134, 0.0667, 0.0334 and 0.0167 mM standard solution.

Positive Control substance solution:
Cinnamaldehyde (26.27 mg for cysteine peptide; 24.65 mg for lysine peptide) was weighed and dissolved to 2 mL of acetonitrile to prepare 100 mM positive control substance solution.

Preparation of test substance solution:
On the experiment day, test substance (68.82 mg for cysteine peptide; 68.62 mg for lysine peptide) was weighed and dissolved to 2 mL of acetonitrile to prepare 100 mM test substance solution.

Creation of calibration curve
Standard solutions of each peptide and the solution of each buffer/acetonitrile (8/2 v/v) were analyzed according to the condition described in 14.2 and a calibration curve for each peptide was made by the concentration of standard solution and the peak area. The coefficients of determination (r2) of both cysteine peptide and lysine peptide were 1.000, which satisfied the acceptance criteria. The concentrations for each peptide described below were calculated by the calibration curves.

Verification of the suitability
For each peptide, 750 μL of the 0.667 mM standard stock solution was mixed with 250 μL of acetonitrile to prepare reference control A (n=3). Reference control A was analyzed. Consequently, the mean concentrations of
reference control A were 0.514 and 0.500 mM for cysteine peptide and lysine peptide, respectively, which satisfied the acceptance criteria.

Verification of retention time of test substance
Cysteine peptide:
To prepare co-elution control, 750 μL of the phosphate buffer was mixed with 200 μL of acetonitrile, and then mixed with 50 μL of the test substance solution prepared in the examination of the solvent. The co-elution control was left at 25°C for 24 hours, and then analyzed. Consequently, no peaks derived from the test substance were detected at the retention time of the peptide.

Lysine peptide
To prepare co-elution control, 750 μL of the ammonium acetate buffer was mixed with 250 μL of the lest substance solution prepared in the examination of the solvent. The coelution control was left at 25°C for 24 hours or more, and then analyzed. Consequently, no peaks derived from the test substance
were detected at the retention time of the peptide.

On the experiment day, 100mM test substance solution (68,82 mg for cysteine peptide and 68.62 mg for lysine peptide) was prepared. Peptide depletion was using HPLC. A calibration curve using the standard solutions for both peptides and suitability of the test method was verified with a reference control (A) for each peptide.

Preparation of reference control B and C, and each reaction solution
For each peptide, reference control B (n=6), reference control C (n=3), positive control reaction solution (n=3) and test substance reaction solution (n=3) were prepared and left at 25°C.

Preparation of reference control B
For each peptide, 750 μL of the 0.667 mM standard stock solution was mixed with 250 μL of acetonitrile to prepare reference control B.

Preparation of reference control C
For each peptide, 750 μL of the 0.667 mM standard stock solution was mixed with 250μL of acetonitrile to prepare reference control C.

Preparation of positive control reaction solution
1) Cysteine peptide
To prepare positive control reaction solution, 750 μL of the 0.667 mM standard stock solution was mixed with 200 μL of acetonitrile, and then mixed with 50 μL of the positive control substance solution.
2) Lysine peptide
To prepare positive control reaction solution, 750 μL of the 0.667 mM standard stock solution was mixed with 250 μL of the positive control substance solution.

Preparation of test substance reaction solution
1) Cysteine peptide
To prepare test substance reaction solution, 750 μL of the 0.667 mM standard stock solution was mixed with 200 μL of acetonitrile, and then mixed with 50 μL of the test substance solution.
Test substance reaction solution was visually inspected immediately after the preparation and 23 hours after the preparation. Consequently, no suspension or precipitation were observed.
2) Lysine peptide
To prepare test substance reaction solution, 750 μL of the 0.667 mM standard stock solution was mixed with 250 μL ofthe test substance solution.
Test substance reaction solution was visually inspected immediately a:fter the preparation and 23 hours after the preparation. Consequently, no suspension or precipitation were observed.
Analysis of reference control B and C, and each reaction solution
For each peptide, the reference control B and C, positive control reaction solution and test substance reaction solution were analyzed.
The analysis of the positive control reaction solution and test substance reaction solution was conducted 24 hours after the preparation or after.
Consequently, the coefficients of variation (CV) of the peak area of the reference control B and C were 1.9% and 0.3% for cysteine peptide and lysine peptide, respectively, which satisfied the acceptance criteria . The mean concentrations of the reference control C were 0.453 mM and 0.501 mM for cysteine peptide and lysine peptide, respectively, which satisfied the acceptance criteria.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

Peptide depletion (percent cysteine peptide): 71.5 % (SD 0.4)
Peptide depletion (percent lysine peptide): 55.8 % (SD 0.7)

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

mean lysine depletion

Value:

0 %

At concentration:

100 mM

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

2 %

At concentration:

100 mM

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean value of the percent cystein and lysine depletion was 1.0% the reactivity class of the test substance was classified to "No or Minimal reactivity" and the skin sensitivity was predicted as "negative".

Executive summary:

The mean value of the percent cystein and lysine depletion was 1.0% the reactivity class of the test substance was classified to "No or Minimal reactivity" and the skin sensitivity was predicted as "negative".