4-acetyl-2-methylbenzoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 06 December 2019, Experimental end: 18 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2019

Deviations:

no

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-acetyl-2-methylbenzoic acid

EC Number:

856-079-4

Cas Number:

55860-35-0

Molecular formula:

C10H10O3

IUPAC Name:

4-acetyl-2-methylbenzoic acid

Test material form:

solid: particulate/powder

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Solutions for cysteine reactivity assay
Stock solution - A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 20.1 mg of SPCC in 40.12 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
SPCC reference control solutions - Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN. In addition, a RCcysCMeOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMeOH sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL MeOH. A SPCC calibration curve was prepared.
Co-elution control, substance and positive control samples - co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.
Solutions for lysine reactivity assay
Stock solution - A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 20.6 mg of SPCL in 39.77 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
SPCL reference control solutions - Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCMeOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMeOH sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL MeOH. A SPCL peptide calibration curve was prepared.
Co-elution control, substance and positive control samples - co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared.
Sample incubation - After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 22.9 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours. Prior to HPLC analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.
HPLC analysis - SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
System 2 (used for Lysine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)

Vehicle / solvent:

other: Methanol

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 71.7% ± 0.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 65.2% ± 3.5%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

lysine depletion

Value:

3.3 %

At concentration:

0.469 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

lysine depletion

Value:

0.8 %

At concentration:

0.481 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

lysine depletion

Value:

0 %

At concentration:

0.485 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

cysteine depletion

Value:

0.6 %

At concentration:

0.502 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

cysteine depletion

Value:

0.2 %

At concentration:

0.504 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 6

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

cysteine depletion

Value:

0 %

At concentration:

0.505 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

All acceptability criteria for the cysteine and lysine reactivity assays were met confirming the suitability of the HPLC system and the stability of the HPLC runs over time.

Applicant's summary and conclusion

Interpretation of results:

other: Negative result is considered in a weight-of-evidence approach

Conclusions:

The substance was negative in the DPRA and was classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The reactivity of the substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was studied under GLP to OECD TG 442c. After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which assigns the test item to one of four reactivity classes used to support the discrimination between sensitizers and nonsensitizers. Methanol was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and CACN:MQ, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA. In the cysteine reactivity assay the test item showed 0.3% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.8% and as a result the test item was considered to be negative in the DPRA and classified in the “No or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.