p-anisic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Nov - 02 Dec 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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted in 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Governement of the United Kindgom, Medicines & Healthcare products Regulatory Agency (MHRA)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

p-anisic acid

EC Number:

202-818-5

EC Name:

p-anisic acid

Cas Number:

100-09-4

Molecular formula:

C8H8O3

IUPAC Name:

4-methoxybenzoic acid

In chemico test system

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

TEST SYSTEM
- Supplier of synthetic peptides: Anaspec
- Peptide stock solution preparation: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
Peptide calibration standrad preparation: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM solution in acetonitrile. A 100 mM stock solution of the test item in acetonitrile/water 90/10 v/v was prepared.

CO-ELUTION CONTROL
- Co-elution controls were set up in parallel to sample preparation without respective peptide solution to verify whether a test chemical co-elutes with the cysteine or lysine peptide.

REFERENCE CONTROLS
Stability controls (Reference Control B and Reference Control C) and precision controls of both peptides were prepared at a concentration of 0.5 mM. Reference Control B samples and the precision control sample contained acetonitrile. The reference control C was prepared with propan-2-ol replacing acetonitrile.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: minimum of 22 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for test item and control C; control B samples were prepared in 6-fold

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Analytical Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Pre-column: Phenomenex, AJO-4286
- HPLC mobile phase:
A: 0.1% trifluoracetic acid in water
B: 0.085% trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Column temperature: 30 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20; MP A (%): 90, 75, 10, 10, 90, 90; MP B (%): 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Injection volume: 2 μL (slow draw rate)
- Retention time cysteine: approximately 11 minutes
- Retention time lysine: approximately 7 minutes

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The peptide depletion of the positive control for the cysteine peptide was 71.7%. The mean peptide depletion of the positive control for the lysine peptide was 58.8%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean cysteine depletion (%)

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

since the cysteine depletion is ≤ 6.38 the prediction for sensitisation is considered negative

Results 2

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean lysine depletion (%)

Value:

1.01

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

since the lysine depletion is ≤ 6.38 the prediction for sensitisation is considered negative

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the DPRA by successfully testing the 10 profiency chemicals outlined in the OECD 442C guideline

ACCEPTANCE CRITERIA:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent positve control value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine depletion,
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine depletion,
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is in the range of 0.45 to 0.55 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

Table 2. Overall achieved depletion values

Test item	Cysteine peptide depletion (%)	Lysine peptide depletion (%)	Overall mean depletion (%)	Reactivity class	DPRA prediction
p-anisic acic	1.40	1.01	1.20	No to minimal	Negative

Table 3. Cysteine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion (%)	Mean Depletion (%)	SD  (%)
Positive control	236103	105.70	71.5^2	71.7	0.08
	234831	105.13	71.6^2
	235271	105.33	71.6^2
p-anisic acic	821285	369.43	1.43^3	1.40	0.43
	825232	371.21	0.953^3
	818128	368.01	1.81^3

SD Standard Deviation

¹  Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²  Calculated against a mean Reference Control B area of 827610 µV.sec (n=6)

³  Calculated against a mean Reference Control C area of 833170 µV.sec (n=3)

Table 4. Lysine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion (%)	Mean Depletion (%)	SD  (%)
Positive control	317845	158.71	59.1^2	58.8	0.29
	322285	160.93	58.5^2
	319492	159.53	58.8^2
p-anisic acic	777172	388.56	-0.209^3	1.01	1.78
	751867	375.89	3.05^3
	774140	387.03	0.182^3

SD Standard Deviation

¹   Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²   Calculated against a mean Reference Control B area of 776330 µV.sec (n=6)

³   Calculated against a mean Reference Control C area of 775550 µV.sec (n=3)

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event "direct peptide binding" of skin sensitisation AOP

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is not predicted a sensitiser in the Direct Peptide Binding Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.