3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From July 9th, 2018 to July 12th, 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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

EURL ECVAM DB-ALM Protocol n.º 154

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:

3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

EC Number:

600-896-9

Cas Number:

109089-77-2

Molecular formula:

C17H18O3

IUPAC Name:

3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

Test material form:

solid: particulate/powder

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
- Cysteine peptide (Ac-RFAACAA-COOH): source RS Synthesis, LLC, lot no. 170622
- Lysine peptide (Ac-RFAAKAA-COOH): source RS Synthesis, LLC, lot no. 170705
- Solvent/vehicle: acetonitrile, based on the results of the preliminary solubility test.
- Preparation of test item stock solution: the test item was dissolved at 100 mM in acetonitrile without sonication.
- Preparation of test item samples for the reactivity with cysteine peptide: 50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5 ± 0.05) and 200 µL of acetonitrile.
- Preparation of test item samples for the reactivity with lysine peptide: 250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2 ± 0.05).

CONTROLS (preparation)
- Positive control: 100 mM cinnamaldehyde (purity ≥ 95%, Sigma-Aldrich, lot no. MKBT8955V).
- Positive control for cysteine peptide: 50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
- Positive control for lysine peptide: 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Reference controls: All the reference control samples were prepared in triplicate at the nominal concentration of 0.500 mM of peptide in the solvent specified below.
- Reference control A: acetonitrile (to check calibration curve accuracy)
- Reference Control B: acetonitrile (included at the beginning and at the end of the sequence, to check the stability of the peptide over time)
- Reference Control C: acetonitrile (solvent used both for the test item and the positive control, to check the influence of the test item solvent on the peptide stability)
- Co-elution controls: 100 mM test item in the appropriate buffer.
- Co-elution control (cys p.): 50 µL test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL acetonitrile.
- Co-elution control (lys p.): 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

HPLC ANALYSIS
- Equipment: HPLC system (Waters e2695) with autosampler, UV detector (200 nm; Waters 2489), Xbridge Peptide BEH C18 (100 x 2.1 mm; 3.5 µm) HPLC analytical column.
- Conditions: sample temperature 25ºC, column temperature 30ºC, injection volume: 7 µL(cys) or 3 µL(lys), flow rate 0.35 mL/min, total analysis time 20 min.
- mobile phase A: trifluoroacetic acid at 0.1% (v/v) in water
- mobile phase B: trifluoroacetic acid at 0.085% (v/v) in acetonitrile

- System suitability: calibration linearity: r2 > 0.9995 for both peptides; mean peptide concentration of Reference control A = 0.503 (cys) and 0.491 (lys) = 0.5 ± 0.005 mM.
- Analysis sequence: The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions). The reference controls C were placed at the beginning of each repetition. The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence. Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.

ACCEPTANCE CRITERIA
- For the positive control, the mean % peptide depletion value must fall within 60.8 - 100.0% (cys) and 40.2 - 69.4 (lys);
- For the positive control, SD (cys) < 14.9% and SD (lys) < 11.6%;
- For the reference controls, CV% of the peak areas for reference controls B, C must be < 15.0%;
- For the reference controls in the analysis sequence, the mean peptide concentration of Reference control C must be 0.5 ± 0.005 mM;
- For the test item replicates, SD (cys) < 14.9% and SD (lys) < 11.6%;

INTERPRETATION OF RESULTS: Cysteine 1:10-only prediction model.
- 0.00 % ≤ mean % depletion ≤ 6.38 % = No or minimal reactivity = Negative DPRA Prediction
- 6.38 % ≤ mean of cysteine and lysine % depletion ≤ 22.62 % = Low reactivity = Positive DPRA Prediction
- 22.62 % ≤ mean of cysteine and lysine % depletion ≤ 42.47 % = Moderate reactivity = Positive DPRA Prediction
- 42.47 % ≤ mean of cysteine and lysine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction

Results and discussion

Positive control results:

The depletion mean of cinnamaldehyde was 49.78 for lysine peptide and 78.17 for cysteine.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

1.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal peptide reactivity

Results 2

Key result

Run / experiment:

mean

Parameter:

other: lysine peptide depletion (%)

Value:

0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal reactivity

Results 3

Key result

Run / experiment:

mean

Parameter:

other: overall average

Value:

0.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Appearance of precipitate (if yes, if precipitate was re-solubilised or centrifuged): no precipitate was observed.
- Co-elution: analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with the cysteine or lysine peptides.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the expected DPRA prediction for the 10 proficiency substances was obtained. The resulted cysteine and lysine depletion values fall within the respective reference range for 8 out of the 10 proficiency substances for each peptide (8 out of 10 expected in OECD guideline).

ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied.
- Acceptance criteria met for reference controls: yes, the mean peptide concentrations of the reference control C samples was 0.506 mM (cys) and 0.482 mM (lys), within ± 10% of the nominal concentration; and the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile was 0.64% (cys) and 1.02% (lys).
- Acceptance criteria met for positive control: yes , for cysteine peptide, the mean percent depletion value was 78.17%, within the acceptance range (60.8 - 100%, SD < 14.9%); and for lysine peptide, the mean percent depletion value was 49.78%, within the acceptance range (40.2 - 69.4, SD < 11.6%).
- Acceptance criteria met for variability between replicate measurements: yes, the maximum SD for the test item replicates was 1.14 < 11.6% for the percent cysteine depletion value, and 0.20 < 14.9%.

Any other information on results incl. tables

Table 1. Test item results: Percent peptide depletion.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	0	1.54
Repetition 2	0.45	0.36		Mean Depletion %
Repetition 3	0.14	1.53
Mean	0.20	1.14		0.67

SD (Standard Deviation)	0.23	0.68
SD Validity criteria	< 11.6%	< 14.9%

Table 2. Positive control results: percent peptide depletion.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	46.11	77.34
Repetition 2	51.03	78.17
Repetition 3	52.20	79.00
Mean	49.78	78.17
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

Table 3. Reference controls.

Lysine Peptide

Cysteine Peptide

	Concentration (mM)	Concentration (mM)		Concentration validity criteria (mM)
Reference A	0.491	0.503		0.500 ± 0.050
Reference C	0.482	0.506

 

	CV %	CV %		CV validity criteria
Reference B/C	1.02	0.64		< 15 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item presents a Cysteine peptide depletion percentage of 1.14% and Lysine peptide depletion percentage of 0.20% i.e. an overall average of 0.67% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Executive summary:

A Direct Peptide Reactivity Assay has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item. The method was performed according to OECD OECD 442C, under GLP. The aim of the study is to evaluate the reactivity of the test item in chemico by quantifying the depletion of synthetic heptapeptides containing either lysine or cysteine.

A preliminary solubility study was conducted for the test item, and based on the results, the test item was prepared in acetonitrile. Peptide solutions were incubated with 100 mM test item solution or 100 mM cinnamic aldehyde solution (positive control), at ratios of 1:10 for cysteine and 1:50 for lysine. Reference controls and co-elution controls were run in parallel. After 24h incubation at 25ºC, the residual peptide concentrations were evaluated by HPLC-UV (220 nm). The test item presents a Cysteine peptide depletion percentage of 1.14% and Lysine peptide depletion percentage of 0.20% i.e. an overall average of 0.67% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. Based on the test results, the test item shows no sensitisation potential.