Dibenzoyl-L-tartaric acid monohydrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05/11/2018 - 20/02/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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:

Dibenzoyl-L-tartaric acid monohydrate

EC Number:

220-374-0

EC Name:

Dibenzoyl-L-tartaric acid monohydrate

Cas Number:

2743-38-6

Molecular formula:

C18H14O8

IUPAC Name:

(2R,3R)-2,3-bis(benzoyloxy)butanedioic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

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, purity > 90%
- Lysine peptide (Ac-RFAAKAA-COOH): source RS Synthesis, LLC, purity > 90%
- 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) 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).

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 ± 0.05) 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 ± 0.05).
- 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 (to check the stability of the peptide over time)
- Reference Control C: acetonitrile (solvent used both for the test item and the positive control)
- 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 with autosampler, UV detector (200 nm), Zorbax SB 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 > 0.990 for both peptides; mean peptide concentration of Reference control A = 0.508 (cys) and 0.498 (lys) = 0.5 ± 0.005 mM.
- Analysis sequence: For each peptide, the analytical sequence included at least: one blank sample (peptide dilution buffer), one calibration curve injected at the beginning of the analytical batch, three reference control A samples, the co-elution control sample, three reference control B samples, reference control C samples (replicate 1), positive control sample (replicate 1), and test item study sample (replicate 1).

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 53.13 for lysine peptide and 74.43 for cysteine.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean / run # 1

Parameter:

other: cysteine depletion (%)

Value:

5.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: mean / run # 1

Parameter:

other: lysine depletion (%)

Value:

2.92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: mean / run # 2

Parameter:

other: cysteine depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: mean / run # 2

Parameter:

other: lysine depletion (%)

Value:

2.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- The test item showed a mean depletion of 5.14% for Lysine and 2.92% for Cysteine, i.e. an overall average of 4.03% reflecting no reactivity and thus a negative prediction of DPRA. However, as the mean percent depletion falls in the range 3% - 10% for the cysteine 1:10 / lysine 1:50 prediction model, a second run was considered. In the second run, the test item showed a mean depletion of 2.14% for Lysine and 0% for Cysteine, i.e. an overall average of 1.07% reflecting no reactivity and thus confirming the negative DPRA prediction.
- 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 (as recommended by the OECD TG).

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. First run: the mean peptide concentrations of the reference control C samples was 0.505 mM (lys) and 0.491 mM (cys), 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% (lys) and 1.09% (cys). Second run: the mean peptide concentrations of the reference control C samples was 0.539 mM (lys) and 0.486 mM (cys), 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 2.20% (lys) and 2.13% (cys).
- Acceptance criteria met for positive control: yes. First run: for cysteine peptide, the mean percent depletion value was 74.43%, within the acceptance range (60.8 - 100%); and for lysine peptide, the mean percent depletion value was 53.13%, within the acceptance range (40.2 - 69.4). Second run: for cysteine peptide, the mean percent depletion value was 68.97%, within the acceptance range (60.8 - 100%); and for lysine peptide, the mean percent depletion value was 55.44%, within the acceptance range (40.2 - 69.4).
- Acceptance criteria met for variability between replicate measurements: yes, the maximum SD for the test item replicates was < 11.6% for the percent lysine depletion value, and < 14.9% for the percent cysteine depletion in both runs.

Any other information on results incl. tables

Table 1. Run 1: Test item results.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	4.46	2.65
Repetition 2	5.21	1.99
Repetition 3	5.75	4.13
Mean	5.14	2.92
Mean depletion %	4.03
SD	0.65	1.10
SD Validity criteria	< 11.6%	< 14.9%

Table 2. Run 1: Positive controls.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	51.26	74.95
Repetition 2	54.80	74.88
Repetition 3	53.33	73.47
Mean	53.13	74.43
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

 

Table 3. Run 1: Reference controls.

	Lysine Peptide	Cysteine Peptide	Concentration validity criteria (mM)
	Concentration (mM)	Concentration (mM)
Reference A	0.501	0.487	0.500 ± 0.050
* Reference C	0.505	0.491

	CV %	CV %	CV validity criteria
Reference B/C	0.64	1.09	< 15 %

Table 4. Run 2: Test item.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	4.92	0
Repetition 2	1.51	0
Repetition 3	0	0
Mean	2.14	0
Mean depletion %	1.07
SD	2.52	0
SD Validity criteria	< 11.6%	< 14.9%

Table 5. Run 2: Positive controls.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	55.70	68.04
Repetition 2	55.19	69.01
Repetition 3	55.43	69.87
Mean	55.44	68.97
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

Table 6. Run 2: Reference controls.

	Lysine Peptide	Cysteine Peptide	Concentration validity criteria (mM)
	Concentration (mM)	Concentration (mM)
Reference A	0.529	0.496	0.500 ± 0.050
* Reference C	0.539	0.486

	CV %	CV %	CV validity criteria
Reference B/C	2.20	2.13	< 15 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria.

Conclusions:

The test item showed mean % depletion of 5.14% for Lysine and 2.92% for Cysteine in a first run and 2.14% for Lysine and 0% for Cysteine in a confirmatory run. Based on these results, the DPRA prediction was negative. The test item showed no sensitisation potential under test conditions.

Executive summary:

A Direct Peptide Reactivity Assay was 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). Under test conditions, no precipitation or co-elution was observed; all the validity criteria were met. In a first run, the test item showed a mean depletion of 5.14% for Lysine and 2.92% for Cysteine, i.e. an overall average of 4.03% reflecting no reactivity and thus a negative prediction of DPRA. However, as the mean percent depletion falls in the range 3% - 10% for the cysteine 1:10 / lysine 1:50 prediction model, a confirmatory run was considered. In the second run, the test item showed a mean depletion of 2.14% for Lysine and 0% for Cysteine, i.e. an overall average of 1.07% reflecting no reactivity and thus confirming the negative DPRA prediction. Based on the test results, the test item shows no sensitisation potential under test conditions.