[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01/02/2018 - 29/03/2018

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:

[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

EC Number:

262-811-8

EC Name:

[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Cas Number:

61477-96-1

Molecular formula:

C23H27N5O7S

IUPAC Name:

[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Test material form:

solid

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 JPT Peptide Technologies GmbH, lot no. 111016HS_MHeW1017
- Lysine peptide (Ac-RFAAKAA-COOH): source JPT Peptide Technologies GmbH, lot no. 120514HSDWW1017
- 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 99.1%, Sigma-Aldrich, lot no. MKCB9907).
- 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 (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: acetonitrile + 0.085% TFA
- mobile phase B: milli-Q water + 0.1% TFA
- 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 ≤ 13.98 % = No or minimal reactivity = Negative DPRA Prediction
- 13.98 % ≤ mean of cysteine and lysine % depletion ≤ 23.09 % = Low reactivity = Positive DPRA Prediction
- 23.09 % ≤ mean of cysteine and lysine % depletion ≤ 98.24 % = Moderate reactivity = Positive DPRA Prediction
- 98.24 % ≤ mean of cysteine and lysine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction

Results and discussion

Positive control results:

The depletion mean of cinnamaldehyde was 51.08 for lysine peptide and 76.99 for cysteine.

In vitro / in chemico

Results

Key result

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

1.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal peptide reactivity

Other effects / acceptance of results:

OTHER EFFECTS:
- Appearance of precipitate (if yes, if precipitate was re-solubilised or centrifuged): precipitate was observed in the positive control samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.
- Co-elution: analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with the cysteine peptide. However, the test item co-eluted with the lysine peptide. The % interference of this co elution could not be evaluated since several peaks were observed at the lysine peptide retention time and therefore no peak integration could have been performed. As a result, the peptide reactivity classification of the test item is only based on the cysteine 1:10 prediction model.

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 9 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 0.504 mM (cys) and 0.493 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 1.0% (cys) and 0.7% (lys).
- Acceptance criteria met for positive control: yes , for cysteine peptide, the mean percent depletion value was 93.69% (SD 0.22), within the acceptance range (60.8 - 100%, SD < 14.9%); and for lysine peptide, the mean percent depletion value was 59.85% (SD 0.86), 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 0.003 < 14.9% for the percent cysteine depletion value, below 15%. Percent lysine peptide depletion value could not be determined due to co-elution.

Any other information on results incl. tables

Table 1. Test item results: Percent peptide depletion.

Sample no.	Cysteine peptide		Lysine peptide		Depletion rate (%) of the test item	Depletion classification
	Peak area(μV/sec)	% depletion	Peak area(μV/sec)	% depletion
1	2684898	1.18	-	-
2	2680963	1.33	-	-
3	2698543	0.68	-	-
Mean	-	1.06	-	-	1.06	No reactivity / minimal reactivity
SD	-	0.34	-	-
CV	-	32.0	-	-

Table 2. Positive control results: percent peptide depletion.

Sample no.	Cysteine peptide		Lysine peptide		Depletion rate (%) of the test item	Depletion classification
	Peak area(μV/sec)	% depletion	Peak area(μV/sec)	% depletion
1	176093	93.52	366445	60.64
2	173530	93.61	372690	59.97
3	164812	93.93	382312	58.93
Mean	-	93.69	-	59.85	76.77	High reactivity
SD	-	0.22	-	0.86
CV	-	0.2	-	1.4

Table 3. Reference control A.

12/03/18 Sample no.	Cysteine peptide			Lysine peptide
	Area (μV/sec)	Concentration (mM)	%Dev	Area (μV/sec)	Concentration (mM)	%Dev
RC A.1	2737214	0.508	1.6	935028	0.496	-0.9
RC A.2	2732757	0.507	1.5	939950	0.498	-0.4
RC A.3	2735744	0.508	1.6	941904	0.499	-0.1
Mean	-	0.508	1.6	-	0.498	-0.5
SD	-	0.000	-	-	0.002	-
%CV	-	0.1	-	-	0.4	-

Table 4. Reference controls B, C.

12/03/18 Sample no.	Cysteine peptide	Lysine peptide
	Area (μV/sec)	Area (μV/sec)
RC B.1	2726083	936771
RC B.2	2716934	938181
RC B.3	2708476	938965
RC B.4	2667294	921984
RC B.5	2667733	932257
RC B.6	2656596	929823
RC C.1	2699563	937539
RC C.2	2725177	933797
RC C.3	2726258	921633
Mean	2699346	932328
SD	28181	6662
%CV	1.0	0.7

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed mean peptide depletion of 0.30% for Lysine, reflecting no or minimal reactivity. Therefore a negative prediction of 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). Under test conditions, the test item co-eluted with lysine, and the % interference could not be determined, so the peptide reactivity classification was only based on the cysteine 1:10 prediction model. The percent cysteine peptide depletion for the test item was 1.06, reflecting no or minimal reactivity, and thus a negative DPRA prediction. 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.