(6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acethyl]amino]-3-(hydroxymethyl)-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-carboxylic acid diphenylmethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 October 2017 - 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

OECD guideline No. 442C: in chemico skin sentitization: Direct Peptide Reactivity Assay (DPRA), adopted on 04 February 2015.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

04 February 2015

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:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

EC Number:

609-461-8

Cas Number:

376653-36-0

Molecular formula:

C44H36N6O6S2

IUPAC Name:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: BAL0001022
CAS number: 376653-36-0
Batch/Lot number: 14024R31D
Appearance: Off-white powder
Purity: 87.7%
Expiry date: 31 January 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤ -15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Specific details on test material used for the study:

Name: BAL0001022
Synonyms:
- BAL0001022-000
- (6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetyl]amino]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diphenylmethyl esther
CAS No.: 376653-36-0
Batch No.: 14024R31D
Storage conditions:
- In the freezer set at -20°C
- Protected from light
- Protected from humidity
- Under nitrogen atmosphere
Purity: 94.0%
Re-test date: January 2020

In chemico test system

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24 hour contact between the test item and synthetic cysteine and lysine peptides. A diluted solution of cysteine or lysine was incubated with the test item for 24 hours. At the end of the incubation, the concentration of residual peptides in the solution was evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percent depletion from the solution based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

Vehicle used: 1:1 mixture DMSO:acetonitrile
Positive control: Cinnamaldehyde (CAS No. 104-55-2), batch No. MKBX8146V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 98.9%.
Cysteine peptide:
- Peptide sequence: AC-RFAACAA-COOH
- Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
- Molecular weight: 750.88 g/mol
- Supplier: JPT Peptide Technologies GmbH
- Batch No.: 111016HS_MHeW0117
- Storage condition: at -20°C
- Description: white powder

Lysine peptide:
- Peptide sequence: AC-RFAAKAA-COOH
- Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
- Molecular weight: 775.91 g/mol
- Supplier: JPT Peptide Technologies GmbH
- Batch No.: 220114HSDWW0117
- Storage condition: at -20°C
- Description: white powder

Criteria: Interpretation of results

Results and discussion

Positive control results:

The acceptance criteria for the positive control satisfied the requirements of the assay.

In vitro / in chemico

Results

Run / experiment:

mean

Parameter:

other: % depletion

Vehicle controls validity:

valid

Remarks:

except for the reference control C, prepared with the cysteine peptide in a mixture of 1:1 mixture DMSO:acetonitrile, which was not within ± 10% of the nominal concentration.

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Due to the limited solubility of the test item, it was considered to have an inconclusive classification for peptide reactivity due to the presence of DMSO in test item formulation leading to inaccurate cysteine peptide % depletion determination.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r2) >= 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
* for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
* for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.

The test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Due to the limited solubility of the test item, under the experimental conditions of this study, the test item was considered to have an inconclusive classification for peptide reactivity due to the presence of DMSO in test item formulation leading to inaccurate cysteine peptide % depletion determination.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides as part of a tiered strategy for skin sensitization assessment.

 

Methods

The reactivity of the test item was evaluatedin chemico by monitoring peptide depletion following a 24‑hour contact between the test item and synthetic cysteine and lysine peptides. A diluted solution of cysteine or lysine was incubated with the test item for 24 hours. At the end of the incubation, the concentration of residual peptides in the solution was evaluated by HPLCwith Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion from the solution based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

 

The test item was dissolved at 100 mM in a1:1 mixture DMSO:acetonitrile.

 

The acceptance criteria for the calibration curve samples, the three reference controls A, B and C (prepared in acetonitrile) as well as positive controls satisfied the requirements of the assay.

However, the reference control C which was prepared with the cysteine peptide in a mixture of 1:1 mixture DMSO:acetonitrile was not within±10% of the nominal concentration and in fact the mean of the cysteine peptide concentration was lower, at 58.2% of the nominal concentration (i.e.0.291 mM instead of 0.500 ± 10% mM). Thus, the acceptance criteria were not fully met with this peptide. It was suggested that the solvent used (1:1 mixture DMSO:acetonitrile) has oxidant properties and therefore could have induced dimerisation of the cysteine peptide. Since the test item was only found soluble in this vehicle/solvent, it was agreed with the Sponsor to consider the solubility of the test item as a limit of this study and therefore that no conclusion could be reported.

 

Therefore, since the mean percent cysteine depletion could not be calculated due to the respective reference control C values with cysteine peptide (i.e. prepared in 1:1 mixture DMSO:acetonitrile which are out of the acceptance criteria) andsince the peptide reactivity could not be evaluated only based on lysine prediction model,no conclusion or classification can be done on the test item peptide reactivity.

Conclusion

Due to the limited solubility of the test item, under the experimental conditions of this study, the test item was considered to have aninconclusive classification for peptide reactivitydue to the presence of DMSO in test item formulation leading to inaccurate cysteine peptide % depletion determination.