benzhydryl (6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-trityloxyiminoacetyl]amino]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

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-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

EC Number:

609-462-3

Cas Number:

376653-37-1

Molecular formula:

C44H34N6O6S2

IUPAC Name:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-formyl-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: BAL0001024
CAS number: 376653-37-1
Batch/Lot number: 013
Appearance: Brown powder
Purity: 63.1% (content by qNMR)
Expiry date: 31 May 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: BAL0001024
Synonyms:
- BAL0001024-000
- (6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenyl methoxy) imino]acetyl]amino]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diphanylmethyl ester
CAS No.: 376653-37-1
Batch No.: 012
Description: brown powder
Storage conditions:
- In the freezer set at -20°C
- Protected from light
- Protected from humidity
- Under nitrogen atmosphere
Purity: 66.0%
Re-test date: May 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: 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

Value:

62.84

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The test item was considered to have a high peptide reactivity and therefore the test item is considered to be positive in the DPRA assay.

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:

other: positive in the DPRA assay

Conclusions:

Under the experimental conditions of this study, the test item was considered to have a high peptide reactivity and therefore the test item is considered to be positive in the DPRA assay.

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 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 basedon 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 inacetonitrile without sonication.

 

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples satisfied the requirements of the assay. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

. for the cysteine peptide, the mean depletion value was 98.91% which was linked to high cysteine dimerisation,

. for the lysine peptide, the mean depletion value was 26.78%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 62.84%. Accordingly, the test item was considered tohave ahigh peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

 

Conclusion

Under the experimental conditions of this study, the test item was considered to have ahighpeptidereactivity and therefore the test item is considered to be positive in the DPRA assay.