2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6-Sept-2013 to 07-Feb-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

EC Number:

815-961-9

Cas Number:

1374760-95-8

Molecular formula:

C17H17N3O2S

IUPAC Name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state : White powder
- Storage condition of test material: Dry place, protected form light, (15 to 30°C)

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. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the Quality Control samples.

Preparation of test item and controls:
- Vehicle: acetonitrile at 100 mM
- Positive control: Name : Cinnamaldehyde (CAS No. : 14371-10-9, Batch No. : STBC8461V, Supplier : Sigma-Aldrich)
- Co-elution control samples: In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared on the day of the assay by incubating the test item formulation with each buffer used to dilute the peptides.
- Quality Control (QC) samples: In order to control the precision of the peptide quantification throughout the analytical sequence, QC samples were prepared on the day of the assay by diluting each peptide at 0.500 mM in acetonitrile.
- Test item and positive control preparation: Each chemical (test item or positive control) was pre-weighed and stored under appropriate conditions until ready to perform testing. The positive control was dissolved in acetonitrile at 100 mM. The test item was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation had the aspect of a yellowish liquid. Each formulation was prepared within 1 hour. Two aliquots of 500 μL were sampled for chemical analysis and stored at -20°C until shipment or destruction. One of the two aliquots was then sent to the Sponsor on dry ice for chemical analysis at the Sponsor’s Test Site; the other aliquot was destroyed after receipt of the finalized analytical phase report.
- Chemical analysis of the dose formulations: The test item quantification in the dose formulation samples was performed at the Sponsor’s Test Site.The analytical method was developed in the Sponsor’s analytical facility (i.e. Test Site). This phase was not under GLP, indeed, this formulation analysis was performed in the spirit of, but not in full compliance with, the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies; however, it was conducted in accordance with the applicable test site’s Standard Operating Procedures (SOPs) and the study protocol and was carried out to high scientific and facility quality standards.

Cysteine peptide:
. Peptide sequence : AC RFAACAA-COOH
. Peptide sequence synonyms : AC RFAACAA-OH; AC-RFAACAA-COOH or RFAACAA-COOH
The cysteine peptide solution was prepared in an aqueous phosphate buffer (pH 7.5) solution.

Lysine peptide
. Peptide sequence : AC-RFAAKAA-COOH
. Peptide sequence synonyms : AC RFAAKAA-OH; AC-RFAAKAA-COOH or RFAAKAA-COOH
The lysine peptide solution was prepared in an aqueous ammonium acetate buffer (pH 10.2) solution.

Study design:
- Preparation of the samples:
On the day of the assay, the following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide.
- Co-elution control samples preparation
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).
- Quality Control samples preparation
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM. The QC samples were prepared using different standard solutions to those used for the calibration curve.
- Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with 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.
For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 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).
- Test item samples preparation
For the reactivity of test item 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.
For the reactivity of test item with lysine peptide: In parallel, 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).
- Incubation of the samples
All samples (co-elution control, QC, test item and positive control samples) were incubated during 24 (± 2) hours at room temperature and protected from light before injection onto the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis. No precipitate was observed in the vials incubated with the cysteine peptide, as a result, these vials were directly transferred onto the HPLC/UV system. However, some crystals were observed in the test item samples vials incubated with the lysine peptide and precipitates were observed in the positive control samples incubated with either cysteine or lysine peptides. Therefore these vials (test item samples incubated with cysteine and positive control samples incubated with lysine and cysteine) and QC sample vials as well, were centrifuged at 400 g for 5 minutes at room temperature. Since precipitates were still observed in the positive control samples incubated with the cysteine peptide, an additional centrifugation was performed on these vials at 2000 g for 10 minutes and at room temperature . Supernatants were then collected and were transferred onto the HPLC/UV system.
- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking both peptides (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.045 to 0.900 mM. The calibration curves were defined by the relationships between the peak area of the peptide signal versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.
- HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis. For each peptide, the analytical sequence included at least:
. one blank sample (peptide dilution buffer),
. one calibration curve (within the range defined during validation) injected at the beginning of the analytical batch,
. one co-elution control sample,
. three QC samples injected at least three times to bracket study samples,
. three cinnamaldehyde depletion control samples preceded and followed by one QC sample,
. three test item samples followed by QC samples.
The HPLC/UV method used for the samples analysis is described in CiToxLAB France internal analytical method. For the cysteine peptide, the whole analytical sequence was injected using 7 μL as the injection volume (as described in CiToxLAB France internal analytical method on DPRA). Since one of the acceptance criteria on QC samples was not reached (back-calculated concentrations out of ± 10% of the nominal concentration 0.500 mM), the obtained results were invalidated and the samples were re-injected on the HPLC/UV using a lower injection volume (5 μL) which is also described in CiToxLAB France internal analytical method on DPRA. Since all acceptance criteria then were met, the analytical sequence was considered as valid and only the results obtained with the injection volume of 5 μL were taken into account for the evaluation.

Results and discussion

Positive control results:

Cinnamic aldehyde was used as a positive control.
- Result of positive control samples in cysteine assay: Mean % depletion = 100.0% and SD = 0.0%
- Result of positive control samples in lysine assay: Mean % depletion = 62.54% and SD = 0.8%
Mean depletion rate of Cinnamaldehyde: 81.27%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Mean % depletion in Lysine assay

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Value set to "0" as the mean % depletion value is <0

Results 2

Key result

Parameter:

other: Mean % depletion in Cysteine assay

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Default value due to concentration below limit of quantification after depletion

Results 3

Key result

Parameter:

other:

Remarks:

Mean depletion rate

Value:

50

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Highly reactive

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Any other information on results incl. tables

See the attached document "Tables of results DPRA"

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item was considered to be highly reactive with peptides and therefore has potential to cause skin sensitization.

Executive summary:

The skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay (DPRA) similarly to the OECD Guideline No. 442C and in compliance with GLP. The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the Quality Control samples.

The acceptance criteria of the samples for the calibration curve, QC and positive control were satisfied. 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 either with the lysine or the cysteine peptides. Mean percentage depletion values were calculated for each peptide:

. 100% for the cysteine peptide since the cysteine concentrations measured in each sample were below the limit of quantification after depletion (< 0.100 mM),

. for the lysine peptide, two out of three individual depletion values were found negative. The mean depletion value was therefore set to 0%.

The mean of the percentage cysteine and percentage lysine depletions was calculated to be 50.00%.

Accordingly, the test item was considered to be highly reactive.

Under the experimental conditions of this study, the test item was considered to be highly reactive with peptides and therefore has potential to cause skin sensitization.