2H-1-Benzopyran, 6-fluoro-3,4-dihydro-2-[(2S)-2-oxiranyl]-, (2R)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-05-24 to 2017-06-08

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))

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:

(2R)-6-fluoro-2-[(2S)-oxiran-2-yl]-3,4-dihydro-2H-1-benzopyran

EC Number:

603-315-7

Cas Number:

129050-26-6

Molecular formula:

C11H11FO2

IUPAC Name:

(2R)-6-fluoro-2-[(2S)-oxiran-2-yl]-3,4-dihydro-2H-1-benzopyran

Test material form:

other: wax

Details on test material:

Physical state: wax form
Appearance: clear, viscous, wax

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16HB3199
- Expiration date of the lot/batch: 2017-30-13 (retest date)
- Purity (GC): 98.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
- The test item should have been heated at 60°C to liquefy before sampling. However, according to the laboratories internal procedures, general test item handling procedures were followed when preparing the test item and it was not heated but partly scraped off.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the cysteine and lysine reactivity assay 31.74 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1634 µL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved.

OTHER SPECIFICS:
- For preparation of the 100 mM test item stock solution a correction factor of 1.00 was used to correct for purity/composition of the test item. On request of the Sponsor this was corrected to 1.15 after the study was performed. This change didn’t have an impact on the study results. In case more test item would be available to react with the peptides (by using a correction factor of 1.15), only a difference in percentage peptide depletion would be observed, however the overall classification in the “high reactivity class” would be the same. Therefore, it was decided that this DPRA should not be repeated.

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC, and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Batch SPCC: 111016HS_MHeW0217
- Batch SPCL: 220114HSDW_W0217
- Storage: The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

EXPERIMENTAL DESIGN
TEST ITEM PREPARATION
see details under "Specific details on test material used for the study"

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- SPCC stock solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 20 mg of SPCC in 38.92 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC reference control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.
- SPCC calibration curve: A SPCC calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- SPCL stock solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 20 mg of SPCL in 38.61 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL reference control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.
- SPCL calibration curve: A SPCL calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

SAMPLE INCUBATIONS
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25 and 26 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 13 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.

HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r²>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.
All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not have been exactly reproduced from the individual data presented.

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
% Peptide Depletion = [ 1 - (Peptide Peak Area in Replication injection at 220 nm / Mean Peptide Peak Area in Reference Controls at 220 nm) ] x 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
There might be cases where the test item absorbed significantly at 220 nm and had the same retention time as the peptide (co-elution). If co-elution of a test item occurred with both the cysteine and the lysine peptide then the analysis was reported as “inconclusive”. In the case where co-elution occurred only with the lysine peptide, the Cysteine 1:10 prediction model was used.

Results and discussion

Positive control results:

Cysteine reactivity assay:
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCC peak area of Reference Controls C, was 73.1% ± 1.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). This was also within the historical positive control data range.

Lysine reactivity assay:
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCL peak area of Reference Controls C, was 58.7% ± 4.3%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). This was also within the historical positive control data range.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: mean of 3 replicates

Parameter:

other: % SPCC depletion

Value:

80.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: mean of 3 replicates

Parameter:

other: % SPCL depletion

Value:

11.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Run / experiment:

other: mean of 3 replicates

Parameter:

other: % Mean of SPCC and SPCL depletion

Value:

45.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive: high reactivity

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The DPRA assay was successfully validated at the laboratory and can be used to support the discrimination between sensitisers and non-sensitisers.

ACCEPTANCE OF RESULTS - Cysteine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.994 was within the acceptance criteria (r²>0.99) (SPCC standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.515 ± 0.012 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.474 mM ± 0.014 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were all within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 3.9% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 73.1% was within the acceptance range of 60.8% to100%
- SD of peptide depletion cinnamic aldehyde: 1.4% was below the maximum (SD <14.9%)

ACCEPTANCE OF RESULTS - Lysine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.991 was within the acceptance criteria (r²>0.99) (SPCL standard calibration curve accepted)
- Mean peptide goh concentration Reference Control A samples: 0.501 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.484 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were all within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 2.6% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 58.7% was within the acceptance range of 40.2% to 69.0%
- SD of peptide depletion cinnamic aldehyde: 4.3% was below the maximum (SD <11.6%)

Any other information on results incl. tables

Solubility assessment:

At a concentration of 100 mM, the test item was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM stock solution in ACN showed that the test itemwas dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. After incubation a precipitate was observed in the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, peptide depletion might be underestimated.

In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the207490/A-cys samples, the mean SPCC A₂₂₀/A₂₅₈area ratio was 20.85. This was outside the 15.77-19.27 range. However, since the test item displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm. Overall, it can be concluded that the test item did not co-elute with SPCC.

Results lysine reactivity assay for the test item:

Preparation of a 100 mM stock solution in ACN showed that thetest item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC andthetest item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, peptide depletion might be underestimated.

In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the207490/A-lys samples, the mean SPCL A₂₂₀/A₂₅₈area ratio was 12.77. Since this was within the 12.22-14.93 range, this again indicated that there was no co-elution of the test item with SPCL.

SPCC and SPCL depletion, DPRA prediction and reactivity classification of the test item:

SPCC depletion		SPCL depletion			Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
Mean	± SD	Mean		± SD		Cysteine 1:10 / lysine 1:50 prediction model
80.3%	± 2.0%	11.4%	± 4.9%		45.9%	Positive: high reactivity

SD = Standard Deviation; NA = not applicable              

Historical control data for DPRA studies

	Positive control - Cinnamic aldehyde
	SPCC depletion	SPCL depletion
Range	71.8 - 77.9%	43.5 - 65.2%
Mean	74.4%	59.5%
SD	1.6%	5.5%
n	19	19

SD = Standard Deviation, n = Number of observations

The above mentioned historical control data were collected over the period of January 2017 to April 2017.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.
The test item was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.