2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethane-1-sulfonyl chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-24 to 2018-11-13

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:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethane-1-sulfonyl chloride

EC Number:

610-161-4

Cas Number:

4403-36-5

Molecular formula:

C10H8ClNO4S

IUPAC Name:

2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethane-1-sulfonyl chloride

Test material form:

solid: particulate/powder

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
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.
Number of replicates: co-elution controls, reference controls and samples were measured in triplicates.
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.
Co-elution control (CC): 750 µL Phosphate buffer pH 7.5, 200 µL ACN, 50 µL CCys 209630/A solution (100 mM)
Positive control: Cinnamic aldehyde (PC): 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL Cinnamic aldehyde solution (100 mM in ACN).
Test item 209630/A: 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL 209630/A test solution (100 mM)
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.
Co-elution control (CC):750 µL Ammonium acetate buffer pH 10.2, 250 µL CClys 209630/A test solution (100 mM)

Positive control: Cinnamic aldehyde (PC): 750 µL Stock solution of 0.667 mM SPCL, 250 µL Cinnamic aldehyde solution (100 mM in ACN)

Test item 209630/A: 750 µL Stock solution of 0.667 mM SPCL, 250 µL 209630/A test solution (100 mM)
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 times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 23.5 hours and 25 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 30 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.
At a concentration of 100 mM, TA-2 was not soluble in MQ, ACN:MQ (1:1, v/v) and isopropanol, but was soluble in ACN, acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v). Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study.
Preparation of a 100 mM TA-2 stock solution in ACN showed that the test item was dissolved completely.
SPCC and SPCL peak areas in the samples were measured by HPLC PDA.
Mobile phase: A: 0.1% (v/v) TFA in Milli-Q water, B: 0.085% (v/v) TFA in ACN
Flow:0.35 mL/min
Injection volume: 3 µL
Sample tray temperature: Set at 25°C
Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 µm (Agilent Technologies, Santa Clara, CA, USA)
Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
Column temperature: Set at 30°C
Detection: Photodiode array detection, monitoring at 220 and 258 nm.

Results and discussion

Positive control results:

% depletion cystein peptide: mean 68.9%; SD 0.3%
% depletion lysine peptide: mean 60.5%; SD 2.1%

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: mean percent cysteine and lysine depletion

Value:

61.7

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Remarks:

high reactivity

Results 2

Key result

Run / experiment:

other: cysteine peptide

Parameter:

other: mean percent depletion

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

high reactivity

Results 3

Key result

Run / experiment:

other: lysine peptide

Parameter:

other: mean percent depletion

Value:

23.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No
Acceptability of the Cysteine Reactivity Assay
The SPCC standard calibration curve exhibited a correlation coefficient (r²) of the SPCC standard calibration curve of 0.998. Since the r² was > 0.99, the SPCC standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.518 ± 0.005 mM while the mean peptide concentration of Reference Controls C was 0.518 ± 0.006 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This 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. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 18.00. The mean A220/A258 ratio ± 10% range was 16.20-19.80. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The Percent SPCC Depletion for the positive control cinnamic aldehyde was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 68.9% ± 0.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the Lysine Reactivity Assay
The correlation coefficient (r²) of the SPCL standard calibration curve was 0.998. Since the r² was > 0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.511 ± 0.018 mM while the mean peptide concentration of Reference Controls C was 0.532 ± 0.013 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This 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. The CV of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 16.85. The mean A220/A258 ratio ± 10% range was 15.17-18.54. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The Percent SPCL Depletion for the positive control cinnamic aldehyde was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.5% ± 2.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Any other information on results incl. tables

Results for Cysteine reactivity

Preparation of a 100 mM TA-2 stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation, no precipitate or phase separation was observed in any of the samples. After incubation, a phase separation was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide. 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 the 209630/A-cys samples, the mean SPCC A₂₂₀/A₂₅₈ area ratio could not be calculated since no SPCC peak was detected. Overall, it can be concluded that the test item did not co-elute with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion forthe test itemwas 100.0%± 0.0%.

Results for Lysine reactivity

Preparation of a 100 mM TA-2stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples. 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 the 209630/A-lys samples, the mean SPCL A₂₂₀/A₂₅₈ area ratio was 18.75.  This was outside the 15.17-18.54 range, however, since the test item displayed reactivity towards SPCL, accurate calculation of the peak purity was not possible due to the low SPCL signal at 258 nm. Overall, it can be concluded that the test item did not co-elute with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion forthe Test Itemwas 23.4%± 1.0%.

Applicant's summary and conclusion

Interpretation of results:

other: positive

Conclusions:

In conclusion, the test item was tested for skin sensitisation in the Direct Peptide Reactivity Assay. This DPRA test is considered valid, thus, 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.