2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethane-1-sulfonic acid-potassium (1/1)

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:

potassium 2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)ethane-1-sulfonate

EC Number:

618-797-4

Cas Number:

91893-72-0

Molecular formula:

C10H9NO5S.K

IUPAC Name:

potassium 2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)ethane-1-sulfonate

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. In addition, a RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MQ.

Co-elution control (CC): 750 µL Phosphate buffer pH 7.5, 200 µL ACN, 50 µL CCys 209629/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 209629/A: 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL 209629/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. In addition, a RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MQ.

Co-elution control (CC):750 µL Ammonium acetate buffer pH 10.2, 250 µL CClys 209629/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 209629/A: 750 µL Stock solution of 0.667 mM SPCL, 250 µL 209629/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.
At a concentration of 100 mM, TA-1 was not soluble in ACN, but was soluble in MQ. Therefore this solvent was used to dissolve the test item in this DPRA study. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.
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:

13.5

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Remarks:

low reacticity

Results 2

Key result

Run / experiment:

other: cysteine peptide

Parameter:

other: mean percent depletion

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: lysine peptide

Parameter:

other: mean percent depletion

Value:

27

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

low reactivity

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

Acceptability of the Cysteine Reactivity Assay
The correlation coefficient (r²) of the SPCC standard calibration curve was 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, the mean peptide concentration of Reference Controls C was 0.518 ± 0.006 mM and the mean peptide concentration of Reference Controls CMQ was 0.506 ± 0.002 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) 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 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, the mean peptide concentration of Reference Controls C was 0.532 ± 0.013 mM and the mean peptide concentration of Reference Controls CMQ was 0.500 ± 0.016 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) 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 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

The test item was found not soluble at 100 mM in acetonitrile. A solution was obtained at 100 mM with MQ water.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples 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 with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide.

The mean of the percent cysteine and percent lysine depletions was equal to 13.5%. Accordingly, the test item was considered to have low peptide reactivity. Therefore, the DPRA prediction would be considered as positive and the test item may have a potential to cause skin sensitization.

 

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 “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.