Sodium diisopropylnaphthalenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

Sodium diisopropylnaphthalenesulphonate

EC Number:

215-343-3

EC Name:

Sodium diisopropylnaphthalenesulphonate

Cas Number:

1322-93-6

Molecular formula:

C16H20O3S.Na

IUPAC Name:

sodium diisopropylnaphthalene-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

white to off-white lumps

In chemico test system

Details on the study design:

Principle of the assay
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

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 and batch
JPT Peptide Technologies GmbH, Berlin, Germany.
Batch No. 111016HS_MHe_W1018
Storage: The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.

Positive control
Cinnamic aldehyde (104-55-2)

Reagents
Acetone HiPersolv, VWR international, Amsterdam, The Netherlands
Acetonitrile (ACN) HPLC-grade, Fisher Chemicals, Loughborough, England
Ammonium acetate (CH3COONH4) Fractopur, Biosolve, Valkenswaard, The Netherlands
Ammonium hydroxide (NH4OH) 25.0% Merck, Darmstadt, Germany
Dimethylsulfoxide (DMSO) Seccosolv, Merck
Disodiumhydrogenphosphate Emsure, Merck
(Na2HPO4.12H2O)
Isopropanol LiChrosolv, Merck
Milli-Q water (MQ) Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion-exchange cartridges
Sodiumdihydrogenphosphate Emsure, Merck
(NaH2PO4.H2O)
Trifluoroacetic acid, >99% (TFA) Sigma Aldrich, Zwijndrecht, The Netherlands

Test item preparation
No correction for the purity/composition of the test item was performed.
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 solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v) and dimethylsulfoxide (DMSO):ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 54.80 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1743 μL MQ after vortex mixing 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. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

HPLC-PDA analysis
SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
* Alliance separations module 2695 (Waters, Milford, MA, USA)
* Dual λ absorbance detector 2487 (Waters)
System 2 (used for Lysine Reactivity Assay):
* Alliance separations module 2695 (Waters, Milford, MA, USA)
* Dual λ absorbance detector 2487 (Waters)

The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) 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.
c) 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.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variability (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:
a) The maximum standard deviation for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Cysteine

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Quantitation of SPCC was not possible because of the lack of a clear distinct peak and as a result the Percent SPCC Depletion could not be calculated.

Results 2

Run / experiment:

other: Lysine

Remarks on result:

not determinable

Remarks:

Quantitation of SPCL was not possible because of co-elution and the Percent SPCL Depletion could therefore not be calculated.

Other effects / acceptance of results:

Please see the attached report for a detailed discussion of the results and the associated HPLC graphs.

ACCEPTANCE OF RESULTS:
Acceptability of the cysteine reactivity assay
Preparation of a 100 mM CH04207 stock solution in MQ 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. 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 SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 209839/A-cys samples, shoulder peaks were observed which indicated that there was interaction of the test item with SPCC. However, quantitation of SPCC in 209839/A-cys samples was not possible because of the lack of a clear distinct peak and as a result the Percent SPCC Depletion could not be calculated.

Acceptability of the lysine reactivity assay
The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9999. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.494 ± 0.004 mM, the mean peptide concentration of Reference Controls C was
0.470 ± 0.005 mM and the mean peptide concentration of Reference Controls CMQ was 0.465 ± 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 SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 2.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 31.58. The mean A220/A258 ratio ± 10% range was 28.42-34.73. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
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 was 69.7% ± 3.6%. This was slightly above the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). As the positive control cinnamic aldehyde displayed significant inhibition while all other acceptability criteria were met, the DPRA was considered to be valid.

Any other information on results incl. tables

At a concentration of 100 mM, CH04207 was not soluble in ACN, isopropanol, acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v), but was soluble in MQ and ACN:MQ (1:1, v/v). Since MQ is preferred over ACN:MQ (1:1, v/v) for the DPRA, MQ was used to dissolve the test item in this study.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, the validation parameters were evaluated and this DPRA was considered to be valid. In the cysteine reactivity assay CH04207 was interacting with SPCC (i.e. a shoulder on the chromatographic SPCC peak was observed in samples containing the test item) while in the lysine reactivity assay CH04207 was co-eluting with SPCL. As no accurate quantification of SPCC or SPCL was possible in test item samples, the DPRA was considered to be inconclusive.

Executive summary:

The objective of this study was to determine the reactivity of CH04207 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm.

The study procedures described in this report were based on the most recent OECD 442C (2015) guideline.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r2) standard calibration curve	>0.99	0.9999	>0.99	0.9999
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.511 ± 0.002	0.50 ± 0.05	0.494 ± 0.004
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.506 ± 0.001	0.50 ± 0.05	0.470 ± 0.005
Mean peptide concentration RC-CMQsamples (mM)	0.50 ± 0.05	0.502 ± 0.005	0.50 ± 0.05	0.465 ± 0.002
CV (%) for RC samples B and C	<15.0	1.1	<15.0	2.4
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	74.8	40.2-69.0	69.71
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.2	<11.6	3.6
SD of peptide depletion for the test item (%)	<14.9	NA	<11.6	NA

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.1This result was outside the acceptance criteria.

The majority of the validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and CMQ, the CV for RC samples B and C, the mean percent peptide depletion values for the SPCC positive control and standard deviation values of peptide depletion values for the SPCC and SPCL positive control, were all within the acceptability criteria for the DPRA. The mean percent peptide depletion value for the SPCL positive control was slightly above the upper acceptance limit of 69.0% (i.e. 69.7%). Because the positive control (cinnamic aldehyde) displayed significant inhibition while all other acceptability criteria were met, the DPRA was considered to be valid. The standard deviation values of peptide depletion values for SPCC and SPCL could not be calculated for the test item.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed interaction with SPCC (i.e. a shoulder on the chromatographic SPCC peak was observed in samples containing the test item) while in the lysine reactivity assay the test item co-eluted with SPCL. As a result, accurate quantification of SPCC or SPCL in test item samples was not possible and the DPRA was considered to be inconclusive.

In conclusion, the validation parameters were evaluated and this DPRA was considered to be valid. In the cysteine reactivity assay CH04207 was interacting with SPCC while in the lysine reactivity assay CH04207 was co-eluting with SPCL. As no accurate quantification of SPCC or SPCL was possible in test item samples, the DPRA was considered to be inconclusive.