Disodium naphthalene-1,5-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium naphthalene-1,5-disulphonate

EC Number:

216-732-0

EC Name:

Disodium naphthalene-1,5-disulphonate

Cas Number:

1655-29-4

Molecular formula:

C10H8O6S2.2Na

IUPAC Name:

disodium naphthalene-1,5-disulphonate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid: crystalline

Details on test material:

Test item: Armstrongsäure-di-Natriumsalz Monohydrat
CAS name: 1,5-Naphthalenedisulfonic acid, sodium salt (1 :2) Monohydrat
CAS number: 207569-02-6
Empirical formula: C10 H6 O6 S2 * 2Na * H2O
Molecular mass: 350.3 g/mol
Batch number: CHNHFC1602
Content monohydrat-form: 99.8 %
Corresponds to 94,7 % anhydrous form, CAS: 1655-29-4

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CHNHFC1602
Disodium-naphthalene-1,5-disulphonate was tested as monohydrate in this test. Calculations of all test article concentrations stated in this report thus include a correction for content (purity) using a factor of 1.056. The correction factor compensates for the crystallisation
water content of the test compound, so that the results are also reliable for Di-sodium-naphthalene-1,5-disulfonic acid CAS: 1655-29-4).

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).

The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5ºC. Relative peptide concentration is measured by reversed phase (C18) high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. The synthetic peptides contain phenylalanine to aid in the detection.
The test item was dissolved and tested according to the given test procedure. Cinnamic aldehyde was used as positive control at a concentration of 100 mmol/L in acetonitrile.

Cysteine and lysine peptide solutions were incubated at 1:10 and 1:50 ratio in glass auto sampler vials with the test item solution for 24 hours at 25±2.5°C in the dark. Samples were visually inspected for precipitation or phase separation before HPLC analysis. The test item was analyzed in triplicate for both peptides. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. HPLC analysis for the cysteine and lysine peptides were performed on separate days with the test item solutions freshly prepared for both assays on each day. The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards. Percent peptide depletion is calculated according to OECD Guideline.

The following criteria should be met for a test chemical’s results to be considered valid: a) the maximum standard deviation for the test chemical replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion, b) the mean peptide concentration of three injections of the reference control C in the appropriate solvent should be 0.50±0.05 mM.

According to the study protocol the mean percent cysteine and percent lysine depletion value was calculated for the test item and the positive control. 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 can be used to support the discrimination between skin sensitizers and non-sensitizers in the framework of an IATA.

Results and discussion

Positive control results:

The positive control cinnamic aldehyde led to a depletion of 71.49% cysteine peptide and 61.72% lysine peptide. The mean cysteine/lysine peptide depletion was calculated with 66.61% leading to a positive results (high reactivity class according to the cysteine 1:10/lysine 1:50 prediction model).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: mean of 2 runs

Parameter:

other: % depletion in the cysteine 1:10/lysine 1:50 prediction model:

Value:

1.08

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA prediction: negative

Results 2

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean % cysteine peptide depletion

Value:

2.16

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean % lysine depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes

The test item visually appeared a clear solution in water at the test concentration of 100 mmol/L. No precipitation occured before or after incubation.
Absorbance at 220 nm was not observed. Retention time similar to peptide was not observed. Co-elution of the test item with the peptides was not observed.

Any other information on results incl. tables

Results of the DPRA:

Test item	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	reactivity class	DPRA prediction
positive control (DNCB)	71.49	61.72	66.61	high	positive
test item	2.16	0	1.08	minimal	negative

Applicant's summary and conclusion

Executive summary:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Since the test item visually appeared a clear solution in water at the test concentration of 100 mmol/L this solvent was used in the DPRA. No precipitaton of the test item occured before or after incubation. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (1.08%). According to the prediction model “minimal reactivity” was derived for the test item in water, leading to a DPRA prediction of “negative“.