Sodium 2-[[4-(cyclohexylamino)-9,10-dihydro-9,10-dioxo-1-anthryl]amino]-5-ethoxybenzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From June 11th to June 20th, 2019

Reliability:

1 (reliable without restriction)

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Blue 280

IUPAC Name:

Acid Blue 280

In chemico test system

Details on the study design:

-Purpose: the DPRA is a chemistry-based assay (based on the OECD guideline for the testing of chemicals, In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD document TG 442C) exploiting the fact that most chenmical allergens have electrophilic properties and are therefore able to reacte with the nucleophilic sidechains of amino acids to form covalent bonds. The underLying rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitizer. The endpoint measured in the assay is the percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures following an approximate 24 hour (22-26 hours) incubation with the test item. The percentage of peptide depletion is calculated by High Performance Liquid Chromatography using ultra-violet detection. The DPRA test allows quantification of a chemical's reactivity and is used to categorize a substance in one of four classes of reactivity to allow discrimination between skin sensitizing and non-sensitizing chemicals and thus assess their sensitization potential.

-Apparatus: HPLC = Waters Alliance 2695 separation module and 2487 dual wavelenght detector; balances fitted with printers = capability of weighing to 5 decimal places; general laboratory apparatus and glassware.
-Assessment of test item solubility: the solubility of test item was assessed at a concentration of 100 mM in acetonitrile and acetonitrile/water 50/50 v/v.

-Preparation of peptide stock solutions: stock solutions of each peptide at a concentration of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 ml aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).

-Preparation of peptide calibration standards: calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

-Preparation of stability controls and precision controls: stability controls (reference control B, n=6) and precision control (reference control A) of both peptides were prepared at a concentration of 0.5 mM. Reference control A and reference control B were prepared with buffer and acetonitrile, reference control C (n=3) was prepared with buffer and acetonitrile/water 50/50 v/v.

-Preparation of positive control solution and test item stock solution: the positive control chemical (cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of test item was prepared in acetonitrile/water 50/50 v/v.

-Preparation of positive control solution and cysteine peptide depletion samples and co-elution controls: triplicate solutions each of test item and the positive control stocks were diluted with the cysteine peptide to prepare final solutions containing 0.5 mM cysteine and 5 mM of either test item or the positive control. For the co-elution control, blank buffer solutions was used in place of the cysteine stock solution.

-Preparation of positive control and lysine peptide depletion samples and co-elution controls: triplicate solutions each of test item and the positive control stocks were diluted with the lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either test item or positive control. For the co-elution control, blank buffer solution was used in the place of the Lysine stock solution.

-Incubation: the appareance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run appearance of the samples in the vials was assessed and documented again.

-Analysis: the concentration of each peptide in the presence of the test item and the associated positive controls were quantified by HPLC using UV detection.

-Calculations: the peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - [Peptide peak area in replicate depletion samples (×100)/ Mean Peptide peak area of reference control samples B or C]

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: mean of 3 run

Parameter:

other: mean depletion cysteine %

Value:

84.7

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Run / experiment:

other: Mean of 3 run

Parameter:

other: mean depletion positive control %

Value:

71.7

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

-Solubility assessment: the solubility of the test item in acetonitrile/water 50/50 v/v at a nominal concentration of 100 mM was confirmed. Test item was insoluble in acetonitrile at 100 mM.

-Reactivity assessment: all analytical acceptance criteria for each peptide run were met, though due to significant co-elution of the test-item with the lysine peptide only the result of the cysteine peptide assay has been used to make the DPRA prediction. The lysine peptide results presented are for information only. Reactivity is classed as "moderate" and the DPRA prediction is positive and test item is thus predicted skin sensitizer by this assay.

Any other information on results incl. tables

	Peptide	Linearity	Positive control depletion ( % )	Reference controls	Test item
Acceptance criteria	Cysteine	>0.99	60.8-100	0.45-0.55 mM	SD <14.9 %
			(SD <14.9 %)	(CV <14.9 %)
	Lysine	>0.99	40.2-69.0	0.45-0.55 mM	SD<11.6 %
			(SD <11.6 %)	(CV <14.9 %)
Achieved results	Cysteine	>0.999	71.7	B: 0.501 mM (CV=0.67 %, n=6)	SD 2.80 %
			(SD 0.44 %, n=3)	C: 0.490 mM (CV=1.23 %, n=3)
	Lysine	>0.999	60.4	B: 0.540 mM (CV=3.56 %, n=6)	SD 1.48 %
			(SD 7.53 %, n=3)	C: 0.539 mM (CV=0.44 %, n=3)

CV = Coefficient of variation

Depletion of peptide in the presence of test item

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide in test samples (µV.sec)	Mean peptide depletion ( % )
Cysteine	B: 898360 (n=6)	134250 (n=3)	84.7
	C: 879920 (n=3)
Lysine	B: 744350 (n=6)	74067^1(n=3)	90
	C: 742560 (n=3)

Note: test item co-elutes with the lysine peptide. The co-elution peak is 10.8 % of the reference control peak area. Therefore, the cysteine 1:10 reactivity prediciton model has been applied.

Depletion

Mean of cysteine % depletion	Reactivity Class	DPRA Prediction
0 %≤ Cys % depletion ≤13.89 %	No or minimal reactivity	Negative
13.89 %< Cys % depletion ≤23.09 %	Low reactivity	Positive
23.09 %< Cys % depletion ≤98.24 %	Moderate reactivity
98.24 %< Cys % depletion ≤100 %	High reactivity

Co-elution occured during the lysine assay

Cysteine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^2(µg/ml)	Peptide Depletion ( % )	Mean Depletion  ( % )	SD ( % )
Positive control^1	250163	104	72.23 ^3	71.7	0.44
	257948	107	71.33 ^3
	255201	106	71.63 ^3
Test Item	160822	66	81.74 ^4	84.7	2.8
	129641	52.9	85.34 ^4
	112290	45.6	87.24 ^4

CV = Coefficient of Variation

¹ Data generated under PV22VQ

² Samples prepared at a concentration of 376µg/ml (0.5 mM)

³ Calculated against a mean reference control B peak area of 898360 µV.sec(n=6)

⁴ Calculated against a mean reference control C peak area of 879920 µV.sec(n=3)

Lysine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^2(µg/ml)	Peptide Depletion^3 ( % )	Mean Depletion  ( % )	CV ( % )
Positive control^1	253561	141	65.93 ^3	60.4	7.53
	271548	151	63.53 ^3
	358374	200	51.93 ^3
Test Item	76716	40.3	89.74 ^4	90	1.48
	83454	44.2	88.84 ^4
	62032	32	91.64 ^4

Data presented for information only

CV = Coefficient of Variation

¹ Data generated under PV22VQ

² Samples prepared at a concentration of 388 µg/ml (0.5 mM)

³ Calculated against a mean reference control B peak area of 744350µV.sec(n=6)

⁴ Calculated against a mean reference control C peak area of 742560µV.sec(n=3)

Applicant's summary and conclusion

Interpretation of results:

other: the test result is used in a global assessment to decide on the classification within the CLP Regulation (EC 1272/2008)

Conclusions:

Based on a depletion of cysteine peptide of 85 %, reactivity of test item is "moderate" and hence the DPRA prediction is positive as based on this assay.

Executive summary:

The study was run according to OECD guideline 442C to assess the reactivity and sensitizing potential of test item.

Solutions of test item were successfully analysed by the validated DPRA analytical method (Covance Analytical Method FIA/M101/15) in just the cysteine containing synthetic peptide. There was significant co-elution (ca 11 %) in the lysine containing synthetic peptide and therefore only the result of the cysteine is reported. With depletion of the cysteine peptide of 85 %, reactivity of test item is "moderate" and hence the DPRA prediction is positive as based on this assay.