Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 2017 - 16 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 (incl. QA statement)

Remarks:

dd 3 November 2015

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

EC Number:

227-497-9

EC Name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Cas Number:

5858-81-1

Molecular formula:

C18H12N2Na2O6S

IUPAC Name:

disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Test material form:

solid: particulate/powder

Details on test material:

Name as cited in the report: D&C Red 6
Appearance: red powder
Storage conditions: at room temperature

In chemico test system

Details on the study design:

TEST ITEM PREPERATION
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), MQ/ACN (1:1, v/v), isopropanol, acetone, 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 63.48 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1475 μL MQ 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.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: 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 for 27 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials'). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 98.4%

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. 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 was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Results and discussion

Positive control results:

The positive control had a mean SPCC depletion of 76.1 ± 1.2% and a mean SPCL depletion of 58.4 ± 1.8%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reacivity Assay

Parameter:

other: SPCC mean depletion (%)

Value:

19

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: SD: 6.7%

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL mean depletion (%)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: SD: 0%

Other effects / acceptance of results:

- In both the cysteine and the lysine assay, a precipitate was observed upon preparation as well as after 27 hours of incubation. In this case one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, a negative result is uncertain and should be interpreted with due care.
- The test item did not co-elute with SPCC and SPCL.

Any other information on results incl. tables

Table 4 Acceptability of the DPRA assay

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPLC
Correlation coefficient (r^2) standard calibration curve	>0.99	0.996	>0.99	0.993
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.503 ± 0.005	0.50 ± 0.05	0.514±0.007
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.489 ± 0.016	0.50 ± 0.05	0.508±0.007
Mean peptide concentration RC-Cwater samples (mM)	0.50 ± 0.05	0.495 ± 0.007	0.50 ± 0.05	0.493±0.014
CV (%) for RC samples B and C	<15.0	1.9	<15.0	2.3
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	76.1	40.2-69.0	58.4
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.2	<11.6	1.8
SD of peptide depletion for the test item (%)	<14.9	6.7	<11.6	0.0

Table 5 SPCC and SPCL depletion and reactivity classification for the test item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	Reactivity class
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
D&C Red 6	19.0%	±6.7%	100.0%	±0.0%	59.5%	High reactivity

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

D&C Red 6 was positive in the DPRA, performed according to OECD 442C and GLP principles, and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In a Direct Peptide Reactivity Assay, performed according to OECD 442C and GLP priniciples, D&C Red 6 was assessed for its reactivity towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). The test item was incubated for 27 hours with either SPCC or SPCL. SPCC and SPCL Percent Depletion Values were calculated using the relative peptide concentrations, determined by HPLC with gradient elution and photodiode array at 220 nm and 258 nm.

In the cysteine reactivity assay D&C Red 6 showed 19.0% SPCC depletion while in the lysine reactivity assay D&C Red 6 showed 100.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 59.5% and as a result D&C Red 6 was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

All acceptability criteria for the DPRA were met and the study was considered to be valid.