1,2-dihydroxyanthraquinone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-06-07 to 2017-09-01

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))

Deviations:

yes

Remarks:

These deviations did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In accordance with Annex VII of REACH regulation, new REACH requirements for skin sensitisation entered into force on 11 October 2016

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dihydroxyanthraquinone

EC Number:

200-782-5

EC Name:

1,2-dihydroxyanthraquinone

Cas Number:

72-48-0

Molecular formula:

C14H8O4

IUPAC Name:

1,2-dihydroxy-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: solid yellow powder

In chemico test system

Details on the study design:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.


PREPARATION OF PEPTIDES
- 18.12 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (33.52 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 21.3 mg lysine peptide with an amino acid sequence of Ac RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.76 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

PREPARATION OF CONTROLS
- Reference controls (RC):
- Reference control A (undiluted) was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
- Reference control B (undiluted) was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
- Reference control C (undiluted) was set up for the test item and the positive control. RC C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
- Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution.. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
- Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetronitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

DOSE GROUPS
- Reference Control C: undiluted
- Test item: 100 mM stock solution
- Positive control: 100 mM stock solution

PRELIMINARY EXPERIMENT
Solubiluty of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100mM). The test item was dissolved in dimethylformamide (DMF)

MAIN TEST
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide).
The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.

Results and discussion

Positive control results:

Mean cysteine peptide depletion: 70.9% and mean lysine peptide depletion: 62.34%

In vitro / in chemico

Results

Key result

Run / experiment:

other: 1

Parameter:

other: Mean cysteine peptide depletion (%)

Remarks:

Prediction Model 2

Value:

80.95

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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

Any other information on results incl. tables

1. Pre-experiments:

Solubility of the test item was determined. The test item was not soluble in acetonitrile but completely soluble in DMF. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepares using DMF.

2. Precipitation and phase separations:

All test item solutions were freshly prepared immediately prior to use. No precipitation, turbidity or phase separation was observed for the samples of the test item. Precipitation was observed for the samples of the positive control, however, since the acceptance criteria for the depletion of the positive contrtol were fulfilled, the observed precipitation and turbidity was regarded as insignificant.

3. Co-elution with the peptide peaks:

Co-elution of the test item with the lysine peptide peak was observed. Therefore prediction model 2 was applied.

4. Results Calibration Curve: Cysteine and Lysine values of the Calibration curve:

Sample	Cysteine peptide		Lysine peptide
	Peak Area 220 nm	Peptide Concentration mM	Peak Area 220 nm	Peptide concentration mM
STD1	4946.3618	0.5340	4453.7617	0.5340
STD2	2473.8633	0.2670	2214.0920	0.2670
STD3	1198.9833	0.1335	1103.6776	0.1335
STD4	613.2764	0.0667	542.1525	0.0667
STD5	313.3556	0.0334	261.3623	0.0334
STD6	155.0795	0.0167	131.8409	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Cysteine Peptide Calibration Curve: y = 9265.42x-5.69 ; R² = 0.9999

Lysine Peptide Calibration Curve: y = 8353.22x-10.69 ; R² = 1.0000

5. Results of Cysteine Peptide Depletion:

Sample	Peak Area 220 nm	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV of Peptide Depletion (%)
Positive Control	1367.6532	0.1482	70.48	70.90	0.44	0.62
	1350.3103	0.1464	70.86
	1327.2146	0.1439	71.36
Test Item	1035.8650	0.1124	77.63	80.95	3.57	4.41
	903.7498	0.0982	80.48
	707.2005	0.0769	84.73

6. Results of Lysine Peptide Depletion:

Sample	Peak Area 220 nm	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV of Peptide Depletion (%)
Positive Control	1562.8215	0.1884	62.85	62.34	0.45	0.72
	1598.4821	0.1926	62.00
	1591.5383	0.1918	62.17
Test Item	4383.3223	0.5267	0.00	1.04	0.96	91.63
	4222.1846	0.5067	1.25
	4195.4971	0.5035	1.88

7. Categorization of the Test item

Co-elution of test item with the lysine peptide peak was observed. Therefore, sensitizing potential of the test item was predicted from the mean peptide depletion of the cysteine peptide using prediction model 2:

Predicition Model	Prediction Model 1 (Cysteine and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	41.00	Moderate Reactivity	sensitiser	80.95	Moderate Reactivity	sensitiser
Positive Control	66.62	High Reactivity	sensitiser	70.90	Moderate Reactivity	sensitiser

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The study alone cannot be used for the classification purpose but in a WoE approach of minimally 2 in vitro assessment representing the key events in AOP

Conclusions:

In a DPRA study, the test item showed moderate reactivity towards the cysteine peptide. Based on these results the test item might be considered to have a sensitising potential (PPD 80.95%).

Executive summary:

In this in chemico study Direct Peptide Reactive Assay (DPRA), the skin sensitisation potential of the test item Mordand Red 11 was assessed according to the OECD 442C and under GLP without signiticant deviations.

Following a pre-experiment to determine the solubility of the test item in different solvent (dissolved in DMF), the test item (in triplicate) was incubated at a concentration of 100 mM (24.022 mg/mL) with the peptides containing either cystiene or lysine for 24 +/- 2h at 25 +/- 2.5 °C. After the incubation periode, the samples were analysed by HPLC in order to quantifyed the percentage of the mean peptide depletion.

Co-elution of test item with the lysine peptide peak was observed. Sensitizing potential of the test item was hence predicted from the mean peptide depletion of cysteine peptide alone by comparing the peptide concentration of the test item treated samples to the corresponding reference control C. (RC C). The mean depletion of the cysteine peptide was > 13.89% (i.e. 80.95%). Based on the prediction model 2 the test item can be considered as skin sensitiser (moderate).

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of positive control on both peptides was 66.62%.

In conlusion, in this study under the given conditions the test item showed moderate reactivity towards the cysteine peptide. Therefore, the test item is considered to have skin sensitising potential.The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.