1,2-dihydroxyanthraquinone

Endpoint summary

Description of key information

Two in vitro studies, DPRA and h-CLAT were ran to assess the skin sensitisation potency of the test substance in a weight of evidence approach. The 2 studies conducted represent 2 key events in the adverse outcome pathway: DPRA representing the 1st key event (peptide reactivity) and h-CLAT the 3rd key event (activation of dendritic cells). Both studies give indication that the test item might be skin sensitizing, therefore it was concluded to regard the substance as skin sensitizing category 1.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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.

Positive control results:

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

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

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03-07-2017 to 12-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

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

Details on the study design:

PREPARATION OF TEST MATERIAL
- The test item was freshly prepared immediately prior use.
- The test item was dissolved in DMSO (dimethyl sulfoxide) at concentration of 250 mg/mL (the highest soluble concentration).
- Stock solution was prepared by diluting the highest soluble concentration seven time with a constant dilution of 1:2.
- Working solution: diluting each stock solution 250 times with cell culture medium

CONTROLS
- Medium control: 0.9% NaCl
- Solvent control: DMSO
- Positive control: 2,4-dinitrochlorobenzene (DNCB) at final concentration of 4µg/mL (alternatively at concentration of the CV75). DNCB was dissolved in DMSO resulting in final concentration of 0.2% (v/v).

TEST SYSTEM
FACS: BD Canto II
Software BD FACS DIVA 6.0

CELL LINE
- THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC.
- Cells were cultured in 75 cm2 culture flasks (Greiner) in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37 ± 1°C and 5% CO2.

DOSE GROUPS
- Medium Control: Cell culture medium
- Solvent Control: 0.2% DMSO (v/v) in cell culture medium
- Positive Control: 4µg/mL DNCB
- Test item concentration:
- Dose finding assay 1 and 2 : 500; 250; 125; 62.5; 31.25; 15.63; 7.81 and 3.91 µg/mL.
- Main experiment 1 and 2: 67.23; 55.83; 46.53; 38.77; 32.31; 26.93; 22.44 and 18.70 µg/mL.

Positive control results:

The positive control led to an up-regulation of the expression of CD54 and CD86 in both experiments. The tresholds of 150 % for CD86 and of 200% for CD54 were exceeded (see details in any other information on results).

Key result

Run / experiment:

other: 1

Parameter:

other: CD86 expression, relative fluorescence intensity

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 expression, relative fluorescence intensity

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: CD54 expression, relative fluorescence intensity

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD54 expression, relative fluorescence intensity

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

1. Dose Finding Assay:

The dose finding assay was performed using stock solutions with a concentration of 250 mg/mL (500µg/mL).

Sample	Experiment 1		Experiment 2
	Concentration applied (µg/mL)	Cell Viability (%)	Concentration applied (µg/mL)	Cell Viability (%)
Medium Control	0.00	97.20	0.00	94.10
Solvent Control	0.00	97.10	0.00	94.40
Test item	3.91	98.0	3.91	94.00
	7.81	97.70	7.81	93.9
	15.63	97.10	15.63	94.0
	31.25	94.10	31.25	90.6
	62.50	74.40	62.50	68.4
	125.0	61.40	125.0	56.3
	250.0	54.90	250.0	49.9
	500.0	52.0	500.0	50.5
Calculated CV75 (µg/mL)	61.19		50.86
Mean CV75 (µg/mL)	56.03
SD CV 75 (µg/mL)	7.31

Based on the mean CV75, the main experiment was performed covering a concentration range from 67.23 - 18.76 µg/mL (33.62 - 9.38 mg/mL stock solution).

2. Results CD54 and CD86 expression experiment 1:

Sample	Concentration (µg/mL)	Cell Viability (%)			Mean Fluorescence Intensity (MFI)			Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype igG1 to (%)
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control		96.0	96.5	96.6	1707	939	434	1273	505	87	84	393	216
Solvent Control	0.20%	96.9	96.5	96.9	1921	1050	450	1471	600	100	100	427	233
DNCB	4.0	88.7	87.3	88.3	4758	1902	486	4272	1416	290	236	979	391
test item	67.23	78.0	77.0	75.9	2918	4160	1486	1432	2674	97	446	196	280
	56.03	78.6	80.4	80.7	3069	3368	1407	1662	1961	113	327	218	239
	46.69	82.1	82.5	81.4	2991	2932	1238	1753	1694	119	282	242	237
	38.91	85.7	86.6	86.9	2746	2169	993	1753	1176	119	196	277	218
	32.42	92.9	93.0	92.2	2472	1752	879	1593	873	108	146	281	199
	27.02	94.6	94.2	94.3	2132	1527	757	1375	770	93	128	282	202
	22.52	95.5	95.7	95.6	2092	1336	679	1413	657	96	110	308	197
	18.76	95.5	95.9	95.6	2003	1290	648	1355	642	92	107	309	199

3. Results CD54 and CD86 expression experiment 2:

Sample	Concentration (µg/mL)	Cell Viability (%)			Mean Fluorescence Intensity (MFI)			Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype igG1 to (%)
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control		95.9	95.8	94.9	1113	1007	706	407	301	89	94	158	143
Solvent Control	0.20%	96.3	96.5	95.7	1127	993	672	455	321	100	100	168	148
DNCB	4.0	89.2	90.7	90.0	2033	1486	681	1352	805	297	251	299	218
test item	67.23	74.7	74.7	72.6	2449	7745	1821	628	5924	138	1845	134	425
	56.03	84.1	84.0	83.0	3225	6031	1623	1602	4408	352	1373	199	372
	46.69	91.9	91.6	90.8	2338	2796	1403	935	1393	205	434	167	199
	38.91	94.7	94.3	94.3	1657	1908	1168	489	740	107	231	142	163
	32.42	95.5	94.3	94.2	1597	1594	1118	479	476	105	148	143	143
	27.02	96.0	95.9	96.0	1779	1445	1082	697	363	153	113	164	134
	22.52	95.9	95.6	95.6	1609	1405	1012	597	393	131	122	159	139
	18.76	95.8	95.5	96.2	1555	1249	797	758	452	167	141	195	157

The expression of the cell surface marker CD86 was upregulated up to 352% in one experiment (experiment 2). The upregulation above the threshold of 150% was observed at a concentration of 56.03 μg/mL and 46.69 µg/mL.

The expression of the cell surface marker CD54 was upregulated to 446% and 1845% in both independent experiments, respectively. The upregulation above the threshold of 200% was observed at a concentrations starting from 46.69 μg/mL in experiment 1 and from 38.91 μg/mL in experiment 2.

Since the expression of both cell surface marker clearly exceeded the threshold in two independent experiments the test item may considered to be a skin sensitiser.

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 this h-CLAT study under the given conditions the test item upregulated the cell surface marker (CD86 and CD54) in at least two independent experiment runs. Therefore, the test item might be considered as skin sensitiser.

Executive summary:

In this in vitro study h-CLAT (Human Cell Line Activation Test), the skin sensitisation potential of the test item Mordand Red 11 was assessed according to the OECD 442E and under GLP without signiticant deviations.

The in vitro h-CLAT enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of these markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensiters.

In the present study, the test item was dissolved in DMSO.

A dose finding assay was performed with stock solutions with concentrations ranging from 250 mg/mL to 1.96 mg/mL. The cells were treated with the different concentration and incubated for 24h at 37°C. After exposure, cells viability was measured by FACS (fluorescence-activated cell sorting) analysis. A cell viability of 75% (CV 75) of 56.03±7.3 µg/mL was determined.

Based on the CV75, two main experiments were performed covering the following concentrations: 67.23; 55.83; 46.53, 38.77; 32.31; 26.93; 22.44 and 18.70 µg/mL. Cells were incubated with the test item for 24h at 37°C. The 3 highest concentrations were applied as stable suspension in both experiments. According to the guideline, stable suspension can be used. An interference of the particles with the fluorescence measurement was not observed. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis and cell viability was assessed in parallel.

Slight cytotoxicity effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was was reduced to 78.0% (CD86), 77.0% (CD54) and 75.9% (isotype IgG1 control) in the first experiment and to 74.7% (CD86), 75.7% (CD54) and 72.6% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 352% in experiment 2. The upregulation above the thresholdof 150% was observed at a concentration of 56.03µg/mL. The expression of the cell surface marker CD54 was upregulated to 446% and 1845% in both independent experiment, respectively. The upregulation above the threshold 200% was observed at a concentration starting from 46.69µg/mL in experiment 1 and from 39.91µg/mL in experiment 2. Since the expression of both cell surface marker clearly exceeded the threshold in two independent experiments the test item may considered to be a skin sensitiser. However, 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.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (290% experiment 1; 297% experiment 2) and 200% for CD54 (236% experiment 1; 251% experiment 2) were clearly exceeded.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Regarding the classification of the substance for skin sensitisation the results of the two individual assays of the in vitro skin sensitisation test battery need to be taken together as they reflect 2 key events in the adverse outcome pathway leading to skin sensitisation. For this reason, a weight of evidence approach is applied for the test battery stating that: 'any two of the three tests determine the overall results, i.e. any two positive results drive the prediction of a sensitizer, while any two negative results drive the prediction of a test substance to be a non-sensitizer'.

Positive results were obtained both in the DPRA and in the h-CLAT studies, leading to the conclusion that the substance is probably skin sensitizer, category 1. The data are not sufficient for further sub-categorization.