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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2017 (Seeding of the cells, 1st experiment of h-CLAT) - 04 April 2018 (study completion date)
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:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The OECD TG 442 E may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]
EC Number:
280-163-4
EC Name:
Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]
Cas Number:
83027-61-6
Molecular formula:
C32H28N2Na2O8S2
IUPAC Name:
Reaction mass of disodium 2-ethyl-3-({4-[(2-ethyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-4-methylbenzene-1-sulfonate, disodium 4-ethyl-3-({4-[(2-ethyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-2-methylbenzene-1-sulfonate and disodium 4-ethyl-3-({4-[(6-ethyl-2-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-2-methylbenzene-1-sulfonate
Test material form:
solid: particulate/powder

In vitro test system

Details on the study design:
Cell line: THP-1 cells
The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB202).

Results and discussion

Positive control results:
The positive control used was 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 µg/mL in 0.2% DMSO in culture medium. The positive and negative and vehicle control data is comparable to the historical data. The results can be seen in Table 2.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Experiment 4
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RFI CD86
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Negative activation of dendritic cells at concentrations of 78-279 µg/mL
Remarks:
viability ≥50%
Key result
Run / experiment:
other: Experiment 4
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RFI CD54
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive activation of dendritic cells at concentrations at concentrations of 233 and 279 µg/mL
Remarks:
viability ≥50%
Key result
Run / experiment:
other: Experiment 5
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RDI CD86
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Negative activation of dendritic cells at concentrations of 78-279 µg/mL
Remarks:
viability ≥50%
Key result
Run / experiment:
other: Experiment 5
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RFI CD54
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive activation of dendritic cells at concentrations of 194, 233, 279 µg/mL
Remarks:
viability ≥50%
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Acceptance criteria
- A tested concentration is not to be further evaluated when relative viability is less than 50%.
- Cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86< 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.  
- The reactivity check of new thawed cells should produce the following result: - Positive response in CD86 and CD54 for NiSO4 and DNCB - Negative response in CD86 and CD54 for LA.
- In addition, positive, negative and vehicle control data should lie within the range of the historic data

Evaluation of results
- A test substance is predicted to activate monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 5000 µg/mL for the vehicle culture medium or 1000 µg/mL for 0.2% DMSO in culture medium) or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested).  
- To be relevant for evaluation, the cell viability must be more than 50% in at least four tested concentrations of an experiment.


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
- Range of historical values if different from the ones specified in the test guideline: Please refer to the historical control data in the 'Attached background material' section

Any other information on results incl. tables

Preliminary cytotoxicity assessment

The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 162 µg/mL.

Experiment 1: The concentrations selected (204 -739 µg/mL) were too high based on the results of a not assessable 1st pre-test (cytotoxicity below 50% was observed in all concentrations) and is not useful for evaluation and not included in the report. A 2nd pre-test was performed.

Experiment 2 : Concentration range selected was 54 -194 µg/mL. The experimen twas considered invalid due to a technical error and is not included in the report.

Experiment 3: This was carried out under the same conditions as the 2nd experiment. There was no cytotoxicity below 50% and no positive response (RFI CD86 ≥ 150% and/or CD54 ≥200%) was observed in the 3rd experiment, further experiments were carried out using the concentration range shown in Table 3. Calculation of the EC15 0% (the concentration resulting in a RFI of 150%) for CD86 and the EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable.

Experiment 4: The calculation of the EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable. The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated by linear regression from the results of the 194 µg/mL and the 233 µg/mL concentration to be 203 µg/mL.

Experiment 5: The calculation of the EC150% (the concentration rsulting in a RFI of 150%) for CD86 was not applicable. The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated by linear regression from the results of the 162 µg/mL and the 194 µg/mL concentration to be 188 µg/mL.

Table 2: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability.

2 valid and evaluable experiments (4thand 5th) were performed. The 1stand3rdexperiments are valid but not useful for evaluation and the 2ndexperiment was invalid and is not included in the report.

4thexperiment

5thexperiment

Concentration (test substance)

 

[µg/mL)

RFI CD86

 

Mean

[%]

RFI CD54

 

Mean

[%]

Viability

 

Relative viability [%]

Concentration (test substance)

 

[µg/mL)

RFI CD86

 

Mean

 [%]

RFI CD54

 

Mean

[%]

Viability

 

Relative viability [%]

78

58

85

100

78

66

139

100

94

57

76

100

94

58

117

100

112

60

112

100

112

61

144

100

135

62

103

100

135

62

131

100

162

62

125

100

162

65

174

99

194

88

166

97

194

92

205

96

233

127

311

90

233

118

370

90

279

137

451

77

279

138

541

80

VC

100

100

100

VC

100

100

100

LA 1000 µg/mL

77

102

100

LA 1000 µg/mL

70

132

100

DNCB 4 µG/mL

284

1222

70

DNCB 4 µG/mL

285

555

84

RFI above 150% (CD86) or 200% (CD54) with relative viability ≥50% are indicated in bold.

VC: vehicle (culture medium); LA : lactic acid, negative control; DNCB : 1 -chloro-2, 4 -dinitrobenzene, positive control

- The 3rd main experiment met the “borderline”-criteria at one concentration. The 4th and 5th experiments were unambigious positive in CD54.

- If the borderline criteria, defined as stated above, would be applied, these results would be considered ambiguous. A differently defined borderline range may yield different outcomes – no borderline range has yet been defined and assigned to the respective OECD test guideline.

Applicant's summary and conclusion

Interpretation of results:
other: positive prediction of skin sensitisation in h-CLAT assay
Conclusions:
Based on the observed results and applying the evaluation criteria in accordance with OECD Guideline 422 E, after 24-hours of exposure, Acid Blue 204 induced CD54 expression in THP-1 cells affording at least 50% viability in at least two independent experiments. Therefore, it can be concluded that Acid Blue 204 induces dendritic cell activation and is therefore predicted to be a skin sensitiser.
Executive summary:

The skin sensitising potential of the test substance, Acid Blue 204, was determined via the in vitro Human Cell Line Activation Test (h-CLAT) that evaluates that ability of Acid Blue 204 to induce the expression of cell membrane markers (CD86 and CD54) and thus activate dendritic cells. The study was conducted following the OECD Guideline for Testing of Chemicals TG. 442E, adopted July 2016 (‘In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)’)

 

Main Assay

The test substance, Acid Blue 204 was weighed and topped up with culture medium to achieve the required 2x concentration of the high concentration, Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution. The test substance was incubated with the human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry. 

In order to determine the concentrations suitable for the main experiment, two pre-tests (non-GLP) were performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 for the 2ndpre-test was 162 µg/mL3, determined by linear regression from the concentration response curve.

 

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-humanCD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid and evaluable experiments were performed (4th and 5th experiment). At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations and final concentrations) and no precipitates were observed at any concentration after 24 hours.

The concentration range (204 -730 µg/mL) used in Experiment 1 was considered too high and was considered not useful for evaluation, therefore a 2nd pre-test was performed. The second experiment (54 -194 µg/mL) was considered invalid due to a technical error. Experiment 1 and 2 are not included in the report. Experiment 3 was conducted under the same conditions as Experiment 2. The calculation of the EC150% for CD86 and the EC200% for CD54 was not applicable. Experiment 4 and 5 were conducted using a concentration range of 78 -279 µg/mL. In Experiment 4, the calculation of the EC150% for CD86 was not applicable and the EC200% for CD54 was calculated by linear regression from the results of the 194 µg/mL and the 233 µg/mL concentration to be 203 µg/mL. In Experiment 5, the calculation of the EC150% for CD86 was not applicable. The EC200% for CD54 was determined by linear regression from the results of the 162 µg/mL and the 194 µg/mL concentration to be 188 µg/mL. The acceptance criteria were met in all experiments and the results for the positive, negative and vehicle controls are comparable with historic data.

 

Conclusion

Based on the observed results and applying the evaluation criteria in accordance with OECD Guideline 422 E, after 24-hours of exposure, Acid Blue 204 induced CD54 expression in THP-1 cells affording at least 50% viability in at least two independent experiments. Therefore, it can be concluded that Acid Blue 204 induces dendritic cell activation and is therefore predicted to be a skin sensitiser.