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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:
03 August 2017 - 22 January 2018 (final study report)
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:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
GLP compliance:
yes (incl. certificate)
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 29-490
- Expiration date of the lot/batch: February 2019
- Purity: 82.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2x concentration of the highest concentration (stock solution).
- Culture medium was chosen as the vehicle, since the test substance is soluble in culture medium.
- Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations in culture medium.
- The test-substance preparations were prepared by stirring and ultra-sonication.
- Visual inspection of each dilution step was performed

In vitro test system

Details on 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
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RFI CD86
Run / experiment:
Experiment 3
Value:
>= 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Activation of dendritic cells positive according to OECD 442E at concentrations of 4.4 µg/mL
Remarks:
viability ≥50%
Key result
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RFI CD54
Run / experiment:
Experiment 3
Value:
>= 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Activation of dendritic cells positive according to OECD 442E at concentrations of 3.7 µg/mL
Remarks:
viability ≥50%
Key result
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RDI CD86
Run / experiment:
Experiment 4
Value:
>= 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Activation of dendritic cells positive according to OECD 442E at concentrations of 4.4 µg/mL
Remarks:
viability ≥50%
Key result
Parameter:
other: Relative fluorescence intensity (RFI) %
Remarks:
RFI CD54
Run / experiment:
Experiment 4
Value:
>= 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Activation of dendritic cells positive according to OECD 442E at concentrations of 2.6, 3.1, 3.7 and 4.4 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

Experiment 1

- Concentrations of 88 µg/mL-315 µg/mL were tested based on the 1st pre-test. The experiment was valid but can not be used for evaluation as all test-substance concentrations were clearly cytotoxic (relative viability well below 50%, details are available with the raw data).

- The results of the 1st pre-test is also not included in this report as the pre-test is considered to be invalid due to the differing results of the 1st main experiment

Experiment 2

- Concentrations of 2.6 µg/mL-9.2 µg/mL were testted based on the 2nd pre-test.

- The experiment is invalid for not meeting acceptance criteria and 4 out of 8 test-substance concentrations had a relative viability below 50%.

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

2 valid and evaluable experiments (3rdand 4th) were performed. The 1stexperiment was not evaluable due to high cytotoxicity and the 2ndexperiment was invalid for not meeting acceptance criteria and is not included in this report.

3rdexperiment

4thexperiment

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 [%]

1.8

62

123

100

1.8

65

159

99

2.1

61

130

99

2.1

57

155

99

2.6

71

142

99

2.6

79

208

98

3.1

86

175

96

3.1

92

273

96

3.7

113

213

90

3.7

106

313

91

4.4

152

181

72

4.4

151

295

70

5.3

175

222

46

5.3

217

306

43

6.4

156

109

31

6.4

205

195

21

VC

100

100

100

VC

100

100

100

LA 1000 µg/mL

60

123

100

LA 1000 µg/mL

64

126

100

DNCB 4 µG/mL

184

407

90

DNCB 4 µG/mL

193

697

81

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 (marker CD86 and marker CD54) and the 4th experiment (marker CD86, only) met the “borderline”-criteria.  

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.

3rdexperiment

Basic Red 29 induced CD86 expression ≥ 150% signified by the RFI CD86 mean 152% result at a concentration 4.4 µg/mL and induced CD54 expression increased ≥ 200%, indicated by the RF1 CD54 mean 213% results at a concentration of 3.7 µg/mL.

4thexperiment

The mean RFI CD86 at 4.4 µg/mL was 151% and the mean RFI CD54 was 208, 273, 313 and 295% at 2.6, 3.1, 3.7 and 4.4 µg/mL respectively.

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, Basic Red 29 induces dendritic cell activation and is predicted to be a skin sensitiser.
Executive summary:

The skin sensitising potential of the test substance, Basic Red 29, was determined via the in vitro Human Cell Line Activation Test (h-CLAT) that evaluates that ability of Basic Red 29 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, Basic Read 29 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 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, 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 7.6 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. 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.

In the first experiment, a concentration range of 88 -315 µg/mL was tested based on the 1st pre-test. The experiment was valid, but almost all of the test-substance concentrations were cytotoxic with relative viability well below 50%. Therefore a 2nd pre-test was performed in order to determine suitable concentrations for further experiments. A concentration range used in Experiment 2 was 2.6 -9.2 µg/mL, based on the 2nd pre-test. The experiment was not invalid for not meeting acceptance criteria and 4 out of 8 test-substance concentrations had a relative viability below 50%.

 

In the third experiment, the EC150% for CD86 was calculated by linear regression from the results of the 3.7-4.4 µg/mL concentration to be 4.4 µg/mL and the EC200% for CD54 was determined to be 2.5 µg/mL. In the 4thexperiment, the EC150% for CD86 was calculated by linear regression from the results of the 3.7 µg/mL and 4.4 µg/mL concentration to be 4.4 µg/mL and the EC200% for CD54 was determined from the results of the 2.1 µg/mL and the 2.6 µg/mL to be 2.5 µg/mL. Both results from the 3rdexperiment and the EC150% CD86 result only met the ‘borderline’ criteria. Therefore, if a differently defined borderline range was applied, it may yield different outcomes and the results would be considered ambiguous. However, no borderline range has yet been defined and assigned to the respective OECD test guideline.

 

In accordance with the evaluation of results, in the 3rdexperiment, Basic Red 29 induced CD86 expression ≥ 150% signified by the RFI CD86 mean 152% result at a concentration 4.4 µg/mL with a relative viability of 72% and induced CD54 expression increased ≥ 200%, indicated by the RF1 CD54 mean 213% results at a concentration of 3.7 µg/mL with a relative viability of 90%. In the 4thexperiment, the mean RFI CD86 at 4.4 µg/mL was 151% with a relative viability of 70% and the mean RFI CD54 was 208, 273, 313 and 295% at 2.6, 3.1, 3.7 and 4.4 µg/mL respectively with a relative viability of 98, 96, 91 and 70% respectively. This thus indicates that the test substance is predicted to activate monocytic THP-1 cells. The results are also relevant for evaluation, since the relative cell viability results are more than 90% in more than four of the test concentrations in the 3rd and 4th experiment.

 

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 taking into account the evaluation criteria, it can be concluded that after 24 hours of exposure to the test substance, Basic Red 29, CD86 and CD54 expression was induced in THP-1 cells with at least 50% viability in at least two independent experiments. Therefore, it can be concluded that Basic Red 29 induces dendritic cell activation and is predicted to be a skin sensitiser.