Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 February 2017 - 06 July 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:
other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
2016
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
Principles of method if other than guideline:
In addition, the h-CLAT was performed according to the methods described in the following publications:
- Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct;26(7):1150-60.
- Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. (2010). A comparative evaluation of in vitro skin sensitization tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug;38(4):275-84.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.

Technical material and conditions:
- Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 µg/mL of penicillin and 100 μg/mL of streptomycin).
- FACS buffer: PBS with 0.1% (w/v) BSA
- Antibodies: Anti - CD86 antibody (Clone: Fun-1), Anti – CD54 antibody (Clone: 6.5B5),
FITC labelled-mouse IgG1 (isotype control)

Controls used:
- Vehicle control: DMSO (final concentration 0.2%)
- Positive control: DNCB in DMSO (diluted with culture medium to a final concentration of 2 and 3 μg/mL DNCB)
- Isotype control: mouse IgG1.

Test procedure:

Dose Finding Assay, XTT Test:
Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). All dose groups (eight concentrations) were tested in 7 replicates for each XTT test. The incubation period was 24 ± 0.5 hours. After incubation with the XTT labelling mixture the absorbance was measured at 450 nm.

Human Cell Line Activation Test (h-CLAT):
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining . The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry.

Acceptance criteria:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.

Evaluation of results:
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.

Results and discussion

Positive control results:
The results of the positive control were within the historical control range.

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: RFI % (CD54)
Run / experiment:
First run_502 µg/ml
Value:
209.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: RFI % (CD86)
Run / experiment:
First run_242 µg/ml
Value:
178.1
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: RFI % (CD86)
Run / experiment:
First run_290 µg/ml
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: RFI % (CD86)
Run / experiment:
First run_348 µg/ml
Value:
182.9
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: RFI % (CD86)
Run / experiment:
First run_418 µg/ml
Value:
158.1
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: RFI % (CD86)
Run / experiment:
First run_502 µg/ml
Value:
240
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: RFI % (CD86)
Run / experiment:
First run_602 µg/ml
Value:
233.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: RFI % (CD54)
Run / experiment:
Second run repetition_all concentrations
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: RFI % (CD86)
Run / experiment:
Second run repetition_all concentrations
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: RFI % (CD54)
Run / experiment:
Third run_all concentrations
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: RFI % (CD86)
Run / experiment:
Third run_all concenctrations
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
The results of the second run were not valid (cell viability < 90% at the highest tested test item concentration with a negative result). Therefore the second run was repeated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Results of the first h-CLAT run for the Test Item

 

Concentration
(µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

230.7*

291.1*

75.0

3.0

245.9*

279.9*

76.6

Test Item

168

130.0

141.4

105.0

202

132.2

148.1

107.2

242

153.0

178.1*

105.9

290

142.2

150.0*

96.3

348

153.0

182.9*

96.9

418

184.8

158.1*

96.4

502

209.6*

240.0*

99.7

602

191.7

233.3*

89.0

 * RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥ 150% and CD54 ≥ 200%)

Table 2: Results of the second h-CLAT run repetition for the Test Item

 

Concentration
(µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

400.9*

652.3*

56.9

3.0

579.4*

564.5*

58.7

Test Item

168

100.0

84.8

102.1

202

105.9

63.4

99.0

242

119.8

81.4

100.9

290

128.7

88.3

96.8

348

102.0

50.3

93.1

418

179.2

72.4

92.4

502

148.5

117.9

99.4

602

158.4

92.4

78.4

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥ 150% and CD54 ≥ 200%)

Table 2: Results of the third h-CLAT run for the Test Item

 

Concentration
(µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

333.0*

592.5*

68.3

3.0

342.7*

656.6*

67.4

Test Item

168

93.6

59.2

97.0

202

90.4

69.6

97.7

242

91.5

72.8

95.4

290

193.6

96.0

102.6

348

110.6

88.8

98.1

418

128.7

64.8

97.4

502

133.0

92.0

83.6

602

158.5

116.8

80.3

 * RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥ 150% and CD54 ≥ 200%)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not activate THP-1 cells up to the highest tested test item concentration (602 μg/mL). Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 168, 202, 242, 290, 348, 418, 502 and 602μg/mL.

The test substance was administered to THP-1 cells for 24 ± 0.5 hours. The test item was tested in 3 independent valid runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in at least 2 of 3 independent run data. Two out of three runs were NEGATIVE, therefore the test item is considered to have no skin sensitisation potential. The controls confirmed the validity of the study.