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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:
From May 30, 2018 to June 13, 2018
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.
Deviations:
no
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT)
Version / remarks:
EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 01 Jul 2015)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
OECD TG 442E cites the h-CLAT model using THP-1 cells as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: paste

In vitro test system

Details on study design:
Description of the test method
Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

Characterisation of the test method
The h-CLAT has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to 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.

Method workflow summary
Solubility was first determined for the test substance using either culture medium (RPMI 1640) or DMSO. Note that for this method, test substances with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test substances with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid. THP-1 cells were pre-cultured for 72 ±2 h. Following this, the cells were dosed with the test substance over an 8 dose range and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test substance that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs. THP-1 cells were pre-cultured for either 48 ±2 or 72 ±2 h. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test substance. This dilution range was used to dose the cells again for 24 ±0.5 h. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.

Test system material source
The human monocytic leukaemia cell line, THP-1, (Cat# TIB-202) were obtained from American Type Culture Collection (ATCC) via their UK subsidiary LGC standards; LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK. E-mail: atcc@lgcstandards.com. Tel: +44 (0)20 8943 8489. Two initial vials of cells were received in April 2016 and data to verify the referenced sources was archived..

OECD test Guideline relating to the test method
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 09 Oct 2017.

XCellR8 SOP
The study was performed using the h-CLAT method as detailed in OECD TG 442E and also in EURL ECVAM DB-ALM Protocol No. 158 (Issued 02 Mar 2017). Methodology is detailed in an XCellR8 SOP (L0094) that covers the h-CLAT. Study data is collected using XCellR8 internal protocols (IPs).

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: CV75 (µg/mL)
Remarks:
A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)
Run / experiment:
Average of two run
Value:
ca. 167.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: CD54 expression measured as RFI (up to 201.12 µg/mL, top dose concentration)
Run / experiment:
Average of two representatives
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: CD86 expression measured as RFI (up to 201.12 µg/mL, top dose concentration)
Run / experiment:
Average of two representatives
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.

Any other information on results incl. tables

Results

 

a) Solvent Selection and CV75 Determination

Prior to the CV75 determination, the test substance was assessed for solubility and was found to be soluble in complete RPMI culture medium at 250 mg/mL. The CV75 value derived from two independent experiments was as follows:

CV75 (Rep1)

CV75 (Rep2)

Average CV75 (µg/mL)

123.7

211.6

167.6

 

b) Acceptance Criteria

Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Note that the cell viability in the medium control in Run 2 for the CD54/86 measurements was a borderline value (86.61%) and was deemed acceptable even though it fell below the acceptance range for the control.

CV75 Determination

Criterion

Run 1

Run 2

Outcome

Cell viability must be ≥ 75% at the lowest dose.

95.24

96.98

PASS

The highest test substance concentration should produce cytotoxicity (< 90% cell viability) unless5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.

51.30

39.17

PASS

Measurement of CD54 and CD86 Expression

Criterion

Run 1

Run 2

Outcome

Cell viabilities of medium and solvent controls should be higher than 90%

97.07

86.61

PASS

In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) compared to the medium control

N/A

N/A

N/A

For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%

CD54: 159.29%

CD86: 139.77%

CD54: 219.67%

CD86: 179.02%

PASS

In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%.

CD54 RFI: 229

CD86 RFI: 158

CD54 Via:

90.20%

CD86 Via:

90.96%

CD54 RFI: 266

CD86 RFI: 155

CD54 Via:

72.06%

CD86 Via:

67.67%

PASS

For each test substance, the cell viability should be greater than 50% in at least four tested concentrations in each run

7/8

8/8

PASS

Negative results are acceptable only for test substances exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.

35.8

90.69

(Test substance is positive)

PASS

RFI - Relative Fluorescence Intensity, MFI - Mean Fluorescence Intensity, DMSO - Dimethyl Sulphoxide

 

c) Results Summary

The CV75 dose informs the dosing range selected for the CD54/86 expression assay. The top dose for the CD54/86 expression assay (main test) is 1.2 x CV75 which was equal to 201.12 µg/mL. The following tables show the expression of CD54 and CD86 against test substance dose with concurrent cytotoxicity measurement:

Run 1 (Valid): Result = Positive

Test Substance Dose (µg/mL)

Cell Viability (%)

Average Cell Viability (%)

CD54 RFI

CD86 RFI

Isotype

CD54

CD86

201.12

30.44

34.90

42.07

35.80

218

160

167.60

45.11

50.45

59.28

51.61

299

206

139.67

50.40

56.94

60.93

56.09

127

108

116.39

54.80

62.05

69.13

61.99

171

123

96.99

62.20

65.90

69.85

65.98

84

74

80.83

54.96

61.37

66.19

60.84

89

98

67.35

62.23

63.06

66.47

63.92

91

108

56.13

58.61

59.35

61.78

59.91

102

74

 

Run 2 (Valid): Result = Positive

Test Substance Dose (µg/mL)

Cell Viability (%)

Average Cell Viability (%)

CD54 RFI

CD86 RFI

Isotype

CD54

CD86

201.12

90.44

90.59

91.05

90.69

1086

1159

167.60

75.29

78.95

75.41

76.55

74

130

139.67

76.50

71.13

76.90

74.84

47

110

116.39

68.33

76.45

74.61

73.13

105

99

96.99

67.61

73.84

74.95

72.13

62

115

80.83

78.53

79.11

72.49

76.71

83

130

67.35

74.22

76.57

77.31

76.03

113

155

56.13

80.29

55.51

80.78

72.19

71

110

 

As can be seen from the data, the expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2 (highlighted in green). The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2 (highlighted in gold). As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%, highlighted in pink) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result is deemed to be valid.

 

The EC200 and EC150 values derived for the test substance were as follows:

Marker

EC200 (Rep1)

EC200 (Rep2)

Final EC200 Value

(µg/mL)

CD54

152

172

172

Marker

EC150 (Rep1)

EC150 (Rep2)

Final EC150 Value

(µg/mL)

CD86

152

66

152

 

For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were 172 and 152 µg/mL respectively, therefore, Test substance was classified as positive as per the prediction model.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was concluded to be skin-sensitiser.
Executive summary:

A study was conducted to determine the skin sensitisation potential of test substance, 'Cocodimonium hydroxypropyl hydrolysed wool (62.8% active)', using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E, in compliance of GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1 (a cell line that mimics DCs). The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells were pre-cultured for 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 µg/mL) in RPMI culture medium and incubated for 24 ± 0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The concentrations tested for CD54/CD86 expression assay were 201.12, 167.60, 139.67, 116.39, 96.99, 80.83, 67.35 and 56.13 µg/mL. The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2. The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2. As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result was considered to be valid. For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were calculated to be 172 and 152 µg/mL respectively; therefore, the test substance was classified as positive as per the prediction model. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under the study conditions, the test substance was concluded to be skin-sensitiser (XcellR8, 2018).