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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

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Diss Factsheets

Administrative data

Description of key information

There is now general agreement on the key chemical/ biological events that lead to skin sensitisation, and this information was summarized and published in 2012 by the OECD in "The Adverse Outcome Pathway (AOP) for skin sensitisation initiated by covalent binding to proteins". The AOP identifies 4 key events in the development of skin sensitisation; the first is the covalent binding of the substance with skin proteins (the molecular initiating event). The second takes place in the keratinocyte and includes inflammatory responses as well as gene expression associated with cell signalling pathways. The third is the activation of dendritic cells and finally, T-cell proliferation.

Knowledge of the skin sensitisation pathway has prompted the development of in vitro methods that address these key events, and at present, 3 in chemico/in vitro tests, each addressing a specific key event of the AOP, have been adopted by the OECD and validated by EURL ECVAM. Annex VII now stipulates that these test methods should be the first step in addressing skin sensitization, and only in the case that these results are not adequate, should an in vivo study be performed. It is on this basis, that the skin sensitisation potential of Bis(hydroxyethyl)methyloleylammonium chloride was assessed, using a combination of in vitro studies as part of a weight of evidence approach and found to be non-sensitizing.

The first key event of the AOP is the covalent binding of the substance to proteins, this was assessed using the In chemico Direct Peptide Reactivity Assay (DPRA) in accordance with OECD guideline 442C. This method quantifies the reactivity between test item and synthetic proteins containing lysine or cysteine by measuring the peptide percent depletion values using HPLC after 24 hr incubation, the Cysteine peptide 1:10 / Lysine 1:50 prediction model is then used to determine sensitisation potential. The final mean percent peptide depletion for Bis(hydroxyethyl)methyloleylammonium chloride was 4.385%, and according to the cysteine 1:10 / lysine 1:50 predication model, this substance is classified as a Non-sensitizer with No or Minimal reactivity.

The second key event of the skin sensitisation AOP is the Keratinocyte inflammatory response. The keratinocyte activation for Bis(hydroxyethyl)methyloleylammonium chloride was assessed using the in vitro KeratinoSens assay according to the OECD guideline 442D. The KeratinoSens cell line is derived from human keratinocytes, transfected with a selectable plasmid containing the luciferase gene under transcriptional control of the Anti-oxidant Response Element from a gene that is known to be upregulated by contact sensitizers. The luciferase signal reflects the activation by sensitizers of the Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows for measurement of luciferase gene induction, as an indicator of the activity of Nrf2 transcription factor in cells. After 48hr exposure of cells with 12 concentrations of Bis(hydroxyethyl)methyloleylammonium chloride, luciferase measurements and MMT viability testing were performed. In this study, Bis(hydroxyethyl)methyloleylammonium chloride did not induce statistically significant luciferase induction when cell viability >70% in any repetition and therefore is classified as negative as per the KeratinoSens predication model.

The final key event assessed for Bis(hydroxyethyl)methyloleylammonium chloride was the activation of dendritic cells, using the In Vitro human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E. This assay quantifies the changes in cell surface marker expression associated with Dendritic Cell activation (CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24-hr exposure to test item using flow cytometry.For Bis(hydroxyethyl)methyloleylammonium chloride the CV75 assay was inconclusive, therefore the highest possible dose (1000µg/ml) was taken forward to the main test. The EC200 and EC150 values for CD54 and CD86 expression were 254 and 286 µg/ml respectively, therefore, Bis(hydroxyethyl)methyloleylammonium chloride was classified as Positive as per the h-CLAT prediction model.

In conclusion, the results from the DPRA assay and the KeratinoSens assay were both unequivocally negative. This shows that Bis(hydroxyethyl)methyloleylammonium chloride does not covalently bind to proteins to cause the molecular initiating event of the AOP, which has been suggested as the main potency-determining step in skin sensitisation, or cause keratinocyte activation. It was therefore determined that Bis(hydroxyethyl)methyloleylammonium chloride is non-sensitising to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Positive control results:
Positive Control (Cinnamic aldehyde) induction >1.5-fold at concentration 16-128µM and EC1.5 was calculated as 8.113 µM.
Key result
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

In this study, Bis(hydroxyethyl)methyloleylammonium chloride was classified as Negative using the KeratinoSens prediction model.

The test item did not induce statistically significant luciferase induction >1.5 in repetitions 2 and 3 but did induce significant luciferase induction in repetition 1. The respective EC1.5 value in rep 1 was calculated as 0.869 µM, however the viability was less than 70% at the inducing concentration and therefore rep 1 was also considered negative.

Sensitisation Potential of the Test Item: Repetition 1

Rep 1

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.500

125.000

250.000

500.000

1000.000

2000.000

Mean fold induction

1.687

1.795

0.694

0.025

0.001

0.002

0.004

0.006

0.012

0.035

0.073

0.006

Viability %

56.329

19.937

-1.582

-6.962

-7.278

-5.063

0.000

-2.848

-3.797

-0.949

2.215

6.329

T-test

4.61E-06

4.08E-07

3.42E-02

2.44E-09

1.27E-09

1.28E-09

1.37E-09

1.43E-09

1.69E-09

3.22E-09

9.37E-09

1.46E-09

SD

0.078

0.220

0.156

0.019

0.000

0.000

0.001

0.001

0.003

0.008

0.021

0.002

IMAX

1.795 at 1.953 µM

EC1.5

0.869 µM

IC30

0.610 µM

IC50

1.147 µM

 

Sensitisation Potential of the Test Item: Repetition 2

Rep 2

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.500

125.000

250.000

500.000

1000.000

2000.000

Mean fold induction

1.020

1.374

1.409

0.409

0.004

0.004

0.006

0.006

0.011

0.043

0.122

0.012

Viability %

83.740

56.325

22.708

8.848

-6.163

-5.071

-0.527

-1.368

-0.176

1.443

3.665

4.268

T-test

8.47E-01

1.17E-02

4.28E-03

8.39E-05

1.37E-09

1.37E-09

1.43E-09

1.44E-09

1.65E-09

4.02E-09

3.75E-08

1.73E-09

SD

0.275

0.380

0.229

0.143

0.001

0.000

0.001

0.001

0.002

0.014

0.051

0.005

IMAX

1.409 at 3.906 µM

EC1.5

N/A - threshold of induction not crossed at any test item concentration

IC30

1.466 µM

IC50

2.320 µM

 

Sensitisation Potential of the Test Item: Repetition 3

Rep 3

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.500

125.000

250.000

500.000

1000.000

2000.000

Mean fold induction

0.644

0.698

0.483

0.040

0.002

0.002

0.003

0.005

0.010

0.033

0.057

0.005

Viability %

75.116

37.728

8.435

-6.187

-5.544

-4.961

1.779

-0.089

0.030

1.398

4.843

9.132

T-test

1.52E-02

4.03E-02

4.82E-04

3.73E-09

1.29E-09

1.30E-09

1.33E-09

1.40E-09

1.60E-09

3.02E-09

5.88E-09

1.40E-09

SD

0.218

0.256

0.140

0.020

0.000

0.001

0.001

0.002

0.003

0.011

0.011

0.002

IMAX

0.698 at 1.953 µM

EC1.5

N/A - threshold of induction not crossed at any test item concentration

IC30

1.111 µM

IC50

1.633 µM

Determination criteria for the skin sensitisation potential ofthe test item

 

REP1

REP2

REP3

Does at least one concentration of Test Item induce luciferase activity >1.5-fold:

Yes

No

No

Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:

No

N/A

N/A

Is p value < 0.05 for at least one concentration that yielded >1.5-fold induction with viability above 70%

Yes

N/A

N/A

Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)

Yes

N/A

N/A

Does the test item induce the luciferase in a dose-dependent manner

No

N/A

N/A

Classification

Negative

Negative

Negative

All of the formal acceptance criteria of the tests were met,except for the average induction of the positive control (cinnamic aldehyde) at 32 µM, which was not within the historical range (1.6 and 3).However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered to be valid.

Assay Acceptance Criteria (Mean of 3 repetitions)

Criteria

Result

PASS or FAIL

1

Positive Control (PC) (Cinnamic aldehyde) induction>1.5-fold in at least one concentration

16-128µM

PASS

2

Average induction of PC at 32µM is [1.6-3.0]

4.773

FAIL

3

EC1.5value is [6-39µM]

8.113

PASS

4

CV% of blank values < 20%

17.0218

PASS

Interpretation of results:
GHS criteria not met
Conclusions:
In a study conducted according to OECD Test Guideline 442D, Bis(hydroxyethyl)methyloleylammonium chloride was found to be not sensitising to the skin.
Executive summary:

The human skin sensitisation potential of Bis(hydroxyethyl)methyloleylammonium chloride was assessed using the validated in vitro method, the KeratinoSens assay, in accordance with the current OECD Test Guideline 442D. This study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

 

All of the formal acceptance criteria of the tests were met, with the exception of the average induction of the positive control (cinnamic aldehyde) at 32 µM, which was not within the historical range (1.6 and 3). However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, the results of this study are considered to be valid.

 

The results from this study found that Bis(hydroxyethyl)methyloleylammonium chloride did not induce statistically significant luciferase induction (>1.5) in repetitions 2 or 3. Significant luciferase induction was induced in repetition 1 and the respective EC1.5 value was calculated as 0.869 µM, however, the cellular viability was less than 70% at the inducing concentration and therefore rep 1 was also considered negative. Bis(hydroxyethyl)methyloleylammonium chloride is therefore classified as Negative according to the KeratinoSens prediction model.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018
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))
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.
Key result
Parameter:
other: Mean peptide depletion (Cys + Lys)
Value:
4.385
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

The test item produced 4.385% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test item was classified as a Non Sensitiser with No or Minimal Reactivity. A single HPLC analysis for both the Cysteine and the Lysine peptide was considered sufficient for the test item as the result was unequivocal.

Test Item ID

% Cysteine Peptide Depletion

% Lysine Peptide Depletion

Mean % Peptide Depletion
(Cys + Lys)

DPRA Prediction

 

DPRA

Reactivity Class

TA1

7.562

1.208

4.385

Non-Sensitiser

No or Minimal reactivity

TA1 = Bis(hydroxyethyl)methyloleylammonium chloride

Acceptance criteria for all controls and the test item were met in both runs.

Criterion

Run 1 (Cysteine)

Run 2 (Lysine)

Outcome

Std Curve r2>0.99

0.995

0.994

PASS

PC 60.8% to 100% depletion Cys

60.846

N/A

PASS

PC 40.2% to 69.0% depletion Lys

N/A

53.202

PASS

SDCys DepletionPC<14.9%

9.578

N/A

PASS

SDLys DepletionPC<11.6%

N/A

0.673

PASS

RefA Mean Conc0.50 ± 0.05mM

0.545

0.480

PASS

Peak Area CV RefB<15.0%

0.596

0.679

PASS

Peak Area CV RefC<15.0%

1.107

0.415

PASS

SDCys DepletionTest Item<14.9%

0.175

N/A

PASS

SDLys DepletionTest Item<11.6%

N/A

0.622

PASS

RefC Mean Conc0.50 ± 0.05mM

0.534

0.515

PASS

Cys = Cysteine, Lys = Lysine, SD = Standard Deviation, CV = Coefficient of Variation, PC = Positive Control.

The validated excel analysis template used to derive the % depletion values was populated with the raw data obtained from the HPLC. The raw data (peak areas at 220nm absorbance) generated from the HPLC system is summarised, with the processed data output from the validated excel analysis template. The standards are used to assign peptide concentrations to each control or test item as appropriate. The peptide depletion for the test item is calculated with respect to the mean RefC peak area value. Identification of the correct peaks corresponding to the peptides was carried out by trained operators by looking at peak areas within the relevant retention time window.

Cysteine Peptide Data

Peak Areas, Peptide Concentration and Peptide Depletion: Replicate Data

Sample ID

Peak Area

Peptide Conc (mM)

Peptide Depletion

Corrected % Peptide Depletion

STD 1

7169147

0.5340

-

-

 

STD 2

2905847*

0.2670

-

-

 

STD 3

1139968

0.1335

-

-

 

STD 4

454473

0.0667

-

-

 

STD 5

199343

0.0334

-

-

 

STD 6

79826

0.0167

-

-

 

STD 7

No peak

0.0000

-

-

 

RefA 1

7072652

0.533

-

-

 

RefA 2

7317324

0.550

-

-

 

RefA 3

7319512

0.551

-

-

 

Co-Elution 1

57478

-

-

-

 

RefB 1

7165468

0.540

-

-

 

RefB 2

7266093

0.547

-

-

 

RefB 3

7179645

0.541

-

-

 

RefB 4

7167517

0.540

-

-

 

RefB 5

7145784

0.538

-

-

 

RefB 6

7165038

0.540

-

-

 

RefC 1

7045117

0.531

-

-

 

RefC 2

7181338

0.541

-

-

 

RefC 3

7045550

0.531

-

-

 

PC 1

3396599

0.270

52.098

52.098

 

PC 2

2881489

0.234

59.362

59.362

 

PC 3

2050660

0.174

71.079

71.079

 

TA1 Rep 1

6563201**

0.497

7.439

7.439

 

TA1 Rep 2

6559896**

0.496

7.486

7.486

 

TA1 Rep 3

6540298**

0.495

7.762

7.762

 

Boxes with “-“ are not applicable for that sample type. *= excluded outlier value for STD2. **= TA1 peak areas minus the peak area from the minimal co-elution value shown in the table (57478).

 

 Peak Areas, Peptide Concentration and Peptide Depletion: Mean, SD and CV

Sample ID

Peak Area

Peptide Conc

% Peptide Depletion

Mean

SD

% CV

Mean

SD

CV

Mean

SD

CV

RefA

-

-

-

0.545

0.010

1.861

-

-

-

RefB

7181590.833

42798.473

0.596

0.541

0.003

0.565

-

-

-

RefC

7090668.333

78522.533

1.107

0.534

0.006

1.050

-

-

-

PC

2776249.333

679113.016

24.462

0.226

0.049

21.470

60.846

9.578

15.741

TA1

6554465.000

12379.769

0.189

0.496

0.001

0.178

7.562

0.175

2.309

Boxes with “-“ are not applicable for that sample type.

 

Lysine Peptide Data

Peak Areas, Peptide Concentration and Peptide Depletion: Replicate Data

Sample ID

Peak Area

Peptide Conc (mM)

Peptide Depletion

Corrected % Peptide Depletion

STD 1

6647604

0.5340

-

-

 

STD 2

2570470*

0.2670

-

-

 

STD 3

1012106

0.1335

-

-

 

STD 4

399215

0.0667

-

-

 

STD 5

157508

0.0334

-

-

 

STD 6

59474

0.0167

-

-

 

STD 7

No peak

0.0000

-

-

 

RefA 1

5837309.5

0.478

-

-

 

RefA 2

5839027

0.478

-

-

 

RefA 3

5903637

0.483

-

-

 

Co-Elution 1

18292.09

-

-

-

 

RefB 1

5880062

0.481

-

-

 

RefB 2

5850722.5

0.479

-

-

 

RefB 3

5927054.5

0.485

-

-

 

RefB 4

5816810

0.476

-

-

 

RefB 5

5832492

0.478

-

-

 

RefB 6

5841858

0.478

-

-

 

RefC 1

6345739.5

0.517

-

-

 

RefC 2

6307118

0.514

-

-

 

RefC 3

6295677

0.513

-

-

 

PC 1

2922488.73

0.254

53.730

53.730

 

PC 2

2941348

0.256

53.432

53.432

 

PC 3

3003716

0.260

52.444

52.444

 

TA1 Rep 1

6262062.41**

0.511

0.857

0.857

 

TA1 Rep 2

6194510**

0.505

1.926

1.926

 

TA1 Rep 3

6263037**

0.511

0.841

0.841

 

Boxes with “-“ are not applicable for that sample type. * = excluded outlier value for STD2. ** = TA1 peak areas minus the peak area from the minimal co-elution value shown in the table (18292.09).

 

Peak Areas, Peptide Concentration and Peptide Depletion: Mean, SD and CV

Sample ID

Peak Area

Peptide Conc

% Peptide Depletion

Mean

SD

% CV

Mean

SD

CV

Mean

SD

CV

RefA

-

-

-

0.480

0.003

0.605

-

-

-

RefB

5858166.500

39789.169

0.679

0.480

0.003

0.637

-

-

-

RefC

6316178.000

26232.398

0.415

0.515

0.002

0.391

-

-

-

PC

2955850.723

42511.139

1.438

0.257

0.003

1.271

53.202

0.673

1.265

TA1

6239869.910

39285.501

0.630

0.509

0.003

0.593

1.208

0.622

51.483

Boxes with “-“ are not applicable for that sample type.

Interpretation of results:
GHS criteria not met
Conclusions:
In a study conducted according to OECD Test Guideline 442C, Bis(hydroxyethyl)methyloleylammonium chloride was found to be not sensitising to the skin.
Executive summary:

The human skin sensitisation potential of Bis(hydroxyethyl)methyloleylammonium chloride was assessed using the validated In Chemico Direct Peptide Reactivity Assay (DPRA) method, in accordance with the current OECD Test Guideline 442C and found to be a Non-Sensitiser with No or Minimal reactivity. This study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

 The DPRA is proposed to address the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of test and reference items towards model synthetic peptides containing either Lysine or Cysteine. Cysteine and Lysine percent peptide depletion values are then used to categorise a substance in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers. The DPRA method has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-led validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and is considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA).

 In this study, Bis(hydroxyethyl)methyloleylammonium chloride and reference samples were incubated with either Cysteine or Lysine containing peptides. After 24hr incubation, a HPLC was run and data analysis carried out using the validated EURL-ECVAM analysis template, in order to determine percentage peptide depletion values. The skin sensitization potential was then assessed using the Cysteine peptide 1:10 / Lysine peptide 1:50 prediction model, where the threshold of 6.38% average peptide depletion is used to discriminate between skin sensitisers and non-sensitisers.

The results from this study show that Bis(hydroxyethyl)methyloleylammonium chloride produced 4.385% mean Cysteine and Lysine peptide depletion, and therefore is classified as a Non-Sensitiser with No or Minimal reactivity as per the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Parameter:
other: CD54 EC200 value
Remarks:
µg/ml
Value:
254
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: CD86 EC150 value
Remarks:
µg/ml
Value:
286
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

The criterion stating “cell viability must be ≥ 75% at the lowest dose” was not met in either run for the CV75 determination. However, the run as a whole was acceptable because the viability was above 75% at both top doses (80.93% Rep 1, 75.3% Rep 2). The data from this assay was not used to determine the dose for the CD54 and CD86 expression, instead the highest possible dose (1000µg/ml) was taken forward into the main test.

Acceptance criteria for all controls and the test item were met in both runs for the measurement of CD54 and CD86 expression.

 

CV75 Determination

Criterion

Run 1

Run 2

Outcome

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

N/A but above 75% at the top dose.

N/A but above 75% at the top dose.

FAIL

The highest test item 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 item.                                                                                                                                                                                                    

80.93

75.3

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%

90.71

97.42

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: 123.08 %

CD86: 113.31 %

CD54: 133.93%

CD86: 121.41%

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: 656

CD86 RFI: 214

CD54 Via:

87.59%

CD86 Via:

85.48%

CD54 RFI: 593

CD86 RFI: 163

CD54 Via:

87.28%

CD86 Via:

87.99%

PASS

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

8/8

6/8

PASS

Negative results are acceptable only for test items 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 item.                                                                                                                                                                                                    

82.75

75.90

PASS

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

The following tables show the expression of CD54 and CD86 against test item dose with concurrent cytotoxicity measurement:

Run 1 (Valid): Result = Positive

Test Item Dose (µg/ml)

Cell Viability (%)

Average Cell Viability (%)

CD54 RFI

CD86 RFI

Isotype

CD54

CD86

1000.00

81.80

83.21

83.25

82.75

-3603

-9140

833.33

80.58

83.67

80.71

81.65

1510

-7228

694.44

71.81

76.15

76.84

74.93

21227

16706

578.70

79.06

73.08

79.38

77.18

36994

51545

482.25

73.87

70.62

66.06

70.18

169197

188416

401.88

69.31

74.37

70.09

71.26

48204

53625

334.90

73.80

72.18

68.43

71.47

12218

2770

279.08

51.18

57.79

52.85

53.94

4450

-202

 

Run 2 (Valid): Result = Positive

Test Item Dose (µg/ml)

Cell Viability (%)

Average Cell Viability (%)

CD54 RFI

CD86 RFI

Isotype

CD54

CD86

1000.00

71.33

79.59

76.78

75.90

191770

240776

833.33

69.49

76.27

74.12

73.29

156776

212741

694.44

61.01

67.20

69.58

65.93

144250

169559

578.70

64.54

69.33

68.88

67.58

198455

200770

482.25

56.60

60.48

58.37

58.48

151163

168880

401.88

53.52

53.59

49.66

52.26

118911

138075

334.90

49.57

45.41

48.42

47.80

112930

124023

279.08

26.15

22.64

25.95

24.91

38225

52836

 

The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at multiple doses in both runs, as did the expression of CD86 (RFI ≥150).

As the CD54/CD86 expression crossed the threshold, Bis(hydroxyethyl)methyloleylammonium chloride is classified as Positive using the h-CLAT prediction model.

Cell viability was 50% in at least 4 of the test item concentrations and therefore the result is deemed to be valid.

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

 

Marker

EC200 (Rep1)

EC200 (Rep2)

Final EC200 Value (µg/ml)

CD54

253

254

254

Marker

EC150 (Rep1)

EC150 (Rep2)

Final EC150 Value (µg/ml)

CD86

286

244

286

Conclusions:
In a study conducted according to OECD Test Guideline 442E, Bis(hydroxyethyl)methyloleylammonium chloride was classified as Positive as per the h-CLAT prediction model.
Executive summary:

The skin sensitisation potential of Bis(hydroxyethyl)methyloleylammonium chloride was assessed using theIn Vitrohuman Cell Line Activation Test (h-CLAT) method according to OECD Test Guideline 442E. This study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

 

For Bis(hydroxyethyl)methyloleylammonium chloride the CV75 assay was inconclusive and therefore the highest possible dose (1000µg/ml) was taken forward to the main test.

 

In the study, the expression of CD54 and CD86, as measured by the RFI crossed the threshold (RFI ≥200 for CD54 and RFI ≥150 for CD86) at multiple doses.

 

The EC200 and EC150 values for CD54 and CD86 expression were 254 and 286 µg/ml respectively, therefore, Bis(hydroxyethyl)methyloleylammonium chloride was classified as Positive as per the h-CLAT prediction model.

 

As the cell viability was 50% in at least 4 of the test item concentrations and therefore the result is deemed to be valid.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

The human skin sensitisation potential of Bis(hydroxyethyl)methyloleylammonium chloride was assessedand found to be non-sensitizing using both the In Chemico Direct Peptide Reactivity Assay (DPRA) in accordance with OECD Guideline 442C and thei n vitro KeratinoSens assay, in accordance with OECD Guideline 442D.

The DPRA is proposed to address the molecular initiating event of the skin sensitisation AOP by quantifying the reactivity of the test item towards synthetic peptides containing either Lysine or Cysteine. Cysteine and Lysine percent peptide depletion values are then used to categorise the substance in 1 of 4 classes of reactivity, where the threshold of 6.38% average peptide depletion is used to discriminate between skin sensitisers and non-sensitisers.  In this study, Bis(hydroxyethyl)methyloleylammonium chloride produced 4.385% mean Cysteine and Lysine peptide depletion, and therefore is classified as a Non-Sensitiser with No or Minimal reactivity as per the Cysteine 1:10 / Lysine 1:50 prediction model.

The KeratinoSens assay measures luciferase gene induction following exposure to test item, which reflectsthe activation of Nrf2 dependent genes by sensitisers.The results from this study found that Bis(hydroxyethyl)methyloleylammonium chloride did not induce statistically significant luciferase induction (>1.5) when cell viability >70% in any repetition and therefore is classified as negative as per the KeratinoSens predication model.

The activation of dendritic cells by Bis(hydroxyethyl)methyloleylammonium chloride was assessed using In Vitro human Cell Line Activation Test (h-CLAT) in accordance with OECD guideline 442E and found to be positive as per the h-CLAT prediction model. However, as the results from both the DPRA and KeratinoSens assay were bothunequivocally negative, showing that Bis(hydroxyethyl)methyloleylammonium chloride does not covalently bind to proteins to cause the molecular initiating event of the AOP or cause keratinocyte activation, it was determined Bis(hydroxyethyl)methyloleylammonium chloride is non-sensitising to skin.