Octan-1-ol, ethoxylated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 - 20 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (in vitro Skin Sensitisation: human Cell Line Activation Test)

Version / remarks:

adopted 20 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Medicines and Healthcare Products Regulatory Agency, Department of Health, London, United Kingdom

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Octan-1-ol, ethoxylated

EC Number:

500-058-1

EC Name:

Octan-1-ol, ethoxylated

Cas Number:

27252-75-1

Molecular formula:

C10H22O2

IUPAC Name:

Octan-1-ol, ethoxylated

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vitro test system

Details on the study design:

TEST METHOD:
The in vitro human Cell Line Activation Test (h-CLAT) is an alternative testing method for the evaluation of the skin sensitization potential of a test compound. It quantifies phenotypic changes, such as cell surface marker expression in cell lines following 24 h treatment with chemicals. The human leukemia cell line THP-1 is used as surrogate for human myeloic dendritic cells, which show enhanced CD86 and CD54 surface protein expression when treated with sensitiziers.
The dose for the h-CLAT assay was determined in two preliminary cytotoxicity test, yielding 75% cell viability (CV75). For the main assay, THP-1 cells were incubated for 24 ± 1 hours at 37 °C with the test compound, as well as the negative and positive controls. Changes of CD86 and CD54 expression were analyzed by flow cytometry, using fluorescently labelled antibodies against the two surface proteins. Relative fluorescence intensities compared to solvent controls are calculated and used in a prediction model to discriminate between sensitizing and non-sensitizing compounds.

TESTS SUBSTANCE PREPARATION:
The test item was dissolved in culture medium.

CONCENTRATIONS:
Pre-experimental dose-finding study: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 µg/mL
Main experiment (h-CLAT): based on the results obtained in the pre-experimental dose-finding study: 595.25, 496.04, 413.37, 344.47, 287.06, 239.22, 199.35, 166.12 µg/mL.

VEHICLE CONTROL: Complete Roswell Park Memorial Institute (RPMI) culture medium containing 10% Human Serum and 0.05 mM 2-mercaptoethanol
POSITIVE CONTROL CV75: 2,4-Dinitrochlorobenzene (DNCB) prepared as 8 µg/mL in DMSO
POSITIVE CONTROL CD54 and CD86 expression: Nickel Sulphate prepared as 100 µg/mL in RPMI medium

TEST CELL LINE: THP-1 cells
- Source: ATCC, #TIB-202

CELL CULTURE CONDITIONS:
- Type and identity of media: RPMI supplemented with 10% Human Serum and 0.05 mM 2-mercaptoethanol

EXPOSURE CONDITIONS:
- Method of application: in medium
- Exposure duration: 24 ± 0.5 h

NUMBER OF REPLICATES: Each concentration was tested in two independent runs

DETERMINATION OF CYTOTOXICITY:
- Method: Propidium iodide, 24 ± 0.5 h exposure with test item, two independent experiments
- Determination of cell viability (= relative aborbance) for calculation of the CV75, which corresponds to the concentration needed to reduce the relative absorbance to 75% of the solvent control.

DETERMINATION OF FLUORESCENCE:
- Flow cytometry
- Antibodies: fluorochrome-tagged CD86 and CD54

Results and discussion

Positive control results:

Relative fluorescence intensities first experiment:
100 µg/mL: CD54 = 212% (93.69% viability), CD86 = 152% (93.04% viability)

Relative fluorescence intensities, second experiment:
100 µg/mL: CD54 = 377% (88.95% viability), CD86 = 210% (86.58% viability)

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: in 2/2 independent experiment data

Results 2

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD86 in µg/mL

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: in 2/2 independent experiment data

Other effects / acceptance of results:

- Acceptance criteria met for CV75 determination: Yes, cell viability is ≥ 75% at the lowest dose and the highest test item concentration produces cytotoxicity (< 90% cell viability)
- Acceptance criteria met for negative control: yes, medium and solvent control RFI values do not exceed the positive criteria CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 90%
- Acceptance criteria met for positive control: yes, RFI values CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%

Any other information on results incl. tables

Table 1: Results of the h-CLAT test, first experiment:

Test Item Dose [µg/mL)	Cell Viability (%)			Average cell viability	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
595.25	55.95	60..92	58.46	58.44	-335	-871
496.04	51.28	53.04	53.36	52.56	-239	-625
413.37	57.3	52.62	53.8	54.57	-145	-425
344.47	60.74	55.92	56.7	57.79	135	120
287.06	71.13	65.12	65.53	67.26	124	126
239.22	78.66	75.32	75.64	76.54	114	105
199.35	86.92	83.85	82.52	84.43	87	87
166.12	85.81	85.22	85.14	85.39	82	65

Table 2: Results of the h-CLAT test, second experiment:

Test Item Dose [µg/mL)	Cell Viability (%)			Average cell viability	CD54 RFI	CD86 RFI
	Isotype	CD54	CD86
595.25	66.59	62.05	61.5	63.38	131	141
496.04	25.18	24.39	25.36	24.98	180	3
413.37	23.39	24.96	25.42	24.59	81	-236
344.47	81.07	84.13	85.74	83.64	2073*	2085*
287.06	42.93	45.66	38.86	42.48	191	173
239.22	64.97	63.79	61.91	63.56	140	81
199.35	81.3	80.15	79.64	80.36	116	89
166.12	89.72	88.53	87.3	88.51	67	90
* artefact, large amount of cell debris (and not live cells) that were picked up by the flow cytometer

Applicant's summary and conclusion

Interpretation of results:

other: negative in the hCLAT

Conclusions:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).