2-benzyloxyethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019

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 442E

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

2-benzyloxyethanol

EC Number:

210-719-3

EC Name:

2-benzyloxyethanol

Cas Number:

622-08-2

Molecular formula:

C9H12O2

IUPAC Name:

2-(benzyloxy)ethanol

Test material form:

liquid

In vitro test system

Details on the study design:

Cell Line: THP-1
Monocytes form peripheral blood
Culture conditions: 37°C +/- 1 °C, 5% CO2, 90+/- 10% relative humidity
Culture medium: RPMI enriched with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine, 0.05 mM 2-mercaptoethanol, 1% penicillin/streptomycin solution
The cells are stored as frozen stocks in liquid nitrogen.
A new batch of THP-1 cell stored in liquid nitrogen was thawed at least 2 weeks prior to start with the test. After thawing, the batch of cells that was emp4loyed in the assay was monitored for the absences of mycoplasma applying yje procedure described in SOP 80. Cells can be propagated up to 2 months after thawing.

h-CLAT is an in vitro method proposed to address the thord key event (dendritic cella ctiviation) of the skin sensitization adverse outcome pathway (AOP) by quantifing changes in the ezpression of cell surface markers associated with the process of activiation of dendrtic cells (i.e. CD54 and CD86) in the human leukemia cell line THP-1, following exposure to sensitizers. The measured expression levels of CD54 and CD86 cell surface markers are the used for supporting the discrimination between skin sensitizers and non-sensitiziers.
THP-1 cells were esposed to the test chemical for 24h and chenages in CD54 and CD86 expression levels were measured by flow cytometric following cell staining with fluorescein isothiocyanate (FITC)-labeled antibodies. In parallel cytotoxity measurement was conducted to assess whether up-regulation of surface marker expression occus at sub-cytotoxic concentrations.
The test was divided in 4 phases:
1) reactivity check
2) pre-feasibility test
3) dose finding assay
4) CD54/CD86 expression assay


CONTROLS
Negative control
- Substance: NC+DMSO culture medium with DMS
Solvent control for positive control
- Substance: DMSO
Positive control
- Substance: DNCB and NiSO 6H2O

TEST CONCENTRATIONS
The final concentration of the test substance in the test was the 1:2 dilution (1000 mcl/mL)

AQUISITION
Expression of cell surface antigen was analysed by flow cytometry. The Excitation and Emission wavelenght of CytoFLEX (Beckman Coulter) are:
Excitation wavelenght: 480 nm
Emission wavelenght: for FITC 525/40 nm; for PI 610/20 nm

DATA ANALYSIS
Collected data were analyzed with Kaluza Flow Cytometry analysis software.

EC200 and EC150 determination
For test substance to be considered sensitizers 2 effective concentrations (EC values) EC 200 for CD54 and EC150 for CD86 were aslo calculated. These values indicate the concentrations at which the test substance induces a RFI of 200 (CD54) or 150 (CD86).
When RFI of CD54 is >= 200% at any test dose contemporary showing cell viability > 50%, in the two indipendent runs , AND/OR if the RFI of CD86 is >= 150% at any tested dose contemporary showing cell viability > 50% in the two independent runs, the test chemical is considered POSITIVE (sensitizer); otherwise it is considered NEGATIVE (non-sensitizer). If the two runs are not concordant for at least one of the markers /CD54 or CD86), it is necessary to perform an additional run and conclusions are based on the majority result of the three individual runs.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: first run

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: second run

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: first run

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: second run

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to test conditions, the test item is to be considered NEGATIVE (non-sensitizer) up to the concentration 1000 mcl/mL.
Since 1000 mcl/mL in DMSO (highest soluble concentration) was used as the maximal test concentration in the CD54/CD86 expression assay, negative result is acceptable even if the cell viability was above 90% at the highest concentration. A third run was not necessary.