3-(heptyloxy)propane-1,2-diol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 November 2019 - 17 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitization tests is the h-CLAT test, which is recommended in international guidelines (e.g. OECD).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E In vitro skin sensitisation: human cell line activation test (h-CLAT)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The purpose of this study was to evaluate the ability of Saskine 70 to activate the dendritic cells. Activation of these cells can lead to skin sensitisation.

Test material

Test material information

Constituent 1

Reference substance name:

3-(heptyloxy)propane-1,2-diol

Cas Number:

10305-39-2

Molecular formula:

C10H22O3

IUPAC Name:

3-(heptyloxy)propane-1,2-diol

Test material form:

liquid: viscous

Details on test material:

Colour: Clear to yellow
Storage temperature: Room temperature (20°C ± 5°C)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test substance was dissolved in DMSO. Highest concentration: 500 mg/mL (stock solution). Compound concentration in the wroking solution (culture medium) 1000 µg/mL

In vitro test system

Details on the study design:

CONTROLS
Vehicle control: DMSO

REFERENCE ITEMS
Dinitrochlorobenzene (DNCB)
Concentration: 4 µg/mL in DMSO
CAS: 97-00-7

Nickel sulfate (NiSO4)
Concentration: 100 µg/mL in PBS
CAS: 10101-97-0

Lactic acid (LA)
Concentration: 1000 µg/mL in 0.9% NaCl
CAS: 50-21-5

TEST SYSTEM
Cell type: THP-1 cells
Source: American type culture collection
Batch: 61077351
Culture conditions: 37 °C, 5% CO2, humidified atmosphere
Culture medium: RPMI-1640, supplemented with 10% Foetal calf serum, 100 units/mL penicillin and 100 µg/ml streptomycin.
Seeding densitiy for testing: 0.2 x E6* (pre-cultured for 72 hours)
*The cell seeding specifications have been modified from the protocol outlined in the guidelines. However, the protocol has been established and validated by Eurosafe as the density is optimal in this condition not exceeding 1 x E6 cells/mL after amplification.

CYTOTOXICITY ASSAYS
Eight concentrations of test item were prepared by a two-fold serial dilution from a maximum final concentration of 1000 µg/mL or lower, depending on its solubility limit.
On the day of testing, cells were harvested and suspended in fresh culture medium at 2 x E6 cells/mL. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µL (1 x E6 cells/well) with various concentrations of test item for 24 ± 0.5 hours at 37 °C. After incubation, cells were stained with 7-AAD (5 µg/mL) and analysed with flow cytometry to measure cell viability.

Log CV75 = ((75-c) x Log (b) - (75-a) x Log (d))/(a-c)
a = minimum value of cell viability over 75%
c = maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively.

ACTIVATION TEST
Each activation test was performed on eight concentrations: 25.1, 30.1, 36.2, 43.4, 52.1, 62.5, 75.0, 90 µg/mL
Top concentration was considered to be be the limit of cytotoxicity based on the cell toxicity assays.
THP-1 cells were plated at 1 X E6 cells/mL/well in 24 well plates and treated for 24 ± 0.5 hours at 37 °C ± under 5 ± 1% CO2. After treatment cells were washed twice with Fb buffer. After washing, blocking the cells with Fb buffer containing 0.01% (w/v) globulin was not performed due to low expression of Fc receptors by THP-1 cells.

Cells were stained for 30 min at 4 °C with the following antibodies: anti-human CD54, anti-human CD86, fluorescein isothiocyanate (FITC) labelled-mouse IgG1 using the manufacturer's recommended dilutions. Then, the cells were stained with 7-AAD for at least 30 min at 4 °C. After washing once and resuspension with Fb, Fluorescence intensities of the THP-1 cell surface markers were analysed by flow cytometry on 10000 living cells.

DATA ANALYSIS AND INTERPRETATION
Relative fluorescence intensity (RFI):
RFI = ((MFI of test item treated cells - MFI of test item treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells)) x 100
MFI: mean fluorescence intensity

Effective concentration (EF):
EC150 (CD86) = Bconcentration + ((150 - BRFI)/(ARFI-BRFI) x (Aconcentration - Bconcentration))
EC200 (CD54) = Bconcentration + ((200 - BRFI)/(ARFI-BRFI) x (Aconcentration - Bconcentration))

Aconcentration: lowest concentration in µg/mL, with RFI > 150 (CD86) or 200 (CD54)
Bconcentration: highest concentration in µg/mL, with RFI < 150 (CD86) or 200 (CD54)
ARFI: RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI: RFI at the highest cncentration with RFI < 150 (CD86) or 200 (CD54)

ACCEPTACE CRITERIA
- In the positive control (DNCB) (performed in all activation experiments) and (NiS04) (performed only in an activation experiment): RFI values of both CD86 and CD54 must be over the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The cell viability must be more than 50%.
- In the negative control (LA) {performed only in an activation experiment): RFI values of both CD86 and CD54 must be under the positive criteria (CD54 < 200% and CD86 < 150%). The cell viability must be more than 50%.
- In the vehicle control(medium, 0.9% NaCI, DMSO, Ethanoletc.): Cell viability must be more than 90%. In the solvent/vehicle control, RFI values of both CD86 and CD54 must not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) compared to the medium control. The MFI ratio of both CD86 and CD54 to isotype control must be > 105%.
- In the test item: Cell viability must be more than 50% in at least four tested concentrations in each run.

EVALUATION CRITERIA
The positive criteria are described as following:
RFI of CD54 ≥ 200% and/or RFI of CD86 ≥ 150% of their respective control, at any tested concentration.

Results and discussion

Positive control results:

Experiment 2 (cell viability ≥ 70%):
- CD54: RFI (%) = 1913
- CD86: RFI (%) = 383
Experiment 3 (cell viability ≥ 70%):
- CD54: RFI (%) = 790
- CD86: RFI (%)= 460

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (%)

Remarks:

CD86

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Top dose: 90.0 µg/mL; RFI: 96 %

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (%)

Remarks:

CD86

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Top dose: 90.0 µg/mL; RFI: 140%

Results 3

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (%)

Remarks:

CD54

Value:

141

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Top dose: 90.0 µg/mL; RFI: 162%

Results 4

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (%)

Remarks:

CD54

Value:

120

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Top dose: 90.0 µg/mL; RFI: 180%

Other effects / acceptance of results:

- Cytotoxicity was induced on THP-1 cells by Saskine 70. According to this cytotoxic profile CV75: 74.1 μg/mL
- One deviation was observed in experiment 1 with a RFI value of CD86 > 150% for the DMSO control.

ACCEPTANCE CRITERIA
- In the positive control (DNCB) RFI values were above the criteria in experiment 2 and 3 (CD54: 1913 and 383; CD86: 383 and 460).
- In the negative control (LA) (performed only in an activation experiment): RFI values of both CD86 and CD54 were under the positive criteria (CD54: 136 and CD86: 59). The cell viability was 98%.
- In the vehicle control (DMSO): Cell viability was ≥ 97%. In the solvent/vehicle control, RFI values of CD86 was 124 and 100 in experiment 2 and 3 and the RFI values of CD54 were 123 and 123 in experiment 2 and 3 (CD86 ≥ 150% and CD54 ≥ 200%) compared to the medium control. The MFI ratio of both CD86 and CD54 to isotype control must be > 105% (198 and 127, respectively).
- In the test item: Cell viability was more than 50% in at least four tested concentrations in each run.

Applicant's summary and conclusion

Interpretation of results:

other: Study can be part of a weight of evidence

Conclusions:

In a h-CLAT test performed according to OECD TG 442E and in accordance with GLP principles, Saskine 70 did not activate dendritic cells and therefore tested negative for skin sensitizing properties under experimental conditions described in this report.

Executive summary:

A h-CLAT test was performed with Saskine 70 according to OECD guideline 442E and in accordance with GLP principles. The study consisted of two seperate experiments. Based on the solvent, negative and positive controls it was concluded that the test conditions were adequate and that the test system functioned properly. Cell viability (CV) was determined to be 75% at a concentration of 74.1 μg/mL. In both experiments, all concentrations tested did not induce a 1.5 or 2-fold increase of CD86 and CD54, respectively. Based on the results, Saskine 70 is not considered to have skin sensitizing properties under the current experimental conditions.