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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-04-24 to 2017-05-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells

Test material

Constituent 1
Chemical structure
Reference substance name:
3-hydroxypropyl octanoate
EC Number:
844-232-8
Cas Number:
102731-54-4
Molecular formula:
C11H22O3
IUPAC Name:
3-hydroxypropyl octanoate

In vitro test system

Details on the study design:
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.

Main test:
The test item was tested in two independent runs.
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75 (first XTT CV75: 384.6μg/mL; second XTT CV75: 256.7μg/mL; mean CV75: 320.65 μg/mL). Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
During flow cytometry acquisition dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).

Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control is adjusted to 100 % and the cell viability of the DMSO control should be more than 90 % in comparison to the medium control.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105 %.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the cell viability should be > 50%.
• For the test chemical, the cell viability should be more than 50 % in at least four tested
concentrations in each run.

Negative results are acceptable only for test items exhibiting a cell viability of < 90 % at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90 % the negative result should be discarded. In such a case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90 %.

Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
− The RFI of CD86 is ≥ 150 % at any tested concentration (with cell viability ≥ 50 %);
− The RFI of CD54 is ≥ 200 % at any tested concentration (with cell viability ≥ 50 %).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: other: 2; 321 μg/mL
Parameter:
other: RFI of CD54
Value:
206.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: other: 2; 223 μg/mL
Parameter:
other: RFI of CD86
Value:
158
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

The CV75 value of the first XTT test: 384.6 μg/mL


The CV75 value of the second XTT test: 256.7 μg/mL


The mean CV75 value of both XTT tests: 320.65 μg/mL


Table 1: Results of h-CLAT runs










































































































































































 Concentration
(μg/mL)
RFI (%)
CD 54 Antibody
RFI (%)
CD 86 Antibody
Cell Viability
(%)
1st h-CLAT run
Medium
Control
-100100100
DMSO
Control
-100100100
Positive
Control
(DNCB)
2219#711.7#80.6
3358.3#734.4#73
Test Item107113.8124.4104
129114.4101.9105.8
155109109.6102.4
186129.3131.499.3
223150.9151.3#97.6
267160.5169.9#97.2
321 E224#549.4#37.5
385 E345.5#3460.3#3.8
2nd h-CLAT run
Medium
Control
-100100100
DMSO
Control
-100100100
Positive
Control
(DNCB)
2229.6#396.4#81.7
3219.9#385.1#77.9
Test Item107115.6101.3105.4
129112.8112.1105.8
155125.6114.6106.4
186146.1136.9105.9
223173.3158#108.3
267188.9179#104
321206.7#343.9#70.9
385 E272.2#1317.2#13.9

E=cell viability below 50 %, are excluded from the evaluation


#=RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %)

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In the h-CLAT study the test item is considered to have a skin sensitization potential.
Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) according to OECD 442E was performed to assess the skin sensitizating potential of the test item dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 625 μg/mL in the first XTT test and of 312.5 μg/mL in the second XTT test up to the highest tested concentration (5000 μg/mL). The mean CV75 value of both XTT tests was calculated as 320.65 μg/mL. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 107, 129, 155, 186, 223, 267, 321 and 385 μg/mL


The test item with a calculated log Pow of 2.84 was tested in 2 independent runs. The cell viability of the two highest tested test item concentrations of the first run and the highest concentration of the second run were below 50 % and therefore excluded from the evaluation. The RFI of CD86 was equal or greater than 150 % in at least one concentration of both independent run data. In addition, the RFI of CD54 was greater than 200 % in one tested concentration of the second run. Both runs were POSITIVE relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to have a skin sensitization potential. In the DMSO solvent control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %. In conclusion, the test item activated THP-1 cells under the test conditions of this study. Therefore the test item is considered to be positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

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