Registration Dossier

Administrative data

Description of key information

Two in vitro / in chemico tests were perfomred to assess the skin sensitization potential of alpha-Lipoic acid.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-29 - 2017-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015-02-04
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
According to REACH regulation Annex VII, 8.3.2 column 2 an in vivo test (LNNA is preferred) will only be performed, when the in chemico / in vitro
methods according to 8.3.1, column 1 are not applicable for the test substance or the are not convincing.

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.

This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”.
Test material information:
Composition 1
Positive control results:
Mean Peptide depletion: 73.04 %
Key result
Parameter:
other: protein depletion [%]
Value:
ca. 20.62
Vehicle controls valid:
not examined
Remarks:
Acetonitrile
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
Cinnamic aldehyde
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed low reactivity towards the peptides. The test item can be classified as “sensitiser” in accordance with UN GHS “Category 1”.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study Thioctic Acid was dissolved in acetonitrile. Based on a molecular weight of 206.3 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. A slight precipitation was observed for the test item samples and the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. Turbidity and phase separation was observed for the samples of the positive control and the respective co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the positive control was fulfilled the validity criteria in both experiments the precipitation, turbidity and phase separation was considered as irrelevant.

No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (10.96%). Based on the prediction model 1 the test item can be considered to possess sensitising potential. Due to the observed precipitation in the test item samples of the cysteine peptide experiment the reactivity might be underestimated.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 62.33%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-30 - 2017-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
other: Activation of THP-1 cells an acute human monocytic leukemic cell line used as a surrogate for denritic cells.
Justification for non-LLNA method:
According to REACH regulation Annex VII, 8.3.2 column 2 an in vivo test (LNNA is preferred) will only be performed, when the in chemico / in vitro
methods according to 8.3.1, column 1 are not applicable for the test substance or the are not convincing.

This test method is able to detect chemicals that cause skin sensitisation and allows hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP.
Test material information:
Composition 1
Details on study design:
Solvent Controls
The solvent control was set up depending on the appropriate solvent previously determined (see chapter 10.6.2).
For chemicals solubilized in DMSO, DMSO was the solvent control.
The solvent controls were diluted according to the procedure described for the test item in chapter 10.7.1, resulting in a final concentration of 1% (v/v) for 0.9% NaCl and 0.2% (v/v) for DMSO.

Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item in chapter 10.7.1, resulting in a final DMSO concentration of 0.2% (v/v).

Test System
FACS: BD Canto II
Software: BD FACS DIVA 6.0

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum (FBS, FBS, Biochrome, Cat No.: S 0615, Lot: 1039D, 0596E), 25 mM HEPES (Gibco Life Science, Cat No.:15630-056 Lot.: 1839003, 1815266), L-glutamine (Gibco Life Science, Cat. No.: 25030-081, Lot: 1839015), 0.05 mM 2-mercaptoethanol (Gibco Life Science, Cat No.: 21985-023, Lot: 1779219) and 100 U/ml penicillin/ 100 μg/mL streptomycin (Gibco Life Science, Cat. No.: 15240-062, Lot: 1786396, 1851591) at 37 ± 1°C and 5% CO2.

Dose Groups
1. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
2. Positive Control: 4 μg/mL DNCB
3. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1 and 2:
1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, and 3.91 μg/mL
main experiment 1 and 2:
500, 416.66, 347.2, 289.4, 241.1, 200.9, 167.4, 139.5 μg/mL
Key result
Parameter:
other: relative fluorescence intensity (RFI), CD86
Remarks:
% of solvent control
Run / experiment:
Main experiment 1 + 2
Value:
>= 271 - <= 409
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: relative fluorescence intensity (RFI), CD86
Remarks:
% of solvent control
Run / experiment:
Sovent control
Value:
ca. 100
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Parameter:
other: relative fluorescence intensity (RFI), CD86
Remarks:
% of solvent control
Run / experiment:
Positive control
Value:
>= 433 - <= 551
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered as skin sensitiser in accordance with UN GHS category 1.
Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study Thioctic Acid was dissolved in DMSO. For the dose finding assay stock solutions with a concentration of 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 963.99 ± 11.58 μg/mL was derived in the dose finding assay

Based on the CV75, the main experiment was performed covering the following concentration steps:

1000; 833.33, 694.44, 579.70, 482.25, 402.88, 335.90, 279.08 μg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Slight cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 68.8% (CD86), 73.5% (CD54) and 72.6% (isotype IgG1 control) in the first experiment and to 52.9% (CD86), 53.3% (CD54) and 53.0% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 409% and to 362% in both independent experiments. The upregulation above the threshold of 150% was observed starting at a concentration of 279.08 μg/mL in both experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item considered as a skin sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (433% experiment 1; 551% experiment 2) and 200% for CD54 (679% experiment 1; 805% experiment 2) were clearly exceeded.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

According to the CLP regulation the test item is classified as "May cause an allergic skin reaction" (UN GHS “Category 1”).