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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Remarks:
OECD 442D
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 March to 6 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Remarks:
Alfalfa, ext was observed to be a very thick dark coloured paste at room temperature.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (4°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: 20 mg/ml in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

In vitro test system

Details on study design:
DESCRIPTION OF THE TEST SYSTEM
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection using a BMG Optima FLUOstar) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

METHOD OF ADMINISTRATION OF TEST ITEM
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

METHOD OF ADMINISTRATION OF REFERENCE ITEMS
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).

EXPOSURE TIMES OF TEST ITEMS AND REFERENCE ITEMS
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.

NUMBER OF REPETITIONS
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT.

NEGATIVE CONTROL
DMSO, 99.7%

POSITIVE CONTROL
Cinnamic aldehyde, 99%

DATA ANALYSIS
The following parameters were calculated in the KeratinoSens test method:
- The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration of the test item and positive control.
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity).
- For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
-The percentage of viability as compared to the negative control.

ASSAY ACCEPTANCE CRITERIA
Test results are acceptable if:
- The positive control (cinnamic aldehyde) produces positive results, i.e., the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
-The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
* Average induction in the three replicates for cinnamic aldehyde at 32 µM is between the XCellR8 historical range (currently 1.6 and 3)
* EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 µM and 39 µM).

Note: At least one of these criteria must be met, otherwise the run is discarded. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
- CV% of blank values < 20%

INTERPRETATION OF RESULTS AND SKIN SENSITISATION PREDICTION MODEL
A test item is considered a sensitiser if the following conditions are met in 2 of 3 repetitions:
- The IMAX is higher than 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test).
- The cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
- The EC1.5 value is < 1000 µM or < 200 µg/mL for test chemicals with no defined MW.
- There is an apparent overall dose-response for luciferase induction (or a biphasic response).

Results and discussion

Positive control results:
Induction (=1.5-fold):
1.518 at 8 µM
2.664 at 16 µM
5.019 at 32 µM
11.866 at 64 µM
20.861 at 128 µM

In vitro / in chemico

Results
Key result
Positive controls validity:
valid
Remarks on result:
other: Negative

Any other information on results incl. tables

See attached additional information on results.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro KeratinoSens test, conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative.
Executive summary:

The in vitro KeratinoSens test, conducted according to OECD test guideline 442D and to GLP, was performed to determine skin sensitisation potential of Alfalfa, ext. by assessing keratinocytes activation.

After 48h exposure of cells to 12 concentrations of Alfalfa, ext. luciferase measurements and MTT viability test were performed.

The sensitisation potential of Alfalfa ext. was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value.

Alfalfa, ext. only caused luciferase induction above 1.5 in repetition 2, with an EC1.5 value below 0.098 µg/ml. Since no clear dose response was observed, this was classified as an inconclusive result for this repetition. In repetitions 1 and 3, the threshold for induction was not exceeded at any of the concentrations tested and, therefore, based on 2 out of 3 concordant results from independent experiments, the result for Alfalfa, ext. in this KeratinoSens test was negative.

The maximum induction for repetition 1 was observed at 200 µg/ml Alfalfa, ext. (1.238). For repetitions 2 and 3 the IMAX values of 3.139 and 0.969, respectively, were observed at 0.391 µg/ml.

All of the formal acceptance criteria of the tests were met except for acceptance criterion 2 (average induction of positive control at 32 µM). However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered as valid.

Therefore, in the in vitro test, conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative for the second key event of the skin sensitisation Adverse Outcome Pathway.