Artemisia pallens, ext.

Endpoint summary

Description of key information

Skin sensitiser, category 1 (OECD 442D, KeratinoSens, GLP, K, rel.1)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1st March 2021 to 12 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No. 442D and under GLP compliance (the exact composition of the test item cannot be exactly determined because the element is an UVCB substance. This deviation to GLP is under the responsibility of the Sponsor and is considered as without impact on the conclusion of the study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Dated June, 25th, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The in vitro ARE-Nrf2 luciferase KeratinoSens™ test method (hereafter called the KeratinoSens™ test method) underwent validation studies followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA, to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard
identification.

Specific details on test material used for the study:

Name of test substance: DAVANA ESSENTIAL OIL - REF : D04035 code ID-21/02708
Description: Yellow to red liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

CONTROLS
- Negative control: Treatment culture medium (DMEM 1 g/L glucose), 1% DMSO, 1% Non-heat inactivated foetal calf serum.
- Positive control: Cinnamaldehyde (SIGMA ALDRICH Ref W228613), CAS#104-55-2

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- The test item was diluted in DMSO. The stock solution was prepared at 40 mg/mL (i.e. 4%). The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from from 0.2 µg/mL to 400 µg/mL.
Then, a 100-fold concentrated dilutions series was prepared in 96-well plate. The test item was placed in one of the rows B to F. 100 µL of DMSO were distributed from columns 1 to 11. 200 µL of the 40 mg/mL stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µL from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Then, the 100-fold DMSO plate was diluted 25-fold in a new plate (4-fold) in treatment medium.

PREPARATION OF THE POSITIVE CONTROL
100 µL of DMSO were distributed in row G from columns 7 to 10. 200 µL of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µL from column 11 to column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
- The positive control stock solution was prepared at 200 mM in DMSO according to the following formula, then diluted to 6.4 mM:
V = 5 x [(p ÷100) x w / MW] - (w/1000)

V is the volume of DMSO in mL to be added
p is the purity of the positive control in %
MW is the molecular weight of the positive control in g/mol
w is the exact weight of the positive control in mg.

NEGATIVE CONTROL
100 µL of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.
- Treatment culture medium, 1% DMSO and 1% Non-heat inactivated foetal calf serum were used as negative control. 6 wells of solvent control (1% DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate were included.

CELL LINE
KeratinoSens™cells (Givaudan) were maintained according to the current working instruction IL 09. Cells are cultured in maintenance medium at 37°C, 5% CO2. Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction IL 07.
Cells were used at passage 17 in repetition 1 and passage 19 in repetition 2.

The cells were trypsinized according to the current working instruction IL 09. Cells suspension were adjusted to a density of 8.10^4 cells/mL in seeding medium.
125 µL of the cell suspension at 8.10^4 cells/mL (i.e. 10^4 cells per well) were distributed in three white plates for the induction measurement (luciferase activity) and two transparent plates to assess the cytotoxicity (cell viability). The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2. Note: the H12 wells were left without cells and allowed the measurement of blanks.

CELL CULTURE
- Maintenance medium: DMEM 1 g/L glucose, 9.1% non-heat inactivated foetal calf serum,
0.05% geneticin - stored at 5°C ± 3°C.
- Seeding medium: DMEM 1 g/L glucose, 9.1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C.
- Treatment medium: DMEM 1 g/L glucose, 1% non-heat inactivated foetal calf serum - stored at
5°C ± 3°C.
- Trypsin (0.5 g/L) - EDTA (0.2 g/L) - stored at -20°C ± 5°C.
- Diluent for the test item and the positive control: DMSO - stored at room temperature 20°C ± 5°C.
- Luciferase substrate: Bright Glo™ Luciferase Assay System (Promega) - stored at -80°C after reconstitution.
- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free with 0.05% EDTA - stored at 5°C ± 3°C.
- Dulbecco’s PBS Ca2+ and Mg2+ free - stored at room temperature 20°C ± 5°C.
- Staining solution: 5 mg/mL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS - prepared extemporaneously and used within the day.
- Desorption solution: 10% SDS in water - stored at room temperature 20°C ± 5°C.

FL REAC 01 and FL REAC 06 forms ensure the traceability of media and reagents used in the study.

EXPERIMENTAL DESIGN
- The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.
- Contact between the cells and the test and reference items: In the 5 seeded plates, the medium was aspirated and replaced with 150 µL of treatment medium. Then the 4-fold plate was replicated 5 times: 50 µL from the 4-fold plate was placed in each of the three white plates and in the two transparent plates. The plates (one-fold) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).
- Luciferase activity: After 48 hours, the medium was aspirated and each well was gently washed once with 200 µL of PBS. Then 100 µL of luciferase substrate (luciferin + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.
- Cell viability assessment with MTT method: After 48 hours, the medium was aspirated and each well was gently washed once with 200 µL of PBS. Then, 225 µL of staining solution diluted at 0.6 mg/mL in treatment medium (from the 5 mg/mL stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37°C, 5% CO2). After this contact time, the staining solution was eliminated, and the cells were treated with 200 µL of 10% SDS overnight in the dark (37°C, 5% CO2). After a 10-minute homogenization, the absorbances were measured at 540 nm.

ACCEPTABILITY CRITERIA
To validate the test, it is essential to check the validity criteria for the test:

Positive Control:
- the gene induction must be statistically significant above the threshold of 1.5 in at least one dose,
- the EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Negative Control
For each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20%.

If for one repetition the validity criteria are not met, a third repetition should be considered.
The validation of the results is carried out by the Study Director in accordance with the current working instruction IL 04.

DATA ANALYSIS
Two parameters are measured in the KeratinoSensTM test method, the luciferase induction and the cytotoxicity:

- Luciferase induction
Imax, maximal fold induction of luciferase activity value observed at any concentration of the test item and positive control. The induction value I is calculated according to the following formula:

I=[Luminescence(Test item) – Luminescence(Blank)]/[Luminescence(Negative control) – Luminescence(Blank)]

The Imax of an item is the average of the Imax calculated for each of the repetitions.

EC1.5, value representing the concentration for which induction of luciferase activity is above 1.5 threshold, is obtained according to the following equation:

EC1.5= (Cb - Ca) × [(1.5 - Ia)/(Ib - Ia)] + Ca

Where:
Ca = the lowest concentration with more than 1.5 fold the induction
Cb = the highest concentration with less than 1.5 fold the induction
Ia = induction factor for the lowest concentration with more than 1.5 fold the induction.
Ib = induction factor for the highest concentration with less than 1.5 fold the induction

The EC1.5 of a test item is the geometric average of the EC1.5 calculated for each of the repetitions.

- Cytotoxicity
The viability V is calculated according to the following formula:
V (%)= [Absorbance(Test item) – Absorbance(Blank)/(Absorbance(Negative control) – Absorbance(Blank)] x 100

IC70, concentration for which we obtained 70% cell viability:

ICx = (Cb - Ca) x [(x) - Va) / (Vb - Va)] + Ca

where
x is the % viability at the concentration to be calculated (70 for IC70
Ca is the lowest concentration for which the % viability is lower than X%
Cb is the highest concentration for which the % viability is higher than X%
Va is the % viability at the lowest concentration for which the % viability is lower than X%
Vb is the % viability at the highest concentration for which the % viability is higher than X%

TEST VALIDATION
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 1000 μM
- an apparent overall dose-response for luciferase induction.
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 μM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

DATA INTERPRETATION
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the KeratinosensTM prediction is considered as negative:

- the Imax is strictly 1.5 fold higher of the basal luciferase activity (if the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated) statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student’s t-test on the raw RLU values)
- the EC1.5 value is strictly below 200 µg/mL,
- at the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e. EC1.5< IC70),
- there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

In rare cases, the test items which induce the gene activity at a concentration very close to the cytotoxic levels, are positive in some repetitions at non-cytotoxic levels, and in other repetitions only at cytotoxic levels. In this case, the test item must be tested again with a narrower range using a dilution factor of 4/3 instead of 2.
Test items that only induce the gene activity at cytotoxic levels are not rated as positive, as it is the case for some non-sensitizing skin irritants.
If, in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, in case of a test item with poor solubility tested at a concentration lower than 200 µg/mL, a negative result obtained should also be considered as inconclusive.

Vehicle / solvent control:

DMSO

Negative control:

other: Treatment culture medium, 1% DMSO and 1% Non-heat inactivated foetal calf serum

Positive control:

cinnamic aldehyde [442D]

Positive control results:

EXPERIMENT 1
- Induction at 8 µM: 2.11
- EC1.5: 4.53 μM

EXPERIMENT 2
- Induction at 8 µM: 1.90
- EC1.5: 5.58 μM

For both repetitions:
- The luciferase activity induction obtained with the cinnamaldehyde is statistically significant above the threshold of 1.5.
- The EC1.5 value of the positive control is within the lab historical range.
- The average induction for cinnamaldehyde at 64 µM is between 2 and 8.
- The average coefficient of variation of the luminescence reading for the vehicule control is below 20 % in each repetition.

All validity criteria are fulfilled, which allows to consider the study valid.

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

3.31

Cell viability:

Greater than 70%

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

4.25

Cell viability:

Greater than 70%

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

2.34 µg/mL

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

2.63 µg/mL

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

In the repetition 1, the maximal fold induction of luciferase activity, Imax, is greater than 1.5, with a value of 4.25. The EC1.5 is less than 200 µg/mL, with a value of 2.63 µg/mL and the EC1.5 viability is greater than 70%. The repetition 1 is considered positive.

In the repetition 2, Imax is greater than 1.5, with a value of 3.31. The EC1.5 is less than 200 µg/mL with a value of 2.34 µg/mL, and the EC1.5 viability is greater than 70%. The repetition 2 is considered positive.

Repetitions 1 and 2 showed reproducible results with a consistent conclusion for both experiments.

For both repetitions:
- The luciferase activity induction obtained with the cinnamaldehyde is statistically significant above the threshold of 1.5.
- The EC1.5 value of the positive control is within the lab historical range.
- The average induction for cinnamaldehyde at 64 µM is between 2 and 8.
- The average coefficient of variation of the luminescence reading for the vehicule control is below 20 % in each repetition.

All validity criteria are fulfilled, which allows to consider the study valid.

Table 1. Results of reference item

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.41	2.11	2.73	3.79	5.82	4.53	5.82
Rep 2	1.24	1.90	2.34	3.10	5.23	5.58	5.23
Mean	1.32	2.01	2.53	3.44	5.52	5.03*	5.52

 

Control solvent	CV % control solvent
Rep 1	9.9
Rep 2	13.5

 

Table 2. Results of test item

 

	VIABILITY	INDUCTION
	IC70 µg/mL	Imax	Linear EC1.5 µg/mL	EC1.5 Lin/Log µg/mL
Rep 1	20.36	4.25	2.63	2.51
Rep 2	14.37	3.31	2.34	2.20
Mean	-	3.78	-	-
Geometric mean	17.10	-	2.48	2.35

 

Table 3. Acceptance Criteria for positive and negative controls

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	9.9	pass	13.5	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	4.0	pass	4.0	pass
EC1.5 PC	7 < x < 34 µM	8 µM	pass	8 µM	pass
Induction PC at 64 µM	2.00 < x < 8.00	5.82	pass	5.23	pass

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the retained experimental conditions DAVANA ESSENTIAL OIL - REF : D04035 code ID-21/02708 may be classified as potential skin sensitizer.

Executive summary:

The sensitizing potential of DAVANA ESSENTIAL OIL - REF : D04035 has been evaluated by an in vitro sensitization assay according to the OECD Guideline 442D: KeratinoSens ™.
The KeratinoSens™ is a test method which quantifies luciferase gene induction as a measure of the activation of the Keap1-Nrf2-antioxidant/electrophile response element (ARE)-dependant pathway in an immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid.

The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.2 µg/mL to 400 µg/mL, to cover a large concentration range. After 48 hours (± 1 hour) of contact at 37°C, 5% CO2, with the test item, the cells KeratinoSens™ were lysed and the induction of luciferase was quantified. In parallel, the cytotoxicity was measured, in order to exclude a false positive generated by a skin irritation. The cinnamaldehyde was used as the positive control. The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent white plates for the measurement of induction and two transparent plates for the measurement of cytotoxicity. Each repetition was performed independently of one another.

Repetitions 1 and 2 showed reproducible results.
In the repetition 1, the maximal fold induction of luciferase activity, Imax, was greater than 1.5, with a value of 4.25. The EC1.5 was less than 200 µg/mL, with a value of 2.63 µg/mL and the EC1.5 viability was greater than 70%. The repetition 1 was considered positive.
In the repetition 2, Imax was greater than 1.5, with a value of 3.31. The EC1.5 was less than 200 µg/mL with a value of 2.34 µg/mL, and the EC1.5 viability was greater than 70%. The repetition 2 was considered positive.
All validity criteria are fulfilled, which allows to consider the study valid.

Based of on results of the study, DAVANA ESSENTIAL OIL - REF : D04035 code ID-21/02708 may be classified as potential skin sensitizer with the in vitro sensitization test: KeratinoSens™.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A key study was identified (IDEA Lab, 2021).

In this study, the skin sensitizing potential of DAVANA ESSENTIAL OIL - REF : D04035 has been evaluated by an in vitro sensitization assay according to the OECD Guideline 442D: KeratinoSens ™.
The KeratinoSens™ is a test method which quantifies luciferase gene induction as a measure of the activation of the Keap1-Nrf2-antioxidant/electrophile response element (ARE)-dependant pathway in an immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid.

The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.2 µg/mL to 400 µg/mL, to cover a large concentration range. After 48 hours (± 1 hour) of contact at 37°C, 5% CO2, with the test item, the cells KeratinoSens™ were lysed and the induction of luciferase was quantified. In parallel, the cytotoxicity was measured, in order to exclude a false positive generated by a skin irritation. The cinnamaldehyde was used as the positive control. The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent white plates for the measurement of induction and two transparent plates for the measurement of cytotoxicity. Each repetition was performed independently of one another.

Repetitions 1 and 2 showed reproducible results.
In the repetition 1, the maximal fold induction of luciferase activity, Imax, was greater than 1.5, with a value of 4.25. The EC1.5 was less than 200 µg/mL, with a value of 2.63 µg/mL and the EC1.5 viability was greater than 70%. The repetition 1 was considered positive.
In the repetition 2, Imax was greater than 1.5, with a value of 3.31. The EC1.5 was less than 200 µg/mL with a value of 2.34 µg/mL, and the EC1.5 viability was greater than 70%. The repetition 2 was considered positive.
All validity criteria are fulfilled, which allows to consider the study valid.

Based of on results of the study, DAVANA ESSENTIAL OIL - REF : D04035 code ID-21/02708 may be classified as potential skin sensitizer with the in vitro sensitization test: KeratinoSens™.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on an in vitro skin sensitisation study (OECD 442D), the registered substance is classified as skin sensitiser: Skin sensitiser Category 1 (H317: May cause an allergic skin reaction) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

No data was available for respiratory sensitisation.