Balmywood

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 March 2022 to 18 March 2022

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 with the following deviation: in repetition 2, the EC1.5 of positive control reference (21.33 µM) is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and allows the study to be validated without impacting on the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Dated June, 25th, 2018

Deviations:

yes

Remarks:

See section 'Principles of method if other than guideline' below for more details.

Principles of method if other than guideline:

The study is performed according to OECD Guideline No. 442D and under GLP compliance with the following deviation: in repetition 2, the EC1.5 of positive control reference (21.33 µM) is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and allows the study to be validated without impacting on the results.

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.

Test material

Test material information

Constituent 1

Reference substance name:

Light fraction extract obtained from the labdanum gum issued from the exudate of Cistus ladaniferus (Cistaceae) by maceration with essential oil of Cedarwood obtained from the wood of Juniperus Mexicana (Cupressaceae)

Molecular formula:

not applicable for NCS substances

IUPAC Name:

Light fraction extract obtained from the labdanum gum issued from the exudate of Cistus ladaniferus (Cistaceae) by maceration with essential oil of Cedarwood obtained from the wood of Juniperus Mexicana (Cupressaceae)

Test material form:

liquid

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

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
- 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.

- Then, 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.

NEGATIVE CONTROL
- Treatment culture medium with 1% DMSO was used as solvent control/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.
- 100 µL of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.

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 25 in repetition 1 and passage 13 in repetition 2.

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 upon opening and 5°C ± 3°C after opening.
- Diluent for the test item and the positive control: DMSO - stored at room temperature 20°C ± 5°C upon opening.
- Luciferase substrate: Lyophilized Bright-Glo™ Substrate: - 20°C ± 5°C, Bright-Glo™ Buffer: Room temperature 20°C ± 5°C and Reconstituted reagent: - 80°C ± 10°C
- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free complemented with 0.05% EDTA - stored at 5°C ± 3°C.
- Staining solution: 5 mg/ml MTT in solution in PBS - extemporaneously prepared 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.
The expiry after opening of the media and reagents used in the study is defined in the form FL REAC 05.

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.

- Cell seeding (first day): The cells culture, 80 - 90% confluent, were trypsinized according to the current working instruction IL 09. After removal of the culture medium from the culture flask, the cell layer was rinsed with PBS 0.05% EDTA, Ca2 + and Mg² + free to remove all traces of serum. The solution of trypsin-EDTA was added and left for a few minutes at 37°C, 5% CO2 until the detachment of cells. The action of trypsin was stopped by addition of maintenance medium.
Cell concentration was determined using a Malassez cell. Cell suspension was 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 cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding. The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.
The H12 wells were left without cells and will enable the measurement of blanks.

- Contact between the cells and the test and reference items (second day): The test item and positive control dilutions were prepared. 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 (1-fold) were incubated for 48 hours ± 1 hour (37°C, 5% CO2). To limit the cross-contamination, an empty line was left between each tested element.

- Luciferase activity (day 4): 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 (constituted by a mix of luciferine + ATP + lysing agent) were added in each well. The formation of foam was avoided by careful pipetting. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plate was placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

- Cell viability assessment with MTT method (day 4): After 48 hours, the medium was aspirated. 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 staining solution) were distributed in each well. The plates were incubated for 4 hours ± 30 minutes (37°C, 5% CO2). The staining solution was then removed and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

DATA ANALYSIS
Two parameters are measured in the KeratinoSens™ 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 a test 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

IC30 and IC50, concentration in µM or µg/ml for which we obtained respectively 30% or 50% viability reduction:

ICx = (Cb - Ca) x [(100-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%

For each concentration showing a luciferase activity induction equal or higher than 1.5 fold, statistical significance is determined (using a two-tailed Student’s t-test) by comparing the luminescence values of the three replicate test item with the luminescence values in the solvent control wells to assess whether the luciferase activity is statistically significant (p<0.05). In addition, at least two consecutive concentrations should have more than 70% viability, otherwise the concentration range should be adjusted.

If the dose-response curve obtained is biphasic (crossing the threshold of 1.5 twice), it is recommended to verify whether this is specific to the test item or due to an experimental artefact. In case the biphasic response is reproducible in an independent repetition, the lower concentration, i.e. when the threshold of 1.5 is crossed the first time, should be recorded.

In case where a statistically non-significant luciferase induction equal or above 1.5 fold is observed, followed by a higher concentration with a statistically significant induction, results from this repetition are only considered as valid and positive if the statistically significant induction equal or above the threshold of 1.5 was obtained for a non-cytotoxic concentration.

If the test item induces a 1.5 fold or higher induction already at the lowest concentration tested (i.e. 0.98 µM or 0.20 µg/ml), the EC1.5 is set to < 0.98 µM or 0.20 µg/ml.

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

Positive Control:
- the gene induction (luciferase induction) must be statistically significant above the threshold of 1.5 in at least one of the tested concentration,
- the EC1.5 value should be between IDEA Lab historical data (3.0 µM ≤ EC1.5 ≤ 20 µM) 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. The OECD validation dataset is between 7 µM and 30 µM.

Negative Control
- The average coefficient of variation of the luminescence reading for the solvent controls (3 x 6 wells) should be below 20% in each repetition.
If for one repetition the validity criteria are not met, or in case of equivocal result additional repetitions 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 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 Keratinosens™ prediction is considered as negative:

- the Imax is equal to or higher than 1.5 times and statistically significantly different as compared to 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 as the test item has no defined molecular weight,
- at the lowest concentration with a gene induction equal to or higher than 1.5, the cell viability must be strictly higher than 70%,
- there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

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, a negative result obtained at a maximal test concentration < 200 µg/ml and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered as inconclusive.

If the test item induces the gene activity at a concentration very close to the cytotoxic levels, it can be positive in some repetitions at non-cytotoxic levels, and in other repetitions only at cytotoxic levels. 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.

Moreover, when testing multi-constituent substances or mixtures, consideration should be given to possible interference of cytotoxic constituents with the observed responses. The presence of a high content of non-sensitizing cytotoxic constituents may mask the response of weakly sensitizing components or sensitizing components present at low concentration. It might be justified to test either single main constituents forming the major fraction or several fractions of the mixture to conclude on the sensitization potential.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

Results for Cinnamaldehyde

EXPERIMENT 1
- Induction at 64 µM: 2.34
- EC1.5: 19.01 μM

EXPERIMENT 2
- Induction at 64 µM: 2.48
- EC1.5: 21.33 μM

Results for control solvent (DMSO)
- CV(%) of experiment 1: 11.4 %
- CV(%) of experiment 2: 13.4 %

For both repetitions:
- the gene induction for cinnamaldehyde is statistically significant above the threshold of 1.5,
- the EC1.5 value of the repetition 1 is within the range of the IDEA Lab historical data. The EC1.5 value of the repetition 2 is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and the criterion is considered to be met.
- the coefficient of variation of the solvent control is less than 20%.

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

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

3.63

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

0.48 µg/mL

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

4.18

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

0.3 µg/mL

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

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 3.63. The EC1.5 is less than 200 µg/mL, with a value of 0.48 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 4.18. The EC1.5 is less than 200 µg/mL with a value of 0.30 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.

In both repetitions:
- the Imax is higher than 1.5 times and statistically significantly different as compared to the negative control,
- the EC1.5 value is below 200 µg/ml,
- at the lowest concentration with a gene induction higher than 1.5, the cell viability is higher than 70%,
- As we can see in the graphs attached in the section 'Overall remarks, attachments', there is an apparent overall dose-response for luciferase induction.

All conditions are met to conclude that both repetitions are positive.

Any other information on results incl. tables

Table 1. Results of reference item

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.04	1.36	1.47	1.61	2.34	19.01	2.34
Rep 2	1.02	1.28	1.40	1.71	2.48	21.33	2.48
Mean	1.03	1.32	1.43	1.66	2.41	20.14*	2.41

Control solvent	CV % control solvent
Rep 1	11.4
Rep 2	13.4

 

Table 2. Results of test item

 

	VIABILITY		INDUCTION
	IC50 µg/mL	IC30 µg/mL	Imax	Linear EC1.5 µg/mL	EC1.5 Lin/Log µg/mL
Rep 1	6.15	5.28	3.63	0.48	0.46
Rep 2	4.62	3.90	4.18	0.30	0.28
Mean	-	-	3.90	-	-
Geometric mean	5.33	4.54	-	0.38	0.36

 

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%	11.4	pass	13.4	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2	pass	2	pass
EC1.5 positive control with IDEA Lab historical data range	2 < EC1.5 < 20 µM	19.01 µM	pass	21.33 µM	not pass
EC1.5 positive control with OECD dataset validation range	7 < EC1.5 < 30 µM	19.01 µM	pass	21.33 µM	pass
Induction PC at 64 µM	2.00 < x < 8.00	2.34	pass	2.48	pass

In repetition 2, the EC1.5 of positive control reference (21.33 µM) is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and allows the study to be validated without impacting on the results.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the retained experimental conditions, BALMYWOOD - REF : ZA3155 code ID-22/01120 may be classified as skin sensitizer.

Executive summary:

The sensitizing potential of BALMYWOOD – REF: ZA3155 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.

 

In the repetition 1, the maximal fold induction of luciferase activity, Imax, is greater than 1.5, with a value of 3.63. The EC1.5 is less than 200 µg/mL, with a value of 0.48 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 4.18. The EC1.5 is less than 200 µg/mL with a value of 0.30 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.

In both repetitions:
- the Imax is higher than 1.5 times and statistically significantly different as compared to the negative control,
- the EC1.5 value is below 200 µg/ml,
- at the lowest concentration with a gene induction higher than 1.5, the cell viability is higher than 70%,
- There is an apparent overall dose-response for luciferase induction in the graph describing dose-response curves for induction of luciferase activity and viability;

All conditions are met to conclude that both repetitions are positive.

 

Based of on results of the study, BALMYWOOD – REF : ZA3155 may be classified as potential skin sensitizer with the in vitro sensitization test: KeratinoSens™.