Rhatany, Krameria triandra, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.06.2018 - 22.06.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed in compliance with the Principle of Good Laboratory Practice, confirmed by Statement of GLP Compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

No deviations from the guideline were observed.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

1

Reference substance name:

Rhatany, Krameria triandra, ext.

EC Number:

283-919-1

EC Name:

Rhatany, Krameria triandra, ext.

Cas Number:

84775-95-1

Molecular formula:

not available

IUPAC Name:

Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Krameria triandra, Krameriaceae.

Test material form:

liquid: viscous

Specific details on test material used for the study:

Name Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
Batch no. PES180014
Appearance reddish brown viscous substance
Composition Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
Purity not applicable, UVCB
Homogeneity inhomogeneous, warm up to about 60°C and stir
Expiry date Jan. 2019
Storage Fridge (2 - 8 °C)
CAS No. 84775-95-1
EINECS-No. 283-919-1
Molecular formula not stated
Molecular weight not applicable, UVCB
Vapour pressure unknown
Stability H2O: 96h; EtOH: 96h; acetone: 96h; CH3CN: 96h;
DMSO: 96h
Solubility in solvents H2O: unknown; EtOH: >1 g/L; acetone: >1 g/L;
CH3CN: >1 g/L; DMSO: >1 g/L
Production date Jan. 2018

In vitro test system

Details on the study design:

Test System:

Reasons for the Choice of the LuSens Cell Line:
The LuSens cell line was specially designed for this test system by the BASF SE (Lud-wigshafen, Germany). It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) up-stream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

Cell Cultures:
The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For my-coplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells (mycoplasma contamina-tion free), which guarantees similar parameters of the experiment and reproducible char-acteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 8 were used. For both main experiments cells of passage 10 were used. After thawing the cells were cultivated in DMEM (9 % FCS (Fetal calf serum) in cell culture flasks at 37 ± 1 °C in a humidified at-mosphere with 5.0 ± 0.5 % CO2.

PERFORMANCE OF THE STUDY
Cytotoxicity Range Finder Test:
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the con-centration range applicable for the main experiments. In the CRFT cytotoxicity was de-termined by measuring the cell viability with MTT. This yellow tetrazole is reduced to pur-ple formazan in viable cells and can therefore be used for assessing the cell metabolic activity and therefore the cell viability. A reduction of the viability below 70 % is defined as a cytotoxic effect.
In the CRFT the following 12 nominal concentrations of the test item were tested:
0.98 µg/mL, 1.95 µg/mL, 3.91 µg/mL, 7.81 µg/mL, 15.63 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL, 2000 µg/mL. The exposure time was 48 h and the exposure date 13. Jun. 2018.

Performance:
At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifuga-tion (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification by cell counter, the cell suspension was adjusted to 83 000 (± 10 %) cells per mL. 120 µL of the cell suspension (≙ 10 000 cells) were seeded in a clear flat bottom 96 well plate. The plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 25 h 30 min.
After the incubation time the medium was removed from the cells and 150 µL medium no. 3 was added to each well. Afterwards, 50 µL of the single test item concentrations as well as controls were added to the cells in triplicates (only test item concentrations). Twelve wells were used as solvent control, 6 wells were used as growth control, 3 wells were used as negative control and 2 wells were used as positive control and 1 well was used as blank control. The plate was sealed with breathable tapes to avoid evaporation of vola-tile compounds and to avoid cross contamination between wells. Afterwards, the plate was incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the viability assay the MTT working solution was prepared by mixing 9 parts of medi-um no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µL MTT working solution was added to each well. The plate was incu-bated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards, the solution was removed and 100 µL MTT-lysis buffer was added to each well. The plate was agitated for 5 min before it was measured at a wavelength of 570 nm and of 690 nm at the photometer.
For calculation of the relative viability a validated Microsoft Excel® file was used.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment I

Parameter:

other: Luciferase induction

Value:

2.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: Experiment II

Parameter:

other: Luciferase induction

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay).Since December 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.
The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.
In the experiments, the highest nominal applied concentration (31.25 µg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.
DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.
A statistically significant and reproducible dose-dependent increase in luciferase induction≥1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both experiments
Under the experimental conditions of this study, the test item,Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Any other information on results incl. tables

Results of Relative Viability [%] in CRFT:

Parameter	Concentration	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	[%]		[%]
Solvent Control	-	100.0	1.68	1.68
Growth Control	-	138.5	2.11	1.52
Negative Control	5000 µM	104.5	1.11	1.06
Positive Control	120 µM	90.9	0.22	0.25
Test item	0.98	94.4	3.64	3.85
Test item	1.95	88.4	4.69	5.31
Test item	3.91	86.2	2.19	2.54
Test item	7.81	88.1	0.46	0.52
Test item	15.63	76.0	4.69	6.17
Test item	31.25	4.7	3.19	67.41
Test item	62.5	1.7	0.16	9.09
Test item	125	3.1	0.09	2.94
Test item	250	5.1*	0.09	1.79
Test item	500	7.9*	0.32	4.00
Test item	1000	14.4*	0.55	3.81
Test item	2000	34.9*	3.26	9.35

*=In these concentrations reddish brown stainings were visible in the wells.

Results of experiment I:

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.06	6.30	100.0	2.78	2.78
Growth Control	-	1.3	0.09	7.20	138.7	3.13	2.26
Negative Control	5000 µM	1.0	0.06	5.64	114.7	4.35	3.79
Positive Control	120 µM	5.9	0.35	5.96	98.8	3.01	3.05
Test item	4.21	2.3	0.12	5.51	87.4	1.31	1.50
Test item	5.05	2.2	0.08	3.81	85.7	0.31	0.37
Test item	6.06	2.4	0.14	5.95	86.7	1.52	1.75
Test item	7.27	2.4	0.06	2.55	85.9	2.35	2.73
Test item	8.72	2.8	0.21	7.42	85.1	2.13	2.51
Test item	10.47	2.8	0.10	3.47	86.4	5.01	5.79
Test item	12.56	3.3	0.23	7.21	86.5	1.48	1.72
Test item	15.07	3.7	0.28	7.52	83.1	2.21	2.66
Test item	18.08	3.9	0.41	10.42	80.2	2.26	2.82
Test item	21.70	4.5*	0.23	5.13	61.1	4.08	6.68
Test item	26.04	3.7*	0.14	3.81	40.1	6.71	16.73
Test item	31.25	1.5*	0.49	31.90	10.3	2.34	22.83

* = Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.

Results of experiment II:

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.06	5.78	100.0	2.38	2.38
Growth Control	-	1.3	0.12	9.06	132.6	3.05	2.30
Negative Control	5000 µM	1.0	0.06	6.02	102.3	3.04	2.97
Positive Control	120 µM	4.9	0.21	4.32	86.0	3.35	3.90
Test item	4.21	2.0	0.25	12.19	83.5	2.70	3.24
Test item	5.05	2.1	0.13	6.33	86.0	1.24	1.44
Test item	6.06	2.2	0.12	5.56	83.2	3.43	4.12
Test item	7.27	2.3	0.11	4.65	83.3	6.19	7.43
Test item	8.72	2.2	0.34	15.29	77.9	5.14	6.60
Test item	10.47	2.7	0.05	1.96	80.2	4.22	5.27
Test item	12.56	3.0	0.28	9.41	78.2	4.11	5.26
Test item	15.07	3.3	0.36	10.73	76.8	4.20	5.47
Test item	18.08	3.4*	0.14	4.06	67.7	2.35	3.47
Test item	21.70	3.3*	0.39	11.67	51.0	2.87	5.64
Test item	26.04	2.8*	0.24	8.69	28.6	4.15	14.48
Test item	31.25	1.0*	0.14	13.58	6.4	2.18	33.77

* = Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II:
4.21 µg/mL, 5.05 µg/mL, 6.06 µg/mL, 7.27 µg/mL, 8.72 µg/mL, 10.47 µg/mL, 12.56 µg/mL, 15.07 µg/mL, 18.08 µg/mL, 21.70 µg/mL, 26.04 µg/mL, 31.25 µg/mL
None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a dis-tinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the posi-tive control.
DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control (table 18-a).
The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.
Since all acceptability criteria of the assay were met the study is valid.
In experiment I cytotoxic effects were observed in the concentrations 21.70 µg/mL, 26.04 µg/mL and 31.25 µg/mL. In experiment II cytotoxic effects were observed in the concen-trations 18.08 µg/mL, 21.70 µg/mL, 26.04 µg/mL and 31.25 µg/mL. Those concentrations were excluded from the evaluation of the luciferase induction.
Finally, the following test item concentrations showed a viability ≥ 70 % and could there-fore be evaluated for luciferase induction:
Experiment I: 4.21 µg/mL, 5.05 µg/mL, 6.06 µg/mL, 7.27 µg/mL, 8.72 µg/mL, 10.47 µg/mL, 12.56 µg/mL, 15.07 µg/mL, 18.08 µg/mL
Experiment II: 4.21 µg/mL, 5.05 µg/mL, 6.06 µg/mL, 7.27 µg/mL, 8.72 µg/mL, 10.47 µg/mL, 12.56 µg/mL, 15.07 µg/mL
A statistically significant (see Annex 8) increase ≥ 1.5 fold in luciferase induction was measured in all non-cytotoxic test item concentrations.
Therefore, both experiments are clearly positive.
In conclusion, it can be stated that under the experimental conditions of this study, the test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction was positive in the LuSens assay and is therefore considered having the potential to activate the Nrf2 transcription factor (sensitizing potential).

Executive summary:

This in vitro study was performed to assess the potential of the test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extractionto activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” (Bauchet al.2012).

It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/AREsignallingpathway (Adeet al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebrielet al. 2010). In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments are performed.

The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Under the experimental conditions of this study, the test item,Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).