L-arabinose

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 18 - October 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

yes

Remarks:

see "Any other information on Material and Methods"

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

L-arabinose

EC Number:

226-214-6

EC Name:

L-arabinose

Cas Number:

5328-37-0

Molecular formula:

C5H10O5

IUPAC Name:

L-arabinose

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Dose Selection for Experiment I and II
In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 μM. For a test chemical which has no defined molecular weight, the final test item concentration 400 μg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). Since no cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment I and II:
269 μM, 323 μM, 388 μM, 465 μM, 558 μM, 670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM
In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction

Experimental Performance
Experiment I and II were performed in the same way. Experiment II serves only to confirm the results of experiment I. The exposure dates were 10. Oct. 2017 and 11. Oct. 2017. 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 centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83000 (±10 %) cells/mL. 120 μL of the cell suspension were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h and 30 min in Experiment I and 24 h and 45 min in Experiment II. The treatment procedure was performed on both 96 well plates identically: After the incubation time the medium was removed from the cells and 150 μL medium no. 3 were added to each well. Afterwards 50 μL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium 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 were added to each well. The plates were incubated 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 of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5- Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability.
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 μL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 μL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometerFor calculation of the luciferase induction as well as the relative viability a validated Microsoft Excel® file was used.

Results and discussion

Positive control results:

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values > 84 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.9 fold, negative control: 1.2 fold). However, the positive control induced a clear effect with an induction value of 4.5 fold in comparison to the solvent control.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment I

Parameter:

other: fold Luciferase induction

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Experiment II

Parameter:

other: fold Luciferase induction

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Acceptability of experiment I and II

Criteria	Found in Experiment I	Found in Experiment II
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.	Positive control Fold induction: 4.5 Relative viability: 84.7 %	Positive control Fold induction: 5.4 Relative viability: 94.2 %
The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.	Negative control: Fold induction: 1.2 Relative viability: 104.9 % Growth control: Fold induction: 0.9 Relative viability: 141.9 %	Negative control: Fold induction: 1.0 Relative viability: 112.0 % Growth control: Fold induction: 1.0 Relative viability: 136.5 %
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.	11.96 %	11.21 %
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.	12 concentrations are analysable	12 concentrations are analysable

Table 2: Summary of the Results of Experiment I

Parameter		Induction of Luciferase			Viability of the Cells
	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[μM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.12	11.96	100.0	5.69	5.69
Growth Control	-	0.9	0.07	7.80	141.9	6.20	4.37
Negative Control	5000	1.2	0.08	6.52	104.9	5.998	5.69
Positive Control	120	4.5	0.13	2.83	84.7	2.20	2.59
Test item	269	1.1	0.07	6.41	110.1	1.66	1.51
	323	1.0	0.08	8.19	104.9	3.07	2.92
	388	1.0	0.08	8.46	100.7	3.18	3.16
	465	1.0	0.04	4.00	101.3	2.14	2.11
	558	1.0	0.11	10.67	103.3	7.22	6.99
	670	1.0	0.03	3.32	101.4	6.13	6.05
	804	0.9	0.05	5.77	105.5	2.48	2.35
	965	1.0	0.05	5.62	94.0	1.14	1.21
	1157	0.9	0.06	6.58	93.4	3.24	3.47
	1389	0.9	0.05	5.86	98.1	0.65	0.66
	1667	0.9	0.06	7.53	95.8	2.08	2.17
	2000	0.9	0.06	6.88	100.2	1.20	1.20

Table 3: Summary of the Results of Experiment II

Parameter		Induction of Luciferase			Viability of the Cells
	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[μM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.11	11.21	100.0	5.89	5.89
Growth Control	-	1.0	0.08	8.22	136.5	4.39	3.22
Negative Control	5000	1.0	0.04	4.04	112.0	4.68	4.18
Positive Control	120	5.4	0.29	5.38	94.2	7.75	8.22
Test item	269	1.2	0.09	7.13	123.0	4.94	4.02
	323	1.1	0.05	4.03	111.3	3.21	2.88
	388	1.1	0.14	13.12	106.7	2.57	2.41
	465	1.1	0.09	8.52	103.8	2.77	2.67
	558	1.1	0.08	7.77	102.5	6.36	6.21
	670	1.0	0.02	2.52	102.3	4.68	4.58
	804	1.0	0.02	1.68	107.2	5.00	4.66
	965	1.0	0.06	6.63	110.7	3.14	2.84
	1157	1.1	0.06	5.27	106.4	1.34	1.26
	1389	1.0	0.07	7.02	102.8	0.88	0.86
	1667	1.0	0.17	17.22	99.8	7.77	7.78
	2000	0.9	0.03	3.46	99.9	2.58	2.58

Applicant's summary and conclusion

Interpretation of results:

other: no activation of keratinocytes

Remarks:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In an in vitro skin sensitisation assay according to 442D, the test item did not induce luciferase activity in at least two independent experiment runs.

Executive summary:

In an in vitro skin sensitisation assay according to 442D, the potential of the test item to activate the Nrf2 transcription factor, by using the LuSens cell line was investigated. 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:

269 μM, 323 μM, 388 μM, 465 μM, 558 μM, 670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM.

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 distinct 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 positive control.D/L-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. The induction of the luciferase of the growth control was < 1.5 fold. Since all acceptability criteria of the assay were met the study is valid. No significant reduction of growth was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction. In all tested concentrations of the test item no substantial and reproducible dose dependent increase of luciferase induction was measured.

In conclusion, it can be stated that under the experimental conditions reported, the test did not have the potential to activate the Nrf2 transcription factor.