1,2,4-triazole-3-thiol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-triazole-3-thiol

EC Number:

221-656-6

EC Name:

1,2,4-triazole-3-thiol

Cas Number:

3179-31-5

Molecular formula:

C2H3N3S

IUPAC Name:

4H-1,2,4-triazole-3-thiol

Test material form:

solid: crystalline

In vitro test system

Details on the study design:

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 2000 µg/mL is used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). In the case of a cytotoxic result, the concentrations for the experiment should be deter-mined so that at least one of them is in the cytotoxic range. Since a cytotoxic reaction was observed in the CRFT, the highest “calculated” concentration for the experiments would be 859.2 µM (1.2*CV75). The aim was to have one cytotoxic and three non-cytotoxic concentrations, so to be save to have at least one cytotoxic concentration, the experiments were started with the concentration of 1400 µM. 12 concentrations for repetition II and III were analysed to cover a range as huge as possible, since the dilutions steps are just 1.2. 188.4 µM, 226.1 µM, 271.3 µM, 325.6 µM, 390.7 µM, 468.9 µM, 562.6 µM, 675.2 µM, 810.2 µM, 972.2 µM, 1166.7 µM, 1400 µM. Repetition II and III were performed in the same way. The exposure dates were 27. Aug. 2019 and 03. Sep. 2019. 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 83 000 (±10 %) cells per mL. 120 µL of the cell suspension (≙ 10 000 cells) 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 23 h 20 min in repetition II and 24 h 0 min in repetition III. 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 hu-midified 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 plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was re-moved 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 colour) to its insoluble formazan (purple colour) in living cells and therefore indicates the amount of living cells. After the measurement of the colour change, the values were transferred in a validated spreadsheet for the calculation of the viability (see chapter 7.3.2, page 20). For the evaluation of the Luciferase induction, the second plate was used: For the evaluation of the Luciferase expression, after the 48 h exposure period, 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 incu-bated 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 shak-en 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 luminometer.

Results and discussion

Positive control results:

Found in Found in
repetition II repetition III
Positive control Fold induction: 5.9 Positive control Fold induction: 6.8
Relative viability: 82.1 % Relative viability: 84.2 %

In vitro / in chemico

Results

Key result

Run / experiment:

other: Repetition II and III

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 188.4 µM, 226.1 µM, 271.3 µM, 325.6 µM, 390.7 µM, 468.9 µM, 562.6 µM, 675.2 µM, 810.2 µM, 972.2 µM, 1166.7 µM

Any other information on results incl. tables

1.1     Results of Repetition II

The results of repetition II are indicated in table 8-a and figures 8-a and 8-b.

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 82 %). 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: 1.0 fold, negative control: 0.9 fold). However, the positive control induced a clear effect with an induction value of 5.9 fold in comparison to the solvent control.

No cytotoxic effect was observed at the test item concentrations 188.4 µM to 1166.7 µM. The viability values were all ≥ 80 % and therefore analysable for luciferase induction. Cytotoxic effects were observed at the concentrations 1400 µM. Due to cytotoxicity this value was not used for the final evaluation of luciferase induction.

In the Luciferase assay, all of the tested non cytotoxic concentrations induced a statistically significant increase in luciferase induction (see Annex 8, chapter20, page46) above the threshold of 1.5 fold in comparison to the solvent control.

Table8‑a        Results of repetition II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.06	5.65	100.0	4.45	4.45
Growth Control	-	1.0	0.07	6.82	137.4	3.94	2.87
Negative Control	5000 µM	0.9	0.03	3.89	107.8	2.40	2.22
Positive Control	120 µM	5.9	0.39	6.66	82.1	3.36	4.09
Test item	188.4	2.0	0.11	5.36	117.7	2.40	2.04
Test item	226.1	2.3	0.48	21.14	116.0	6.76	5.83
Test item	271.3	2.7	0.39	14.30	115.4	7.01	6.07
Test item	325.6	2.8	0.28	9.89	118.2	6.54	5.53
Test item	390.7	3.8	0.45	11.72	118.6	5.60	4.72
Test item	468.9	5.1	0.15	2.88	120.4	7.82	6.50
Test item	562.6	6.9	1.18	17.02	107.3	5.40	5.04
Test item	675.2	8.9	1.16	13.09	105.4	7.21	6.84
Test item	810.2	11.2	0.61	5.47	97.4	3.61	3.71
Test item	972.2	14.0	0.48	3.40	90.4	2.99	3.30
Test item	1166.7	17.7	0.29	1.66	80.1	2.82	3.52
Test item *	1400*	17.5*	0.50*	2.85*	54.1*	3.81*	7.05*

* = Due to cytotoxicity this value was not used for the final evaluation of luciferase induction.

1.1     Results of Repetition III

The results of repetition III are indicated in table 8-b and figures 8-c and 8-d.

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: 1.0 fold, negative control: 0.9 fold). However, the positive control induced a clear effect with an induction value of 6.8 fold in comparison to the solvent control.

No cytotoxic effect was observed at the test item concentrations 188.4 µM to 1166.7 µM. The viability values were all ≥ 77 % and therefore analysable for luciferase induction. Cytotoxic effects were observed at the concentrations 1400 µM. Due to cytotoxicity this value was not used for the final evaluation of luciferase induction.

In the Luciferase assay, all of the tested non cytotoxic concentrations induced a statistically significant increase in luciferase induction (see Annex 8, chapter20, page46) above the threshold of 1.5 fold in comparison to the solvent control.

Table8‑b        Results of repetition III

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.09	9.25	100.0	3.82	3.82
Growth Control	-	1.0	0.09	8.95	147.7	4.59	3.11
Negative Control	5000 µM	0.9	0.09	9.85	110.6	3.83	3.47
Positive Control	120 µM	6.8	0.38	5.66	84.2	3.07	3.65
Test item	188.4	1.8	0.16	8.84	126.7	1.61	1.27
Test item	226.1	2.0	0.23	11.47	123.7	5.01	4.05
Test item	271.3	2.1	0.17	7.91	118.8	1.60	1.34
Test item	325.6	2.6	0.16	6.15	114.4	3.41	2.99
Test item	390.7	3.3	0.05	1.38	113.7	3.87	3.40
Test item	468.9	4.5	0.25	5.58	107.7	7.34	6.81
Test item	562.6	6.8	0.16	2.33	100.8	5.40	5.36
Test item	675.2	9.1	1.11	12.21	99.9	3.39	3.40
Test item	810.2	13.9	1.03	7.45	93.5	3.34	3.57
Test item	972.2	20.3	1.68	8.28	85.8	1.05	1.23
Test item	1166.7	22.3	1.60	7.17	77.2	2.37	3.08
Test item *	1400*	23.0*	0.81*	3.53*	64.6*	3.43*	5.30*

* = Due to cytotoxicity this value was not used for the final evaluation of luciferase induction.

 

Acceptability of repetition II and III

Criteria	Found in repetition II	Found in repetition III
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.	Positive controlFold induction: 5.9 Relative viability: 82.1 %	Positive controlFold induction: 6.8 Relative viability:  84.2 %
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: 0.9 Relative viability: 107.8 % Growth control: Fold induction: 1.0 Relative viability: 137.4 %	Negative control: Fold induction: 0.9 Relative viability: 110.6 % Growth control: Fold induction: 1.0 Relative viability: 147.7 %
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.	5.65 %	9.25 %
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.	11 concentrations are analysable	11 concentrations are analysable

 

All validity criteria were met. Therefore, the study is valid.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

This in vitro study was performed to investigate the potential of 3-Mercapto-1,2,4-triazole to activate the Nrf2 transcription factor, by using the LuSens cell line. A detailed listing of all measured and calculated values of the assay is given in Annex 2 (values of CRFT), Annex 3 (values of repetition II) and Annex 4 (values of repetition III). In addition, the final results of both repetitions are summarized in table 8-a and 8-b and graph-ically illustrated in figure 8-a to 8-d. In total three repetitions were performed of which one was invalid due to a technical error (one repetition I) and had to be repeated. This repetition is not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. In the end two valid repetitions were performed and evaluated.
12 concentrations of the test item were evaluated. The exposure time was 48 h. The follow-ing nominal concentrations of the test item were investigated in repetition II and III: 188.4 µM, 226.1 µM, 271.3 µM, 325.6 µM, 390.7 µM, 468.9 µM, 562.6 µM, 675.2 µM, 810.2 µM, 972.2 µM, 1166.7 µM, 1400 µM. None of the real treatment concentrations in all repetitions deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration in the repetitions.
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 de-tected. This luciferase induction is well within the historical data range of the positive 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. Cytotoxic effects were observed at the concentrations 1400 µM. Due to cytotoxicity this value was not used for the final evaluation of luciferase induction. Finally, the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction: Repetition II and III: 188.4 µM, 226.1 µM, 271.3 µM, 325.6 µM, 390.7 µM, 468.9 µM, 562.6 µM, 675.2 µM, 810.2 µM, 972.2 µM, 1166.7 µM. A statistically significant (see Annex 8) increase ≥ 1.5 fold in luciferase induction in compar-ison to the solvent control was measured in all non-cytotoxic test item concentrations. Therefore, both repetitions are clearly positive. In conclusion, it can be stated that under the experimental conditions of this study, the test item, 3-Mercapto-1,2,4-triazole, was positive in the LuSens assay and is therefore consid-ered to have the potential to activate the Nrf2 transcription factor.

Executive summary:

This in vitro study evaluates the potential of the test item 3-Mercapto-1,2,4-triazoletoactivate the Nrf2 transcription factorby using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. The LuSens test is an ARE Reporter Gene Assay performed according to the OECD 442D Guidelinewith 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 one experiment, consisting of three independent repetitions (repetition I, II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on these results, the concentrations to be tested in the repetitions were determined. Repetition I was invalid, due to a technical error. This repetition is not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. In the experiment (repetition II and III), the highest nominal applied concentration (1400 µM) 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 repetitions. 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. The evaluated experimental points and the results are summarised in chapter 7.1.2, page 19. 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 repetitions. Conclusion: Under the experimental conditions of this study, the test item,3-Mercapto-1,2,4-triazole, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.