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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.01.2018 – 26.01.2018
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:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Adopted in February 2015
GLP compliance:
yes (incl. QA statement)
Type of study:
other: gene induction of luciferase activity above 1.5 treshold

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrazol-5-ylamine
EC Number:
224-581-7
EC Name:
Tetrazol-5-ylamine
Cas Number:
4418-61-5
Molecular formula:
CH3N5
IUPAC Name:
1H-tetrazol-5-amine
impurity 1
Reference substance name:
Unknown impurities
Molecular formula:
Not available
IUPAC Name:
Unknown impurities
Test material form:
solid: crystalline
Details on test material:
white crystals

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

This method specifically addresses second key event of the Adverse Outcome Pathway of the skin sensitisation through induction of cyto-protective pathways in cells in response to electrophiles and oxidative stress.

The in vitro assay quantifies luciferase gene induction as a measure of the activation of the Keap1-Nrf2-antioxidant/electrophile response element (ARE)-dependent pathway in an immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid (KeratinoSensTM). The involvement of this regulatory pathway in skin sensitisation has been demonstrated in a number of in vivo studies.
KeratinoSensTM cell line was maintained in 96-well plate and exposed to test item over a two-fold range of twelve concentrations for 48 hours. After time of exposure the cells from the first plate were stained by MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 3 – 4 hours. After dying time, the MTT taken up by the viable cells were then extracted and the absorption was measured by spectrophotometer. The cells from the second plate are mixed with luciferase substrate and the luminiscence was measured. The viability of the cells and the gene induction of the luciferase activity were then calculated.
The amount of the viable cells in the presence of the test item concentrations was compared to the amount of the viable cells in the negative control cultures. The percentage of the viability was calculated and the IC50 was determined.
The maximal gene induction of the luciferase activity (Imax) in the presence of the test item concentrations was calculated and compared to the gene induction in the blank and solvent (negative) control cultures. The EC1.5 was then calculated.

Results and discussion

Positive control results:
Positive control (EC1.5 in μM) =23.4 μM
Historical range: EC1.5 = 4 - 29 µM

In vitro / in chemico

Results
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5
Remarks:
in µM
Value:
2 000
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The doubling time of KeratinoSensTM cells (supplier Givaudan, Schweiz) was about 20 hours. It is in accordance with cell characterisation in scientific literature.
The Mycoplasma contamination was verified on 11.12.2017. The absence of contamination was confirmed.

ACCEPTANCE OF RESULTS:

All presented results met criteria of the acceptability:
1.The luciferase activity induction obtained with the positive control (Cinnamic aldehyde) should be statistically significant above the threshold 1.5 in at least one of the tested concentration (4 – 64 µM).
In the 1st experiment the value above the threshold 1.5 was 5 (mean value) in concentration 64 µM and in the 2nd experiment was 2 in concentration 64 µM.

2.EC1.5 value of the positive control should be in historical range control (of laboratory dataset: 4 - 29 µM). In addition, the average induction in 3 replicates for cinnamic aldehyde in concentration 64 µM should be between 2 – 8.
EC1.5 in the 1st experiment was 21.3 µM and in the 2nd experiment 25.6 µM, which is in the historical range.

3.The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.
In the 1st experiment the coefficient of variation was 20.4 % and in the 2nd experiment the coefficient of variation was 13.2 %.
Overview of historical control values of positive control are stored in testing laboratory.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental design described above, the results were negative, so the test item 5-aminotetrazole did not have sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method.
Executive summary:

Sensitisation Test In Vitro: ARE-Nrf2 Luciferase Method assayed sensitisation potential of the test item 5-aminotetrazole. This method specifically addresses second key event of the Adverse Outcome Pathway of the skin sensitisation through induction of cyto-protective pathways in cells in response to electrophiles and oxidative stress. The test was performed according to OECD Test Guideline No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, Adopted in February 2015 and is used for supporting the discrimination between skin sensitisers and non-sensitisers.

The cells of the line KeratinoSensTM were seeded into 96-well plates and incubated overnight. The test item was dissolved in DMSO to prepare stock concentration 200mM. Then the stock concentration was dissolved into final concentration two-fold range 0.98 – 2000 µM in medium for exposure. The plates with cells were incubated for next 48 hours. After time of exposure the cells from the first plate were stained by vital dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and incubated next 4 hours. After dyeing time, the MTT was desorbed by fixative solution and the absorbance was measured. The cells from the second and third plate were mixed with luciferase substrate (supplemented by Promega) and the luminescence was measured. The gene induction of sensitisation pathways is compared to amount of luminescence.

Under the experimental design described above, the results were negative, so the test item 5-aminotetrazole did not have sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method.