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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.10. – 02.11.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted: July 29th, 2016
Deviations:
yes
Remarks:
The medium during MTT direct reduction test changed colour from red to yellow by low pH of the test substance. This deviation had no impact on the outcome of study.
GLP compliance:
yes (incl. QA statement)

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

Test system:
human skin model
Remarks:
a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek)
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Cell source:
other: Keranocyte strain: 00267
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SR)
- Tissue batch number(s): Lot No. 25851, kit B

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
tissues were thoroughly rinsed and blotted to remove the test substance (controls)
Detailed procedure is described in internal SOP M/46/3.
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
tissues were transferred to 24-well plates containing MTT medium (1 mg·mL-1)
- Incubation time: 3 hr
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570±30 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Direct MTT reduction - functional check in tubes: the test substance does not reduce MTT directly.
2. Colour interference: colour of the test substance did not interfere with evaluation
3. MTT test

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
test substance C2: 25 mg of the test substance was placed directly atop to the previously moistened tissue (50 μL of H2O) and it was spread to match size of the tissue.
NC: 50 μL H2O tested with every exposure time
PC: 50 μL 8N KOH tested with every exposure time
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
180 minutes (in MTT medium)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
99.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct MTT reduction - functional check in tubes:
The test substance did not change colour from red to blue, so other steps were not employed.

- Colour interference
The test substance did not change colour, so other steps were not employed.

ACCEPTANCE OF RESULTS: All assay acceptance criteria have been met.
Negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.790 (3 min) and 1.747 (60 min) which is ≥ 0.8 and ≤ 2.8.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 4.6 % which is ≤ 15%.
Coefficient of variance: CV values in all triplets of tissues were ≤ 0.3

Any other information on results incl. tables

MTT test:

OD570values obtained at the MTT test, their averages, standard deviations (%), coefficients of variance and relative viabilities

Treatment 3 min OD570     mean SD CV %NC
water (NC) 1.873 1.766 1.731 1.790 0.060 0.034 100.0 
413/17 (C2) 1.790 1.772 1.809 1.790 0.015 0.008 100.0
8N KOH (PC) 0.156 0.126 0.120 0.134 0.016 0.118 7.5
Treatment 60 min OD570     mean SD CV %NC
water (NC) 1.898 1.647 1.697 1.747 0.108 0.062 100.0 
413/17 (C2) 1.734 1.743 1.751 1.743 0.007 0.004 99.7
8N KOH (PC) 0.066 0.083 0.091 0.080 0.010 0.130 4.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, the test substance 5-aminotetrazole was non-corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.
Executive summary:

The test substance 5-aminotetrazole was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to OECD Test Guideline 431, In Vitro Skin Corrosion: Human Skin Model Test, Adopted: 29th July 2016.

The test substance is white powder. There was no colour change after incubation with isopropanol and water.

Direct reduction test in test tubes was performed simultaneously. MTT medium changed colour from red to yellow. It was caused by low pH of the test substance. Hovewer the direct reduction of the MTT medium by the test substance was not observed.

In the MTT test, the test substance (25 mg) was placed atop the previously moistened tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (C2), three for the positive control (PC) and three for the negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol overnight at refrigerator without shaking and than for two hours at room temperature with shaking. OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, the average viability of tissues treated with the test substance 5-aminotetrazole was 100.0 % of the negative control average value after 3 minutes treatment and 99.7 % after 60 minutes treatment.

According to test guidelines, the test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

In the experiment arrangement described above, the test substance 5-aminotetrazole was non-corrosive in the EpiDermTM model.