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Description of key information

The health hazard evaluation of the skin sensitisation potential of 5-aminotetrazole:

ARE-Nrf2 Luciferase test:

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.

Result: the test item 5-aminotetrazolePropatylnitrate did not have sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method.

Human Cell Line Activation Test (h-CLAT):

The h-CLAT assay was performed according to OECD Guideline for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT). The relative fluorescence intensity of surface markers compared to solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

Result: 5-Aminotetrazole revealed sensitising properties in the h-CLAT test method

DPRA test:

The test was performed according to OECD TG 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity assay (DPRA) (Adopted: 4 February 2015)

Mean of percent Cysteine and percent Lysine depletion values is 0.8.

Result: Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used. Test item, 5-aminotetrazole, was classified as negative (skin non-sensitizer) in DPRA prediction; it was assigned to reactivity class – No or minimal reactivity.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.01. - 12.03.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
adopted 09 October 2017
Deviations:
yes
Remarks:
see any other informations (copy/paste minor deviations)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Details on the study design:
2. Dose finding assay
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value was used to determine the concentration of test items for the CD86/CD54 expression measurement.
2.1. Preparation of test and control items
100 mg of the test item were suspended in 1 mL medium to a stable dispersion at a concentration of 100 mg/mL. The test and control items were prepared on the day of testing.
For the h-CLAT assay, the stock solution of the test item in medium was diluted as follows: Eight dilutions (eight concentrations) were prepared, by two-fold serial dilution with medium. These dilutions were then further diluted 50-fold into culture medium containing 1% serum (working solutions). Additionally, two further working solutions (10 and 4 mg/mL) were prepared directly from the 100 mg/mL stock solution. The final concentration in plate should not exceed 5000 µg/mL even if this concentration is non-toxic. The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to achieve a further two-fold dilution and a final range of concentrations in the plate of 7.81 - 5000 µg/mL. The solvent medium was used as vehicle control.
The control dimethyl sulfoxide (DMSO) was tested at a single final concentration in the plate of 0.2%. The final concentration of DMSO in the culture system did not affect cell viability or growth rate and corresponds to the same concentration of DMSO found in the positive control.
2.2. Application of the test and control items
The culture medium or working solutions described in section 2.1 were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well flat-bottom plate. The treated plates were incubated for 24 ± 0.5 hours at 37°C under 5% CO2.
2.3. Propidium iodide (PI) staining
After 24 ± 0.5 hours of exposure 250 µL of each cell-preparation was transferred into a 96-well round-bottom plate and cells were collected by centrifugation (at 250 g for 5 minutes at 4°C). The supernatants were discarded and the remaining cells were washed twice with washing buffer (0.1% bovine serum albumin (BSA) in Dulbecco’s phosphate-buffered saline (DPBS)). Cell pellets were resuspended in 400 µL washing buffer. Shortly prior to analysis 20 µL propidium iodide solution (12.5 µg/mL) were added (final concentration of PI: 0.6 µg/mL).

3. Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The propidium iodide (PI) uptake was analysed using flow cytometry with the acquisition channel FL-3 (620 nm). A total of 10 000 living cells (PI negative) were acquired. The cell viability was calculated using the following equation by the cytometer analysis program. When the cell viability is low, up to 30 000 cells including dead cells were acquired. Alternatively, data can be acquired for one minute after the initiation of the analysis.
Cell viability = (Number of living cells /Total number of acquired cells) x 100

The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = (75 - c) x log (b) - (75 - a) x log (d) /a - c
a is the minimum value of cell viability over 75%; c is the maximum value of cell viability below 75%; b and d are the concentrations showing the value of cell viability a and c respectively.

3.1. Preparation of test and control items
The solvent/vehicle culture medium as determined in the dose finding assay was used to dissolve the test item. If the CV75 could not be determined (i.e. if sufficient cytotoxicity is not observed in the dose finding assay), the highest soluble or stably dispersed concentration of test item prepared with each solvent/vehicle would have been used as the starting concentration. The final concentration in the plate should not exceed 5000 µg/mL. The test item was first diluted to the 100 mg/mL stock solution. Based on this solution the concentration corresponding to 2-fold of the 1.2 × CV75 determined in the dose finding assay was prepared. Then, 1.2-fold serial dilution was made using the corresponding solvent/vehicle to obtain the dilutions (eight concentrations ranging from 2 x 1.2 x CV75 to 2 x 0.335 x CV75. These working solutions were further diluted two-fold for use for exposure in the plate. Only 24-well plates were used for CD86/CD54 expression measurement.
The solvent/vehicle control was prepared as described in section 2.1.
DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate. To obtain a 4.0 µg/mL concentration of DNCB in the plate, a 2 mg/mL stock solution of DNCB in DMSO was prepared and further diluted 250-fold with culture medium to a 8 µg/mL working solution.
3.2. Application of test and control items
For each test and control item, one experiment was needed to obtain a prediction. Each experiment consists of at least two independent runs for CD86/CD54 expression measurement. Each independent run was performed either on a different day or on the same day provided that for each run:
a) independent fresh stock solutions and working solutions of the test item and antibody solutions were prepared and
b) independently harvested cells were used (i.e. cells were collected from different culture flasks); however, cells may come from the same passage.
Test and control items prepared as working solutions (500 µL) were mixed with 500 µL of suspended cells (1 x 10e6 cells) at 1:1 ratio, and cells were incubated for 24 ± 0.5 hours. In each run, a single replicate for each concentration of the test and control items were sufficient because a prediction was obtained from at least two independent runs.

3.3 Cell staining and analysis
After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, collected by centrifugation (at 250 g for 5 minutes at 4°C), and then washed with washing buffer. After washing, cells were blocked with 600 µL of blocking solution (washing buffer containing 0.01% (w/v) globulin (Cohn fraction II, III, Human) ) and incubated at 4°C for 15 minutes. After blocking, cells were divided into three aliquots of 180 µL into a 96-well round-bottom plate or microtube.
After centrifugation, cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies at 4°C for 30 minutes. The antibodies described in the h-CLAT DB-ALM protocol no. 158 were used (3:25 (v/v, for CD86 (BD-PharMingen, #555657; Clone: Fun-1)) or 3:50 (v/v, for CD54 (DAKO, #F7143; Clone: 6.5B5) and IgG1 (DAKO, #X0927)) with staining buffer). After washing with 200 µL of washing buffer twice, cells were resuspended in 400 µL washing buffer and stored until analysis at 4°C. Shortly prior to analysis, 13 µL of PI solution (final concentration of approximately 0.6 µg/mL) were added. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.
Positive control results:
DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.
Mean viability was 65.10% for 1st experiment and 52.50% for 2nd experiment (>50).
Run / experiment:
other: 1
Parameter:
other: Viability Mean of IgG, CD54 and CD86 %
Value:
50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
in at least 4 concentrations from range 716.7- 2568.2 µg/mL
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
The following acceptance criteria should be met when using the h-CLAT assay.
• The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
• In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). RFI values of the solvent/vehicle control are calculated by using the formula ("MFI of test item" should be replaced with "MFI of solvent/vehicle", and "MFI of solvent/vehicle" should be replaced with "MFI of (medium) control").
• For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
• For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.

All quality criteria required were fulfilled.
Interpretation of results:
other: sensitising properties in the h-CLAT test method
Conclusions:
In conclusion, 5-Aminotetrazole revealed sensitising properties in the h-CLAT test method.
Executive summary:

5-Aminotetrazole was examined for sensitising properties in the h-CLAT assay. The h-CLAT assay is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

The human monocytic leukaemia cell line, THP-1 (TIB-202™, ATCC) was used for performing the h-CLAT assay.

The test item was suspended in culture medium to a stable dispersion.

A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. Ten dilutions (Ten concentrations) were prepared, by two-fold serial dilution with medium and a final range of concentrations in the plate of 7.8 - 5000 µg/mL culture medium. Culture medium was used as solvent control. The CV75 value was used to determine the concentration of test item for the CD86/CD54 expression measurement. In this preliminary experiment (consisting of two independent runs) an average CV75 of 2140.2 µg/mL and a maximum test concentration for the main experiment of 2568.2 µg/mL was calculated. Hence, 5-Aminotetrazole was tested at 8 concentrations in the range from 716.7 to 2568.2 µg/mL.

DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate. The solvent of DNCB dimethyl sulfoxide (DMSO) was tested as an additional control tested at a single final concentration in the plate of 0.2%. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.

The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control.

The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) was also calculated.

Acceptance criteria and evaluation of the h-CLAT assay parameters in the first and second experiment:

Parameter

RFI [%]

MFI relative to IgG [%]

Viability

Mean of IgG, CD54 and CD86

[%]

CD54#1

CD86#1

CD54

CD86

Medium control

<150

<150

>105

>105

>90

Vehicle control

100

100

>105

>105

>90

5-Aminotetrazole

Range for 716.7-

2568.2 µg/mL

 

<200

 

>150#2

 

 

 

>50#3

Positive control (DNCB)

>200

>150

>50

#1            compared to the vehicle control

#2            in both experiments at the non-cytotoxic concentration of 2140.2 µg 5-Aminotetrazole mL-1.

#3            >50% in at least 4 concentrations

- no acceptance criteria defined regarding the MFI

The h-CLAT prediction was considered positive as:

•The RFI of CD86 was above 150% at the non-cytotoxic concentration of 2140.2 µg 5-Aminotetrazole/mL (with a mean cell viability of 70.9 or 60.10% in the first or second experiment, respectively).

The vehicle control and the positive control DNCB were run in both experiments. All quality criteria for the vehicle control and the positive control required were fulfilled.

In conclusion, 5-Aminotetrazole revealed sensitising properties in the h-CLAT test method.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15.02.-16.03.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
Adopted: 4 February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:
The DPRA is a chemistry-based assay. Nucleophile-containing synthetic peptides (cysteine peptide­Ac-RFAACAA-COOH; lysine peptide- Ac-RFAAKAA-COOH) are used to screen for skin sensitisation potential by measuring peptide depletion following incubation with allergens and non-allergens.
Synthetic heptapeptides containing either cysteine or lysine are incubated with the test substance for 24 hours. Depletion of the peptide in the reaction mixture is measured by high pressure liquid chromatography (HPLC) using UV detection. Average peptide depletion data for cysteine and lysine are then calculated.

According to DB-ALM (INVITTOX) Protocol No.154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing 2012, acetonitrile is the preferred solvent for test chemicals. From the previous testing, it was known that the test item is not soluble at this concentration in acetonitrile.
An approximately 100 mM solution of 5-aminotetrazole in ultra pure water was prepared. Test item was dissolved completely, therefore ultra pure water was used for further tests. The weighed amount of 5-aminotetrazole (approximately 25.8 mg) was dissolved in 3 ml of ultra pure water.

Positive control: The weighed amount of Cinnamic aldehyd (approximately 39.7 mg) was dissolved in 3 ml of acetonitrile.
Negative control: The weighed amount of 1-butanol (approximately 22.7 mg) was dissolved in 3 ml of acetonitrile.

Reference Control is a peptide solution where the test chemical is replaced by the solvent used to dissolve it.
• Reference Control A was made with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
• Reference Control B was made with acetonitrile and its replicates are injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
• Reference Controls C was made with acetonitrile for positive and negative control and was made with ultra pure water (solvent used to solubilise the test chemicals) for test item. They are used to verify that the solvent does not impact the Percent Peptide Depletion. The appropriate Reference Controls C for each chemical are used to calculate Percent Peptide Depletion.
• Co-elution control was constituted by the test chemical alone without the addition of peptides. Instead of the addition of peptides, the appropriate amount of buffer was added. The co-elution test serves to verify that the test substance does not have the same retention time and does not absorb at 220 nm as the Cysteine and Lysine peptides.

Preparation of peptide stock solution (0.667 mM)
The necessary amount of peptide was estimated. For each analysis with addition of peptide, 800 µl of stock solution is needed. 30 ml of the stock solution of Cysteine or Lysine peptide (0.667 mM) was prepared. Based on the amount of peptide stock solution needed, was weighed an appropriate amount of peptide into a volumetric flask. The appropriate amount of peptide was calculated.
The appropriate amount of buffer to individual peptides was added just before testing itself.
Approximately 15.0 mg of the Cysteine peptide was added to 30 ml of phosphate buffer, pH 7.5. Approximately 15.5 mg of the Lysine peptide was added to 30 ml of ammonium acetate buffer, pH 10.2.

Standard preparation for the determination of the calibration curve
Using serial dilution was prepared standards of the peptide stock solution covering the range from 0.534 – 0.0167 mM.
The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours.

Preparation of samples for analysis:
1:10 ratio, Cysteine peptide (0.5 mM Peptide, 5 mM test chemical)
750 µL Cysteine peptide solution (or 750 µL Phosphate buffer, pH= 7.5 for co-elution controls)
200 µL Acetonitrile
50 µL test chemical solution
or 50 µL solvent (ACN, UPW) for reference controls
or 50 µL positive (negative) control solution for positive (negative) controls

1:50 ratio, Lysine peptide (0.5 mM Peptide, 25 mM test chemical)
750 µL Lysine peptide solution (or 750 µL Ammonium acetate buffer, pH= 10.2 for co-elution controls)
250 µL test chemical solution
or 250 µL solvent (ACN, UPW) for reference controls
or 250 µL positive (negative) control solution for positive (negative) controls

The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours. Then, samples were visually inspected prior to HPLC analysis. Each test chemical was analysed in triplicate for both peptides.

The validated HPLC method - conditions:
Column: Agilent Zorbax SB-C18, 100x2.1 mm, 3.5µm
Precolumn: Phenomex security guard C18, 4.0 x 2.0 mm
Mobile phase A: 0.1% Trifluoracetic acid in water
Mobile phase B: 0.085% Trifluoracetic acid in acetonitrile
Time programmer:
0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5-20 min 10 % B
Column temperature: 30 °C
Sample temperature: 25°C
Flow rate: 0.35 mL/min
Injection volume: 10 μL
Detection: 220 nm
Positive control results:
Cinnamic aldehyd was used as positive control at a concentration of 100 mM in acetonitrile.
The mean Percent peptide depletion value of three replicates for Cinnamic aldehyd was calculated.

Percent Cystein depletion 71.9 %
Percent Lysine depletion 64.7 %
Key result
Run / experiment:
other: 1
Parameter:
other: mean of percent Cysteine and percent Lysine depletion values
Value:
0.8
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All acceptance criteria have been successfully met.
The following criteria should be met for a run to be considered valid:
a) the standard calibration curve should have an r2˃0.99
b) the mean percent peptide depletion value of the three replicates for the positive control Cinnamic aldehyde should be between 60.8% and 100% for the Cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be <14.9% for the percent Cysteine depletion and <11.6% for the percent lysine depletion
c) the mean peptide concentration of reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%. If one or more of these criteria is not met the run should be repeated.
The following criteria should be met for a test item results to be considered valid:
a) the maximum standard deviation for the test item replicates should be <14.9% for the percent Cysteine depletion and <11.6% for the percent lysine depletion
b) the mean peptide concentration of the free Reference controls C in the appropriate solvent should be 0.50±0.05 mM.
Interpretation of results:
GHS criteria not met
Conclusions:
Mean of percent Cysteine and percent Lysine depletion values is 0.8. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used.
Test item, 5-aminotetrazole, was classified as negative (skin non-sensitizer) in DPRA prediction; it was assigned to reactivity class – No or minimal reactivity.
Executive summary:

The purpose of the test is to contribute to the evaluation of the skin sensitisation potential of 5-aminotetrazole. The study is a part of test item health hazard evaluation.

The test was performed according to OECD TG 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity assay (DPRA) (Adopted: 4 February 2015)

All acceptance criteria have been successfully met.

Mean of percent Cysteine and percent Lysine depletion values is 0.8. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used.

Test item, 5-aminotetrazole, was classified as negative (skin non-sensitizer) in DPRA prediction; it was assigned to reactivity class – No or minimal reactivity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification