4H-1,2,4-triazol-4-ylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.06. - 24.06. 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

Adopted: 26 June 2020, Appendix I: The Direct Peptide Reactivity Assay (DPRA)

Deviations:

yes

Remarks:

error by typo, with no influence on result (see Overall remarks, attachments)

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4H-1,2,4-triazol-4-ylamine

EC Number:

209-533-5

EC Name:

4H-1,2,4-triazol-4-ylamine

Cas Number:

584-13-4

Molecular formula:

C2H4N4

IUPAC Name:

4H-1,2,4-triazol-4-ylamine

Test material form:

solid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions
Preparation of peptide stock solution (approx. 0.667 mM)
The necessary volume of peptide solution was estimated. (800 µl of peptide stock solution is needed for preparation of one analysed solution). 20 ml of the stock solution of Cysteine and Lysine peptide (0.667 mM) were prepared.

20 ml of phosphate buffer, pH = 7.5 was added to 10.06 mg of the Cysteine peptide. The concentration of Cysteine peptide in the stock solution was 0.6699 mM. 20 ml of ammonium acetate buffer, pH = 10.2 was added to 10.60 mg of the Lysine peptide. The concentration of Lysine peptide in the stock solution was 0.6831 mM. The amount of buffer was added to individual peptides just before testing itself.

Using serial dilution, standard solutions were prepared of the Cysteine peptide stock solution covering the range from 0.0167 - 0.5359 mM.
Using serial dilution, standard solutions were prepared of the Lysine peptide stock solution covering the range from 0.0171 - 0.5465 mM.




- Preparation of the test chemical solutions
Theoretical weight of 4H-1,2,4-triazol-4-amine is 25.48 mg ± 10%.
The weighed amount of 4H-1,2,4-triazol-4-amine was dissolved in 3 ml of ACN.
The weighed amount of 4H-1,2,4-triazol-4-amine was 42.49 mg for Cysteine peptide analysis. The weighed amount of 4H-1,2,4-triazol-4-amine was 42.57 mg for Lysine peptide analysis.



- Preparation of the positive controls, reference controls and co-elution controls
A positive control: 100 mM solution of cinnamaldehyde. The weighed amount of cinnamaldehyde (approximately 40.13 ± 10 % mg) was dissolved in 3 ml of acetonitrile.
A negative control: 100 mM solution of 1-butanol. The weighed amount of 1-butanol (approximately 22.5 ± 10 % mg) was dissolved in 3 ml of acetonitrile.

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 were 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 were made with acetonitrile (solvent used for dissolving the positive, negative controls and test item). They are used to verify that the solvent used to dissolve the test item does not any impact the percent peptide depletion. The Reference Controls C are used to calculate percent peptide depletion.

Co-elution control was constituted by the test item 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 item does not have the same retention time as peptides and does not absorb at 220 nm as the Cysteine and Lysine peptides.




INCUBATION
- Incubation conditions
The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 ± 2 hours.
- Precipitation noted: no

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
A standard calibration curve was generated for both the Cysteine and the Lysine peptides.
Linearity was determined by mathematical treatment of results obtained by analysis of standard solutions ( report chapter 3.5.6. and 3.5.7.) The concentration interval of the standard solutions was 0.0167 - 0.5359 mM for Cysteine peptide and 0.0171 - 0.5465 mM for Lysine peptide.
Each prepared calibration solution was analysed by HPLC with DAD detection.


- Verification of the suitability of the HPLC for test chemical and control substances
The criteria of quality in accordance with the OECD TG 442C were fulfilled.


DATA EVALUATION
The Cysteine and Lysine peptide concentrations were determined at a 220 nm by evaluating of their peak area in samples and calculating from the linear calibration curve constructed from the standard solutions

The Cysteine 1:10/Lysine 1:50 prediction model was used based on the experimental results .
The mean of Cysteine and Lysine percent depletion value is 1.4 %.
The test item 4H-1,2,4-triazol-4-amine was included in the reactivity class – No or minimal reactivity and was determined as Negative in the DPRA prediction.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

The mean percent peptide depletion value at 220 nm for the positive control Cinnamaldehyde is 70.8 % for the Cysteine peptide.
The mean percent peptide depletion value at 220 nm for the positive control Cinnamaldehyde is 65.3 % for the Lysine peptide.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

2.3 %

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0.5 %

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method, another in vitro test is needed

Conclusions:

All study acceptance criteria have been successfully met.

The test item, 4H-1,2,4-triazol-4-amine, has been found negative in the DPRA prediction; it was assigned to reactivity class “No or minimal reactivity”.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The purpose of the test is to contribute to the evaluation of the skin sensitisation potential of 4H-1,2,4-triazol-4-amine. This study is a part of this test item health hazard evaluation for registration of industrial chemicals.

 

The test was performed according to OECD TG 442C, In Chemico Skin Sensitisation: Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins, Adopted: 26 June 2020, Appendix I: The Direct Peptide Reactivity Assay (DPRA)

 

In the study, 4H-1,2,4-triazol-4-amine was dissolved in acetonitrile, based on the results of the pre-experiments. The stock solution at concentration 167 mM was prepared based on a molecular weight of 84.08 g/mol.

 

The test item solutions were tested by incubating the samples with synthetic heptapeptides containing either cysteine or lysine at 25 °C for 24 ± 2 hours. Depletion of the peptide in the reaction mixture was measured by HPLC with UV detection. Average peptide depletion data for cysteine and lysine were calculated. Cinnamaldehyde (100 mM) was used as a positive control and 1-butanol (100 mM) was used as a negative control. Each test chemical was prepared and analysed in triplicate for both peptides.

Samples were visually inspected prior to the start of incubation and prior to HPLC analysis.

Immediately after addition of the Cysteine peptide to the solutions, there were not any turbity or cloudiness, any colour changes in the vials. There were not any turbity or cloudiness, any colour changes in the vials after 24-hour incubation.

Immediately after addition of the Lysine peptide to the positive control, slight whitish turbidity was observed in the vial. The other vials were clear. The yellowish sediment on the bottom and on the wall of the vials with positive control was presented after 24-hour incubation. The other vials were clear with no color change, no turbidity and no precipitate in the vial.

All study acceptance criteria have been successfully met.

 

The mean of Cysteine and Lysine percent depletion values is 1.4 %. Based on the obtained results, the prediction model the Cysteine 1:10/Lysine 1:50 was used for evaluation. The test item, 4H-1,2,4-triazol-4-amine, has been found negative in the DPRA prediction; it was assigned to reactivity class “no or minimal reactivity” for both peptides.

 

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.