2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 May 2016 to 10 May 2016

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

EC Number:

248-387-7

EC Name:

2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

Cas Number:

27287-73-6

Molecular formula:

C6H7N5O

IUPAC Name:

2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

Test material form:

solid: particulate/powder

Details on test material:

-Storage conditions: room temperature in the dark
-Colour: beige

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™ reconstructed human epidermis

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 16-EKIN-018
- Delivery date: 03 May 2016
- Date of initiation of testing: 05 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) with Ca and Mg. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: no reference filter

NUMBER OF REPLICATE TISSUES: three per treatment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure period followed by the 42-Hour post-exposure incubation period is less than 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure period followed by the 42-Hour post-exposure incubation period is equal to or more than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl Sodium Dodecyl Sulphate
- Concentration (if solution): 5% v/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three per treatment

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Sample 1

Value:

95.2

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Sample 2

Value:

103.7

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue Sample 3

Value:

104.4

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (of 3 test tissue samples)

Value:

101.1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No. Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert and also that the temperature indicator colour and agar medium colour were satisfactory so as to confirm the tissues were acceptable for use in the study. The tissue model supplier also demonstrated the batch of tissues was acceptable by determining the barrier function and also performed histological examination demonstrating appropriate morphology of the tissues.
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple. Therefore the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to use additional colour correction tissue controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%. The mean OD562 for the negative control treated tissues was 0.730 and the standard deviation value of the viability was 5.0%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 4.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.1%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

The mean OD₅₆₂values and viabilities for the negative control, positive control and test item are presented in the table below:

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD Relative mean viability (%)
Negative Control Item	0.701	0.730	0.037	N/A	100*	N/A
	0.718			N/A
	0.771			N/A
Positve Control Item	0.072	0.058	0.032	9.9	7.9	4.4
	0.022			3.0
	0.081			11.1
Test Item	0.695	0.738	0.037	95.2	101.1	5.1
	0.757			103.7
	0.762			104.4

* The mean viability of the negative control tissues is set at 100%

OD = Optical Density

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this EPISKIN(TM) assay according to OECD Guideline 439, the results indicate that the test item is non-irritant to the skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN(TM) reconstructed human epidermis model according to OECD Guideline 439. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to a 96-well plate. The optical density was measured at 562 nm. Data was presented as percentage tissue viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of test item treated tissues was 101.1% compared to the negative control after the 15-minute exposure period and 42-hours post-exposure incubation period. This is above the 50% threshold for classification and therefore the test item was considered to be non-irritant to the skin under the conditions of this assay. The quality criteria required for acceptance of results in the test were met, therefore the study is considered valid.