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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

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

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

other: 0.9% w/v sodium chloride solution.

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% w/v

VEHICLE
- Concentration (if solution): 0.9% w/v

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three per treatment

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
Three per treatment

NEGATIVE CONTROL USED
0.9% w/v sodium chloride solution

POSITIVE CONTROL USED
Imidazole, as a 20% w/v solution in 0.9% w/v sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of either the test item, negative, control or positive control were applied to the appropriate corneas for 240 minutes.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three rinses with Eagle's Minimum Essential Medium
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in corneal opacity (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG were used

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Measured at 240 minutes.

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Measured at 240 minutes.

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

94.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Measured at 240 minutes.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The positive control IVIS (94.7) was within the historical control range for the laboratory and thus the positive control acceptance criterion was satisfied.
- Acceptance criteria met for positive control: The negative control gave a mean opacity of 1.7 and a mean permeability 0.019 and was within the historical control for the laboratory and thus the negative control acceptance criteria were satisfied.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The eye irritation/ corrosivity potential of the test substance was evaluated in a test according to the OECD Guideline 437. The in vitro irritancy score (IVIS) score for the test substance at 240 minutes was 0.1. Therefore, the test substance was found to be non-irritant therefore not assigned a UN GHS classification category.

Executive summary:

The purpose of this test was to evaluate the eye irritation/ corrosivity potential of the test substance according to the OECD Guideline 437. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method is used to identify test items (both chemicals and mixtures) which are potential serious eye corrosives or irritants as well as those which are non-irritant, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items that are non-irritants are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The test substance was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes to the surface of the corneas. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS score for the test substance at 240 minutes was 0.1. IVIS scores for the negative and positive control were 2.0 and 94.7, respectively. Therefore, the test substance was found to be non-irritant and was not assigned a UN GHS classification category.