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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Test guideline 1

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)

Version / remarks:

26 July 2013

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

248-383-5

EC Name:

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

Cas Number:

27277-00-5

Molecular formula:

C9H13N5O

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Physical state/Appearance: Beige powder
- Storage condition of test material: Room temperature in the dark

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Test animals / tissue source

Species:

cattle

Strain:

other: bovine

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Characteristics of donor animals: Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Storage, temperature and transport conditions of ocular tissue: 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.
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% w/v solution in 0.9% w/v sodium chloride solution.

VEHICLE
- Amount applied: 0.75 mL - Concentration (if solution):
- Concentration: 0.9% w/v sodium chloride solution

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- 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. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
- The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
- Three corneas with opacity values close to the median value of all corneas were allocated to the vehicle control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
3

VEHICLE CONTROL USED
sodium chloride 0.9% w/v

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

APPLICATION DOSE AND EXPOSURE TIME
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation (20% w/v solution) or control items were applied to the appropriate corneas.
- The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), which was calculated as follows: IVIS = mean opacity value + (15 x mean permeability OD492 value)

DATA EVALUATION
- The change in opacity for each cornea (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.
- The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- The condition of the cornea was visually assessed post treatment.

DECISION CRITERIA: according to OECD Guideline 437

CRITERIA FOR AN ACCEPTABLE TEST
For an acceptable test the following positive and negative control criterion should be achieved:
- 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2014 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 66.9 to 101.4.
- 0.9% w/v sodium chloride solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤4.1 and for permeability ≤0.105.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean in vitro irritancy score

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

deviation: see 'acceptence of results'

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean opacity

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

deviation: see 'acceptence of results'

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein leakage

Run / experiment:

mean permeability

Value:

0.005

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

deviation: see 'acceptence of results'

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was 110.7, and therefore not within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore not satisfied. However, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. This deviation was considered to have not affected the integrity or validity of the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this BCOP test according to OECD 437 and EU Method B.47, the test item was not found to be irritant to the eye.

Executive summary:

The purpose of this GLP compliant test according to OECD 437 was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in 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 can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, 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 not classified for eye irritation or serious eye damage 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.

750 µL of the test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Vehicle only control and positive control (20% w/v imidazole in 0.9% w/v sodium chloride solution) 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 2.8 and 110.7 for the vehicle control and positive control, respectively. Acceptance criteria for the negative control were met. The IVIS of the positive control was higher than the criteria range set for an acceptable test. However, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. This deviation was considered to have not affected the integrity or validity of the study. The IVIS of the test item was 1.0, indicating that the requirements for classification as UN GHS/ EU CLP Category 1 or 2 are not met. In conclusion, classification for eye irritation is not required for the test item.