2,2'-iminobis[4,6-diamino-1,3,5-triazine]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-Apr-2011 to 14-Apr-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.47 Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants "

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Melam

IUPAC Name:

Melam

Constituent 2

Reference substance name:

2,2'-iminobis[4,6-diamino-1,3,5-triazine]

EC Number:

222-695-1

EC Name:

2,2'-iminobis[4,6-diamino-1,3,5-triazine]

Cas Number:

3576-88-3

Molecular formula:

C6H9N11

IUPAC Name:

N~2~-(4,6-diamino-1,3,5-triazin-2-yl)-1,3,5-triazine-2,4,6-triamine

Details on test material:

- Name of test material (as cited in study report): Melam
- CAS 3576-88-3
- Substance type: White powder
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter on the same day.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco,
's Hertogenbosch, The Netherlands), where the eyes were excised by a
slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (304 to 305 mg)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole

Duration of treatment / exposure:

240 minutes

Details on study design:

TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by immediate opacity measurement and permeability evaluation of the cornea.
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS = 55.1 is defined as a corrosive or severe irritant

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In vitro irritancy score (IVIS)

Basis:

mean

Time point:

other: 240 minutes

Score:

6.3

Remarks on result:

other: Test substance

Results 2

Irritation parameter:

other: In vitro irritancy score

Basis:

mean

Time point:

other: 240

Score:

0

Remarks on result:

other: negative control

Results 3

Irritation parameter:

other: In vitro irritancy score

Basis:

mean

Time point:

other: 240

Score:

87

Remarks on result:

other: positive control

Results 4

Irritation parameter:

other: opacity

Basis:

mean

Score:

0

Remarks on result:

other: test substance

Results 5

Irritation parameter:

other: opacity

Basis:

mean

Score:

0

Remarks on result:

other: negative control

Results 6

Irritation parameter:

other: opacity

Basis:

mean

Score:

66

Remarks on result:

other: positive control

Results 7

Irritation parameter:

other: permeability

Basis:

mean

Score:

0.422

Remarks on result:

other: test substance

Results 8

Irritation parameter:

other: permeability

Basis:

mean

Score:

0

Remarks on result:

other: negative control

Results 9

Irritation parameter:

other: permeability

Basis:

mean

Score:

1.365

Remarks on result:

other: positive control

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.1.

The individual positive control in vitro irritancy scores ranged from 85.9 to 87.5. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with Melam showed opacity values ranging from -1 to 1 and permeability values ranging from 0.172 to 0.743. The corneas were clear after the 240 minutes of treatment with Melam. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 2.6 to 10.1 after 240 minutes of treatment with Melam.

Applicant's summary and conclusion

Interpretation of results:

other: not severe irritant or corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Since the mean in vitro irritancy score for Melam was below 55.1 after 240 minutes treatment Melam is considered to be not severely irritant or corrosive.

Executive summary:

An in vitro test for the eye irritancy potential of Melam using the Bovine Corneal Opacity and Permeability test (BCOP test) was performed.

This report describes the ocular irritation properties of Melam on an isolated bovine cornea. The possible ocular irritancy of Melam was tested through topical application for 240 ± 10 minutes.

The study procedures described in this report were based on the most recent OECD and EC guideline.

Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (304 to 305 mg).

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 87 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Melam induced mild ocular irritation through one endpoint (permeability), resulting in a mean in vitro irritancy score of 6.3 after 240 minutes of treatment.

Since the mean in vitro irritancy score for Melam was below 55.1 after 240 minutes treatment Melam is considered to be not severely irritant or corrosive.