4-amino-2-methylpyrimidine-5-methylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June - August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully Guideline- and GLP-compliant

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD-Guideline 437, Bovine Corneal Opacity and Permeability Test Method (BCOP), 7th of September 2009.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-2-methylpyrimidine-5-methylamine

EC Number:

202-384-7

EC Name:

4-amino-2-methylpyrimidine-5-methylamine

Cas Number:

95-02-3

Molecular formula:

C6H10N4

IUPAC Name:

5-(aminomethyl)-2-methylpyrimidin-4-amine

Details on test material:

- Name of test material (as cited in study report): "Grewe-diamin".
Chemical name: 4-amino-2-methyl-5-Pyrimidinemethanamine.
Molecular formula: C6H10N4.
Charge No.: M-622-035-200.
CAS No.: 95-02-3.
Appearance: White-yellow crystals in pellet form.
Solubility: 430.000 mg/l in water.
Conditions of storage: In the dark at room temperature.
Stability at conditions of storage: Hygroscopic after pulverization. Stable in water.
Expiry date: 01 February 2011.

Test animals / tissue source

Species:

other: in vitro system

Strain:

other: Isolated corneas from the eyes of cows and bulls aged between 24 30 month and free of macroscopically visible defects.

Details on test animals or tissues and environmental conditions:

Corneas: Isolated corneas from the eyes of cows and bulls aged between 24 30 month and free of macroscopically visible defects.
Supplier: Slaughterhouse Klaus Grandits, Ungerbachstr. 10, A-2860 Kirchschlag.
Number of corneas: Total of 9 corneas: 3 for the test substance, 3 for the negative control and 3 for the positive control.

Test system

Vehicle:

water

Controls:

other: n.a.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 ul
- Concentration: 20 % (w/v) in deionised water (sterile)
POSITIVE CONTROL
- Amount applied: 750 ul
- Concentration: 20 % imidazole (w/v) in deionised water (sterile)
NEGATIVE CONTROL
- Amount applied: 750 ul
- Concentration: deionised water (sterile)

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

n.a.

Number of animals or in vitro replicates:

Total of 9 corneas: 3 for the test substance, 3 for the negative control and 3 for the positive control.

Details on study design:

Test design
Bovine corneas were isolated and mounted in cornea holders and equilibrated for one hour to achieve normal metabolic activity.
After exclusion of corneas which did not achieve quality criteria, the corneas were distributed into groups (3 per group) and exposed to the
test substance, the negative control and the positive control for 4 hours. Then the substances were removed and the corneas were accurately
washed and the opacity and permeability of each cornea were recorded. Opacity was measured quantitatively with the aid of an opacitometer.
Permeability was determined by the amount of sodium fluorescein dye that penetrated all cornea layers. For this purpose fluorescein solution
was filled into the anterior chambers of the cornea holders followed by an incubation period of 90 minutes. The amount of sodium fluorescein
that crossed into the posterior chambers was quantitatively measured with a spectrophotometer at OD490. Using opacity and permeability
data an IVIS was calculated. The positive and negative control groups were simultaneously used for other, concurrently performed studies.

Application of test and control substances
750 µl test substance preparation were introduced into the anterior chamber through the dosing holes on the top surface of the chamber,
and the holes were subsequently sealed during exposure (close-chamber method). Then the corneas were exposed in a horizontal position for
4 hours while ensuring that the test substance adequately covered the epithelial surface. Incubation temperature, monitored with a min / max
thermometer, was 31.2 °C 33.0 °C.

Post-Exposure Incubation
After the exposure period substances were removed from the anterior chamber and the epithelium was washed three times with
EMEM+ to determine the effectiveness of rinsing acidic or alkaline materials and to remove substance residues. cEMEM was used as a
final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Both chambers were then
refilled with fresh cEMEM.

Opacity Measurement
Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively in Lux with
the aid of an opacitometer-kit BASF-OP2.0 (see Appendix 2), which was calibrated with a standard filter set before the corneas were measured.

Application of Fluorescein
1 ml sodium fluorescein solution was added to the anterior chamber of the cornea holder and then incubated in a horizontal position for
90 minutes. Incubation temperature, monitored with a min / max thermometer, was 31.2 °C 33.0 °C.

Permeability Measurement
Permeability was determined by the amount of sodium fluorescein dye that penetrated all corneal cell layers. The amount of sodium
fluorescein that crossed into the posterior chamber was quantitatively measured with the aid of a Bio-Tek EL800 microtiter plate reader
at 490 nm. For measurement 360 µl of cEMEM from the posterior chamber were transferred into the wells of a 96-well microtiter plate (triplicates).
Data were recorded as OD490 values which were equivalent to the OD490 values based upon a visible light spectrophotometer using a
standard 1 cm path length. To proof that the measurement was performed in the linear range wells containing
5 concentrations (ranging from 12.5 µg/ml to 0.78 µg/ml) of fluorescein solutions were additionally prepared.

Calculations
Opacity
Opacity values were calculated as follows:
Opacity value = a x (I0/I) + b
I illuminance (Lux) with the cornea
I0 illuminance (Lux) without cornea
a, b equipment-specific variables
The standard deviation for each group was calculated as well.

Permeability
To ensure that the permeability values measured were in the exponential range the linearity was determined by triple measurement of fluorescein solutions of 5 concentrations. A linear calibration function and the correlation coefficient thereof was calculated.
As the OD490 values of corneas treated with 20% imidazole lied outside of the linear range dilutions of 1:5 were measured.
As the OD490 values of corneas treated with the test substance lied outside of the linear range dilutions of 1:5 were measured.
The standard deviation for each group was calculated as well.

IVIS (In Vitro Irritancy Score)
The mean opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity and permeability values for each treatment group were combined in an empirically-derived formula to calculate the IVIS for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The standard deviation for each group was calculated as well.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

84

Vehicle controls validity:

valid

Remarks on result:

other:

Remarks:

ocular corrosive

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The IVIS of "Grewe-diamin" was 84.0. An important increase in opacity as well as in permeability was observed.

Thus "Grewe-diamin" is regarded to be an ocular corrosive or severe irritant, according to the OECD Guideline 437 for the testing of
chemicals Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants.
According to the results of this study and the Directive 2001/59/EC for classification, the test substance "Grewe-diamin" needs to
be labelled as R41 (EU), Category 1 (EPA and GHS).

Executive summary:

Aim

TheBovine Corneal Opacity and Permeability Study (BCOP Test Method)was performed to reveal possible ocular corrosivity and severe irritation of"Grewe-diamin", according tothe OECD Guideline 437 for the testing of chemicals Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, 07 September 2009.

Method

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µl of the test substance preparation were topicallyadministered to 3 isolated bovine corneas to the epithelial surfaces for 4 hours andthe final opacity was measured. Then 1 ml of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances:

• Negative control:       sterile aqua dest.
• Positive control:        20 % imidazole.

Finally the IVIS (In Vitro Irritancy Score) was calculated as follows:

IVIS = mean opacity value + (15 x mean permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS=55.1 is defined as ocular corrosive or severe irritant.

Results

General

The IVIS for"Grewe-diamin"was 84.0.

IVIS of the negative control was 6.5 and for the positive control 111.8, thus demonstrating the validity of the experiment.

Conclusion

According to the results of this study and the OECD Guideline 437, the test substance
"Grewe-diamin" is considered to be an ocular corrosive or severe irritant.

According to the guidelines "Grewe-diamin" needs to be labelled as R41 (EU),
Category 1 (EPA and GHS).