Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine
EC Number:
216-122-4
EC Name:
1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine
Cas Number:
1502-47-2
Molecular formula:
C6H6N10
IUPAC Name:
1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine
Test material form:
solid
Details on test material:
Test Item Name: Exolit 775
Sample Number: Ba-Ha-19-025-1; Deflam 20 20151106 JOB D02441
CAS No.: 1502-47-2

Chemical name (IUPAC): 1,3,4,6,7,9,9b-Heptaazaphenalene-2,5,8-triamine/Melem
CAS No.: 1502-47-2
Sample No.: Ba-Ha-19-025-1; Deflam 20 20151106 JOB D02441
Manufactured Date: 2019-09-06
Expiry Date: 2034-09-06
Purity as per Certificate of Analysis: 95.75%
Physical Appearance: Solid White
Storage Conditions: Cool and Dry (+2 to +8 °C)

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 µg/mL) in an ice box.

Test system

Amount / concentration applied:
100 mg/cornea
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
4 hours
Number of animals or in vitro replicates:
3 corneas/group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Procurement of corneas: The bovine eyes were procured from local abattoir and transported in a jar x containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 μg/mL) in an ice box. On day of experiment 27 numbers were found suitable for experiment.

- Dissection: On day of experiment, post pretest examination, eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container of fresh HBSS (Hank Balanced Salt Solution).

- Mounting: The isolated corneas were mounted in the cornea holder, which separated holder into anterior and posterior chambers. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. The EMEM medium was added in to both the chambers and kept in an incubator (32°C) for 1 hour.

- Opacity and Permeability: All the selected corneas for the study were tested for their opacity and permeability to calculate IVIS (Invitro irritancy score).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean of three measurements
Value:
0.114
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

The assay results are found to be accepted because the positive control i.e. Chlorhexidine used in this study has exhibited an IVIS score of 165.878 which fall between two standard deviations of the historical mean i.e 154-172.
Also, the negative control i.e. Distilled water response resulted in opacity and permeability values that are less than established upper limits for negative control opacity and permeability values.

Any other information on results incl. tables

Table 1.            Details of Opacity Calculation

Cornea

Holder Number

Blank value*

Io (lux)

Pre-treatment Cornea reading#

I (lux)

Initial opacity

(t0)*

Treatment group

Post-treatment Cornea reading$

I (lux)

Final opacity

(240 mins)*

Change of Opacity

 

 

Corrected Opacity

 

 

Mean Opacity

2016-123#01

501

434

7

Negative control: Distilled water

435

6

0

NA

0

2016-123#12

501

441

6

440

6

0

NA

2016-123#15

501

445

5

442

6

1

NA

2016-123#17

501

434

7

Positive control: Chlorhexidine

109

144

137

137

131

2016-123#18

501

460

4

116

133

129

129

2016-123#26

501

434

7

115

134

127

127

2016-123#22

501

505

0

Test item:

Exolit 775

506

0

0

0

0

2016-123#24

501

485

2

499

1

-1

-1

2016-123#16

501

494

1

494

1

0

0

* Blank value for each cornea holder with medium but without cornea.

#Reading of each cornea holder with cornea and medium (pre-test incubation period).

$240 minutes post reference item exposure incubation period

·     Initial/Final opacity = ((I0/I)-b)/a, where I = individual reading, a=0.0251 & b=0.9894 (a and b values are instrument specific constants).

·     Change of opacity = Final opacity – Initial opacity

·     Corrected opacity = Change in opacity of each treated cornea – average change of opacity value of negative corneas i.e. “0” of Distilled water

·     Mean opacity = Mean of corrected opacity

Table 2. Details of Permeability Calculation

 

Cornea

Holder Number

Treatment group

OD490reading

(90 mins)

Blank corrected OD490*

Negative control corrected OD490#

Mean Permeablity

 

 

NA

 

Blank OD reading

 

0.0391

NA

 

 

NA

0.0406

0.0407

0.0382

0.0456

0.0416

0.0385

 

 

2016-123#01

Negative control: Distilled water

0.0373

-0.0033

 

NA

0.0033

0.0414

0.0008

2016-123#12

0.0474

0.0068

0.0508

0.0102

2016-123#15

0.0357

-0.0049

0.0510

0.0104

 

 

2016-123#17

Positive Control:

Chlorhexidine

2.5078

2.4672

2.4639

2.3252

2.5036

2.4630

2.4597

2016-123#18

2.0068

1.9662

1.9629

1.9837

1.9431

1.9398

2016-123#26

2.6033

2.5627

2.5594

2.6098

2.5692

2.5659

 

2016-123#22

Test item:

Exolit 775

0.0729

0.0323

0.0290

0.0076

0.0711

0.0305

0.0272

2016-123#24

0.0425

0.0019

-0.0014

0.0492

0.0086

0.0053

2016-123#16

0.0384

-0.0022

-0.0055

0.0348

-0.0058

-0.0091

* Blank corrected OD490= Individual corneal reading OD490– Mean OD Blank reading

#Negative control corrected OD490= Individual blank corrected OD490– Mean of Negative control OD490reading (Distilled water)

 

Table 3. Calculation ofIn VitroIrritation Score (IVIS*)

Treatment group

Mean Opacity value

Mean Permeability value

IVIS

Negative control

(Distilled water)

0

0.0033

0.050

Chlorhexidine

131

2.3252

165.878

Exolit 775

0

0.0076

0.114

*IVIS = Mean Opacity value + (15 × Mean Permeability value)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The registration substance was investigatied for its eye irritation property accroding to the OECD Guideline 437 (BCOP). The registration substance is a non-irritant for the eye.
Executive summary:

The registration substance was investigatied for its eye irritation property accroding to the OECD Guideline 437 (BCOP). 100 mg of of the test item (Exolit 775) and positive control (Chlorhexidine) was applied to each cornea by means of open chamber method. A volume of 0.75 mL of negative control (Distilled water) were applied undiluted on the corneal surface by means of open chamber method. The control and treated corneas were then subjected to the opacity and permeability measurements. The test item Exolit 775 exhibited an IVIS score of 0.114. The registration substance is a non-irritant for the eye.

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