1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Eye irritation

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

Test material information

Constituent 1

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.