Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 14 to 18, 1988.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedures cannot be subsumed under testing guideline and according to GLP, nevertheless are well documented and scientifically acceptable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Bovine Eye and the Chicken Chorioallantoic Membrane (BECAM ASSAY).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

EC Number:

255-217-5

EC Name:

Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Cas Number:

41098-56-0

Molecular formula:

C40H44N12O18S6.6Na

IUPAC Name:

hexasodium 2,2'-(ethene-1,2-diylbis{(3-sulfonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino})dibenzene-1,4-disulfonate

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Calf eyes were obtained from a local slaughterhouse (Viguurs, 's-Hertogenbosch). The eyes were excised by standard procedures within 15 minutes after killing the animal. They were transported to the laboratory and examined for injuries. This was done by holding the eyes submersed in saline; eyes with corneal damage or other abnormalities were discarded. The selected eyes were preserved at ambient temperature (approximately 20 °C) under saline not longer than 4 hours.
White eggs were obtained from a chicken breeding centre.
Date of arrival: April 5, 1988.
They were transported at a temperature between 15 and 20 °C.
After approximately 6 hours of rest, the incubation was started by placing the eggs in the incubator with the air chamber upwards.
The temperature was maintained at 37-38 °C and the relative humidity between 50 and 70 %. The eggs were turned once every hour.
They were candled at the seventh day of incubation; unfertilized eggs or eggs containing a dead embryo were removed.

Test system

Amount / concentration applied:

Calf eyes: 0.1 ml
eggs: 0.2 ml

Duration of treatment / exposure:

calf eyes: 30 sec
eggs: 20 sec

Observation period (in vivo):

calf eyes: 10 min
eggs: 30 sec, 1.5 and 4.5 min

Number of animals or in vitro replicates:

5 calf eyes

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks on result:

other: See "Any other information on results"

Any other information on results incl. tables

BE ASSAY

Treatment	Opacity	Detachment	Fluorescence	Total
Neg. control	0	0	0	0
Toluene	0	0	1	1
Acetone	2	0	1.5	3.5
Eye1	0	0	0	0
Eye2	0	0	0	0
Eye3	0	0	0	0
Eye4	0	0	0	0
Eye5	0	0	0	0
			Mean	0

CAM assay

		30 sec			2 min			5 min			Total
Time	Egg	I	H	C	I	H	C	I	H	C
09:37	1	+	-	-	+	-	-	+	+ (a)	-	3
09:40	2	+	-	-	+	-	-	+	-	-	2
09:43	3	+	-	-	+	-	-	+	+ (a)	-	3
09:46	4	+	-	-	+	-	-	+	+ (a)	-	3
										Mean:	2.75

^{a: Bleeding by 2 min}

The total scores are not consistent with the above mentioned scoring system (table 2). According to this scoring in table 2 the total score for egg 1 -4 would be: 4.5, 3.5, 4.5, 4.5, respectively, which would lead to a mean score of 4.25. The score of 4.25 would lead to moderate irritation.

REFERENCE SUBSTANCES

For toluene, a score of 1.0 was obtained in the BE assay whereas the average score in the CAM assay was 1.25.

Acetone treatment resulted scores of 3.5 in the BE assay and an average score of 2.5 in the CAM assay.

As the effects, produced by acetone and toluene, fell within the historical range and the untreated eyes and eggs showed no signs of irritation, the results were considered to be valid.

To evaluate the effects observed in both assays the documented in vivo effects of the two reference substances toluene and acetone were taken into consideration. Toluene produces slight transient conjunctival irritation with negligible to slight corneal epithelial damage without opacity in the rabbit eye, while no serious effects have been reported in case of accidental exposure of human eyes.

Acetone causes temporary edema and moderate corneal opacity with epithelial damage to exposed rabbit eyes while in man reversible corneal opacity has been observed.

The test substance induced no effects in the bovine eye.

The effects on the CAM consisted of vascular injection almost instantly after application. In three eggs (no. 1, 3 and 4) haemorrhages were scored 2 minutes after application. On account of these results it is expected that eye contact with the test substance may lead to slight irritation. Acute corneal effects may be negligible.

Applicant's summary and conclusion

Interpretation of results:

other: BE assay: not irritating (according to the CLP Regulation). CAM assay: inconclusive (according to the CLP Regulation).

Conclusions:

Today a validated methods and procedures protocol about the Bovine Eye assay is available. The differences between the present test and the validated protocol are not suspected of impairing the results. Therefore, it can be concluded that the substance is not irritating, based on the BE assay.
In the case of the CAM assay the protocol is not validated yet and the re-evaluation of the result obtained is not appicable. Therefore, the CAM assay can be considered as inconclusive.

Executive summary:

Method

The eye irritation potential was assessed according to the in vitro Bovine Eye and the Chicken Chorioallantoic Membrane (BECAM ASSAY) method.

Results

The test substance induced no effects in the bovine eye.

The effects on the CAM consisted of vascular injection almost instantly after application. In three eggs (no. 1, 3 and 4) haemorrhages were scored 2 minutes after application. On account of these results it is expected that eye contact with the test substance may lead to slight irritation. Acute corneal effects may be negligible.

Conclusion

Today a validated methods and procedures protocol about the Bovine Eye assay is available. The differences between the present test and the validated protocol are not suspected of impairing the results. Therefore, it can be concluded that the substance is not irritating, based on the BE assay.

In the case of the CAM assay the protocol is not validated yet and the re-evaluation of the result obtained is not appicable. Therefore, the CAM assay can be considered as inconclusive.