Reaction mass of sodium amino-bis{[4-(ethenylsubstituted)phenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-{[4-(ethenylsubstituted)phenyl]diazenyl}-{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-bis{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate

Eye irritation

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Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorption. The deviation was signed and assessed by the study director on 07. Aug. 2014.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

None

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes (Bos primigenius Taurus (Fresh bovine corneas)) were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2 -4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Test system

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

750 μL of the test item were tested as suspension at 20% concentration in 0.9% sodium chloride solution.

Duration of treatment / exposure:

Incubation time: 4 hours

Observation period (in vivo):

No data

Number of animals or in vitro replicates:

No data

Details on study design:

Refer to "Any other information on materials and methods incl. tables".

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

-0.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

None

Other effects:

None

Any other information on results incl. tables

 Opacity and Permeability Values

The absorption (570 nm) and opacity values which were measured before and after exposition are given in the following table:

Parameter	Negative Control
Absorption before exposition	0.1703	0.1803	0.1790
Absorption after exposition	0.2574	0.2851	0.5227
Opacity before exposition	1.4801	1.5146	1.5101
Opacity after exposition	1.8088	1.9280	3.3320
Opacity Difference	0.3287	0.4134	1.8219

 

Mean opacity difference of the negative control is 0.8547.

 

Absorption and Opacity Values Test Item and Positive Control

Parameter	Test ItemFAT 40865/A TE			Positive Control
Absorption before exposition	0.1908	0.2072	0.1664	0.1583	0.1376	0.1975
Absorption after exposition	0.2762	0.3804	0.4183	1.7073	1.6212	1.6112
Opacity before exposition	1.5517	1.6114	1.4669	1.4398	1.3728	1.5758
Opacity after exposition	1.8889	2.4010	2.6200	50.9683	41.8023	40.8507
Opacity Difference	0.3372	0.7897	1.1531	49.5285	40.4295	39.2750

 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

Repl.	Negative Control			Test ItemFAT 40865/A TE			Positive Control
Meas.	0.0058	0.0065	0.0051	0.0022	0.0029	0.0045	0.3828	0.3462	0.4711
Corr.	0.0290	0.0325	0.0255	0.0110	0.0145	0.0225	1.9140	1.7310	2.3555
Mean	0.0290			--

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9% NaCl	0.764	1.290	61.6 %
	0.901
	2.204
Test Item FAT 40865/A TE	- 0.788	- 0.290	170.4 %
	- 0.283
	0.201
Positive Control 20% imidazole solution	76.949	71.790	8.5 %
	65.105
	73.318

 

Classification

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation

or serious eye damage.

IVIS	UN GHS Category Eye Damage
≤ 3	No Category
> 3;≤ 55	No prediction can be made
> 55	Eye damage Category I

 

In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category 1. The test item FAT 40865/A TE showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is - 0.290

Validity

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The negative or solvent control has to show an IVIS between 0 and 3.

The validity criteria and findings are given in the following table:

Parameter	Criterion	Found	Assessment
IVIS of negative control 0.9% NaCl	0 - 3	1.290	ok
IVIS of positive control 20% imidazole solution	35.6 – 130.4	71.790	ok

 Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

Under the conditions of this test, the test item FAT 40865/A TE showed no effects on the cornea of the bovine eye. Hence, it was considered to be non-irritating to eyes.

Executive summary:

An OECD 437 guideline study was performed in accordance to GLP to assess the irritation potential of test item FAT 40865/A to eyes using bovine corneas. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item FAT 40865/A TE was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea. 20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea. The calculated IVIS (in vitro irritancy score) was - 0.290. According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS≤3 requires no classification for eye irritation or serious eye damage. Hence, under the conditions of this test system, the test item FAT 40865/A TE showed no effects on the cornea of the bovine eye and considered to be non-irritating to eyes.