Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 April 2014 to 18 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Vivimed, lot no. 20140101
- Expiration date of the lot/batch: January 2016
- Purity test date: 16th January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature in the dark
- Stability under test conditions: stable within two hours
- Solubility and stability of the test substance in the solvent/vehicle: stable within two hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Remarks:

adult cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: local abattoir
- Age at study initiation: typically 12 to 60 months old
- Weight at study initiation: not applicable
- Housing: not applicable
- Diet (e.g. ad libitum): not applicable
- Water (e.g. ad libitum): not applicable
- Acclimation period: not applicable

Test system

Vehicle:

other: 0.9% sodium chloride solution

Controls:

yes

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL of test item in 0.9% w/v sodium chloride applied
- Concentration (if solution): 0.9%w/v sodium chloride
- Lot/batch no. (if required): 300999 104
- Purity: 0.9%

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

triplicate

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders

QUALITY CHECK OF THE ISOLATED CORNEAS
The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control.

NUMBER OF REPLICATES
Three corneas were also allocated to the test item and three corneas to the positive control item

NEGATIVE CONTROL USED

SOLVENT CONTROL USED (if applicable)
0.9% w/v Sodium chloride solution

POSITIVE CONTROL USED
20% w/v Imidazole solution in 0.9%w/v sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of the test item preparation or control items were applied to the appropriate corneas for 240 minutes exposure time

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: three times
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader](OD490)
- Others (e.g, pertinent visual observations, histopathology): Histopathology

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The test item is classified according to the prediction model below:
IVIS
CLASSIFICATION
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Main Test : Negative Control

Value:

2.9

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Main Test : Positive Control

Value:

75

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Main test : Test item

Value:

68.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No but histopathology showed necrosis.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Imidazole sued as positive control and induce good positive response to the test system.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

The In Vitro irritancy score for the test item, 68.1, suggested that classification as serious eye damage was appropriate. However the intensely colored Test Item had interfered with the study parameter of opacity and therefore histopathology was conducted as a confirmatory parameter. The results of histopathology identified necrosis to all cell layers of the cornea indicating that the In Vitro irritancy score, although artificially increased by the staining of the cornea, did not produce an over prediction of ocular irritancy potential.

Any other information on results incl. tables

Table1     Individual and Mean Corneal Opacity and PermeabilityMeasurements

 

Treatment	Cornea Number	Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment  Pre-Treatment	Corrected Value		Corrected Value
Negative   Control	10	2	5	3		0.046
	11	2	3	1		0.071
	12	2	4	2		0.058
				2.0*		0.058		2.9
Positive   Control	1	3	51	48	46.0	1.558	1.500
	2	1	60	59	57.0	1.224	1.166
	3	3	68	65	63.0	1.322	1.264
					55.3		1.310	75.0
Test Item	4	2	31	29	27.0	2.265	2.207
	5	1	50	49	47.0	1.916	1.858
	6	3	44	41	39.0	2.086	2.028
					37.7		2.031	68.1

 

 

 

 

OD =Opticaldensity* = Mean of the post-treatment pre-treatmentvalues                                            =MeanValue

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the experimental condition of the study, the registered substance Acid Violet 43 induced an in vitro irritancy score of 68.1 and histopathology showed necrosis to all cell layers of the cornea. Hence, the test substance was classified as Category 1 for Eye Irritation, causes serious eye damage according CLP regulation.

Executive summary:

The purpose of this GLP study was to identify Acid Violet 43 eye effect that can induce serious eye damage using OECD Guideline 439 method for Bovine Corneal Opacity and Permeability Assay (BCOP).

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). Histopathological evaluation of the corneas at the end of the treatment was performed as an additional endpoint.

Histological evaluation revealed that the test item induced much loss of the epithelium and any remaining cells were necrotic. The In Vitro irritancy score for the test item, 68.1.

Under the experimental condition of the study, the registered substance Acid Violet 43 induced an in vitro irritancy score of 68.1 and histopathology showed necrosis to all cell layers of the cornea. Hence, the test substance was classified as Category 1 for Eye Irritation, causes serious eye damage according CLP regulation.