9-[bis(2-hydroxyethyl)amino]-5-[(3-methylphenyl)amino]-7-phenylbenzo[a]phenazin-7-ium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

EC Number:

941-946-2

Molecular formula:

C33H31ClN4O2

IUPAC Name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

purity; 89.7 % (w/w) (main component)

Test system

Vehicle:

physiological saline

Amount / concentration applied:

Each 0.75 mL of a 20% (w/v) suspension of the test item in physiological saline were applied to three bovine corneas.

Duration of treatment / exposure:

240 minutes

Details on study design:

Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

SCORING SYSTEM:
Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups and after rinsing the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA INTERPRETATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; < 55 No prediction can be made
≥ 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)

Results and discussion

In vivo

Other effects:

Relative to the negative control, the test item caused a significant increase of the corneal opacity but not of the permeability. The calculated mean IVIS was 171.91 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is classified as seriously eye damaging.

Any other information on results incl. tables

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	GHS
		Mean		Mean
Negative Control	0	0	0.050	0.050	0.75	0.75	No Category
	0		0.053		0.80
	0		0.047		0.71
Positive Control	111.00		0.168*		113.52*	115.93*	Category 1
	125.00		0.037*		125.56*
	108.00		0.048*		108.72*
Test item	211.00		0.049*		211.74*	171.91*	Category 1
	170.00		0.000*		170.00*
	134.00		0.000*		134.00*

*corrected values

 

Applicant's summary and conclusion

Interpretation of results:

Category I

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is seriously eye damaging (Category 1 according to CLP/EPA/GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline of the test item , the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.75).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 115.93) corresponding to a classification asseriously eye damaging (Category 1 according to CLP/EPA/GHS).

Relative to the negative control, the test item caused a significant increase of the corneal opacity but not of the permeability. The calculated mean IVIS was 171.91 (threshold for serious eye damage: IVIS≥ 55). According to OECD 437 the test item is classified as seriously eye damaging.