Aluminium Bordeaux

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The test was performed on 11 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

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

Principles of method if other than guideline:

Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
EC Commission Directive 2004/10/EC

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

1

Reference substance name:

disodium 2,4-dihydroxy-5-[(2-hydroxy-3-nitro-5-sulphonato-phenyl)azo]benzoate, reaction products with chromium triacetate

Molecular formula:

not applicable

IUPAC Name:

disodium 2,4-dihydroxy-5-[(2-hydroxy-3-nitro-5-sulphonato-phenyl)azo]benzoate, reaction products with chromium triacetate

Test animals / tissue source

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three cornea were exposed to each 0.75 mL of the test item, the negative, and the positive control for 10 minutes.

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.
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 was 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.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation

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 (t130 – t0).
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 IVIS score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

33.16

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

Relative to the negative control, the test item did not cause an increase of the corneal permeability, but opacity effects were observed. The calculated mean IVIS was 33.16 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Other effects:

None

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	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0		0.068		1.02
	0	0.33	0.068	0.071	1.02.	1.39	Not categorized
	1		0.076		2.14
Positive Control	124.67*		0.050*		125.42
	114.67*		0.015*		114.90	122.96	Category 1
	127.67*		0.059*		128.56
test item	33.67*		-0.011*		33.51
	31.67*		-0.015*		31.45	33.16	No hazard prediction can be made
	34.67*		-0.010*		34.52

*corrected values

Applicant's summary and conclusion

Interpretation of results:

other: The calculated mean IVIS was 33.16 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the test item 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 = 1.39).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity of the corneae (mean IVIS = 122.96) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item did not cause an increase of the corneal permeability, but opacity effects were observed. The calculated mean IVIS was 33.16 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Conclusion:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).