1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The test was performed on 21 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

EC Number:

309-269-1

EC Name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Cas Number:

100208-66-0

Molecular formula:

not applicable

IUPAC Name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Test material form:

solid: particulate/powder

Details on test material:

Test item: Brown 3
Appearance: black powder
CAS No. 100208-66-0
EC No. 309-269-1
Storage: room temperature

Specific details on test material used for the study:

The test item was supplied by or on behalf of the Sponsor including the following information:
Identification: Brown 3
CAS Number: 100208-66-0
Batch: 106135842
Appearance: Black solid (powder), becomes brown in solution
Expiry Date: 16 June 2020
Storage Conditions: At room temperature, under light protection
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

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

Vehicle:

physiological saline

Controls:

other: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline); negative control: Saline

Amount / concentration applied:

EThe test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes. Prior to treatment of the corneae the pH-value of the test item suspension was determined (6.65).

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 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, after rinsing and further incubation of the corneae for two hours, 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 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 (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)


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

Run / experiment:

mean IVIS of three runs

Value:

1.19

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Results after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score	Standard Deviation ofin vitroIrritancy Score
		Mean		Mean
Negative Control	1	0.33	0.075	0.065	2.13	1.31	Not categorized	0.71
	0		0.069		1.04
	0		0.052		0.78
Positive Control	115.67*		0.005*		115.74	111.77	Category 1	3.9
	107.67*		0.018*		107.93
	111.67*		-0.001*		111.65
Brown 3	0.67*		-0.008*		0.54	1.19	Not categorized	0.57
	1.67*		-0.007*		1.56
	1.67*		-0.012*		1.48

*corrected values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not categorized as an eye irritant (GHS).

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 the different corneae and incubated for 240 minutes at 32± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae and opacity 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. For the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS =1.31).

The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =111.77) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The acceptance criteria were met. The test item was tested as suspension. Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.19 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.