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EC number: 276-059-3 | CAS number: 71838-68-1 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53090.
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Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 October 2017 - 05 March 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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 (incl. QA statement)
Test material
- Reference substance name:
- Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives
- EC Number:
- 276-059-3
- EC Name:
- Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives
- Cas Number:
- 71838-68-1
- Molecular formula:
- not applicable
- IUPAC Name:
- Reaction product of formamide, N,N'-1,4-phenylenebis- with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives
- Test material form:
- solid: particulate/powder
- Details on test material:
- Test Item: Leuco Sulfur Brown 26
Appearance: Dark, near black powder
Constituent 1
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- - Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: at least 9 month old donor cattle
Test system
- Vehicle:
- physiological saline
- Controls:
- yes, concurrent vehicle
- yes, concurrent positive control
- Amount / concentration applied:
- The 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 as 7.62. - 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
Resultsopen allclose all
- Irritation parameter:
- in vitro irritation score
- Remarks:
- Replicate 1
- Run / experiment:
- 1
- Value:
- 1.44
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Remarks:
- Replicate 2
- Run / experiment:
- 1
- Value:
- 0.41
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Remarks:
- Replicate 3
- Run / experiment:
- 1
- Value:
- 1.43
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
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 |
Proposed in vitro Irritancy Score |
Standard deviation of in vitro score |
||
|
|
Mean |
|
Mean |
|
|
|
|
Negative Control |
1 |
0.33 |
0.068 |
0.073 |
2.02 |
1.43 |
No Category |
0.51 |
0 |
0.078 |
1.17 |
||||||
0 |
0.073 |
1.10 |
||||||
Positive Control |
123.67* |
0.035* |
124.19 |
124.00 |
Category 1 |
5.86 |
||
129.67* |
0.006* |
129.76 |
||||||
117.67* |
0.025* |
118.04 |
||||||
Test item |
1.67* |
-0.015* |
1.44 |
1.09 |
No Category |
0.59 |
||
0.67* |
-0.017* |
0.41 |
||||||
1.67* |
-0.016* |
1.43 |
*corrected values
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- According to the current study and under the experimental conditions reported, the test item is not categorised as an eye irritant (GHS).
- Executive summary:
This in vitro study according to OECD guideline 437 was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspension in saline of the test item, the positive, and the negative controls were applied to the different corneas 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 corneas and opacity was measured again (t240). After the opacity measurements, permeability of the corneas 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 corneas could be observed (mean IVIS = 1.43). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 124.00) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The IVIS of the positive control fell within two standard deviations of the HCD. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control. Therefore the acceptability criteria were fulfilled. The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 1.09 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorised as an eye irritant (GHS).
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