Registration Dossier

Administrative data

Description of key information

pH: extreme, acid reserve: low, in vitro skin corrosion: negative, in vitro skin irritation: method not suitable, LLNA: no signs of skin irritation, BCOP: positive.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes
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)


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.

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

 

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.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For assessment of Skin Irritation / Corrosion and Eye Irritation data coming from:

- pH Determination (Company Guideline),

-  Acid Reserve Determination (Young),

- In Vitro Membrane Barrier Test Method for Skin Corrosion (OECD Guideline 435),

- In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (OECD TG 439),

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

- Skin Sensitisation: Local Lymph Node Assay (LLNA, OECD Guideline 429)

were used. 

            

                                              

Contributions of these studies for the assessment of skin and eye effects are as follows:

Endpoint

Effect / Value

Suggestion Derived

pH

1,6 (10%, 20°C)
1,1 (10%, 40°C)

Extreme pH raises suspicion of corrosive properties which however were not approved by the acid reserve. Thus data do not suggest that Duasyn Blue T 48 is corrosive to skin.

Acid Reserve
(g NaOH/100g)

1,37

In Vitro Skin Corrosion

No color change of CDS after >60 min

Does not suggest that Duasyn Blue T 48 is corrosive to skin

In Vitro Skin Irritation

Method failed to produce valid results, thus the test method is not suitable for determination of the irritation potential of
Duasyn Blue T 48.

No suggestion derivable

LLNA

No signs of local skin irritation up to test item concentrations of 20% (w/w) (i.e. the highest dose tested)

Does not suggest that Duasyn Blue T 48 is corrosive or irritant to skin

BCOP

IVIS: 172

Seriously eye damaging (Cat 1, CLP)

 

Taking all these findings together it is concluded:

- that Duasyn Blue T 48 has to be classified as “Seriously eye damaging (Cat 1, CLP)”, and

- that classification as “Causes skin irritation (Cat 2)” seems to be the most appropriate classification for potential local skin effects,

- that there is no need for further clarification by means of additional animal testing.


Justification for selection of skin irritation / corrosion endpoint:
Justification for classification is based on several studies thus no singele key study was selected for endpoint conclusion.

Justification for selection of eye irritation endpoint:
Sole study available

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Eye irritation: In vitro data from a BCOP Test correspond with the criteria for classification as “Seriously eye damaging (Cat 1)”.

Skin irritation/corrosion: Conclusive data on skin irritation/corrosion are not available. However data coming from several studies provide no evidence for corrosive properties. Since due to the extremely low pH and the serious effects seen in a BCOP test irritant properties to skin cannot entirely be excluded classification as “Causes skin irritation (Cat 2)" seems to be warrantable.