Registration Dossier

Administrative data

Description of key information

Skin: The test item was assessed by means of the Human Skin Model Test according to OECD TG 439.

The test item reduced MTT (pre-test for direct MTT reduction), and it dyed water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was intensive. Consequently, additional tests with freeze-killed or viable tissues were necessary. Three tissues of the human skin model EpiSkin™ were treated with the test item, the negativecontrol.Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 86.8%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential to the skin.

Eye: In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.

The test item was investigated by means of the BCOP assay using fresh bovine corneae.

Relative to the negative control, the test item caused an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 58.55.

According to OECD 437 the test item is classified as serious eye damaging (EU CLP/UN GHS Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2018 until 28 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD 439 and GLP
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: UN GHS (2003, last rev. 2017)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 640/2012, L 193, Part B. 46. “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (06 July 2012).
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015
Test system:
human skin model
Details on test system:
EpiSkin™ Kit Lot No.: 18-EKIN-039
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Test material:
5 µL of deionised water were topically applied to the epidermal surface in order to
improve further contact between the solid and the epidermis. Each approximately 10 ± 2 mg (26 mg/cm² according to guideline) of the test item were applied to the wetted tissues.

Negative Controls:
Each 10 µL were applied to each of triplicate tissues for 15 minutes.

Positive Controls:
Each 10 µL were applied to each of triplicate tissues for 15 minutes.
Duration of treatment / exposure:
15 minutes.
Number of replicates:
3 tissues each for the test substance treatment and the controls.
Species:
other:
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissues
Value:
86.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
viable tissues
Value:
2.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Freeze killed tissues
Value:
2.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water showed an intense intrinsic colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent showed dark colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 86.8% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
The acceptance criteria were met:
• the mean OD of the three negative control exposed tissues is greater than or equal to 0.6 till ≤ 1.5 (range: 0.618 to 0.672).
• the standard deviations between tissues of the same treatment group was ≤ 18 (range: 0.1 to 5.0).
• the mean relative tissue viability of the positive control was ≤ 40% (25.9%).
• the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SDS (2.2 mg/mL).
• the results for the negative control are within the historical data (means, standard deviation, and ranges) of Envigo CRS GmbH. The viability of the positive control is slightly above the historical control data but still meets the acceptance criterion.

 

Tissue

OD

OD

Mean OD

Mean OD

Mean

Rel.

SD

Mean

Treatment Group

No.

570 nm

570 nm

of

of

OD

Viablility [%]

 

Relative

 

 

Well 1

Well 2

2 Wells

2 wells

of3tissues

Tissue

 

Viability

 

 

 

 

 

blank corrected

blank corrected

1, 2 + 3

 

[%]

Blank

 

0.038

0.038

0.038

 

 

 

 

 

 

1

0.651

0.662

0.656

0.618

 

95.6

 

 

Negative Control

2

0.706

0.714

0.71

0.672

0.647

103.9

4.2

100

 

3

0.696

0.68

0.688

0.65

 

100.5

 

 

 

1

0.185

0.186

0.185

0.147

 

22.7

 

 

Positive Control

2

0.191

0.187

0.189

0.15

0.167

23.3

5

25.9

 

3

0.248

0.239

0.243

0.205

 

31.7

 

 

 

1

0.605

0.607

0.606

0.568

 

87.8

 

 

Test Item

2

0.6

0.602

0.601

0.563

0.583

87

4.8

8.6*

 

3

0.665

0.649

0.657

0.618

 

95.6

 

 

 

1

0.043

0.043

0.043

0.005

 

0.7

 

 

Neg.Cont. Viable Tissues

2

0.046

0.046

0.046

0.008

0.006

1.2

0.3

0.9

 

3

0.043

0.043

0.043

0.005

 

0.7

 

 

 

1

0.058

0.055

0.057

0.018

 

2.8

 

 

Test Item Viabletissues

2

0.057

0.051

0.054

0.016

0.016

2.5

0.4

2.5

 

3

0.052

0.051

0.052

0.013

 

2.1

 

 

Neg.Cont. Freeze killed Tissues

1

0.047

0.046

0.046

0.008

 

1.2

 

 

 

2

0.05

0.05

0.05

0.011

0.012

1.8

0.6

1.8

 

3

0.054

0.053

0.054

0.015

 

2.4

 

 

 

1

0.058

0.055

0.056

0.018

 

2.8

 

 

Test Item Freeze killed Tissues

2

0.055

0.055

0.055

0.017

0.017

2.6

0.1

2.7

 

3

0.055

0.056

0.056

0.017

 

2.7

 

 

 

SD = Standard Deviation

* corrected value

Interpretation of results:
GHS criteria not met
Remarks:
Regulation EC 1272/2008
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Aluminium Orange G is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD TG 439.

The test item reduced MTT (pre-test for direct MTT reduction), and it dyed water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was intensive. Consequently, additional tests with freeze-killed or viable tissues were necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negativecontrol (PBS) orthe positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 86.8%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.

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
Study period:
The test was performed on 17 September 2018
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.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The positive control is 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline since the laboratory historical control data is established with this chemical.
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) No 1152/2010: B. 47. Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (Official Journal of the European Union, L 324, 9.12.2010).
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (Reach).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13 until 16 April 2015, Date of signature: 14 September 2015
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: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was tested as a 20% suspension (w/v) in saline.
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
The anterior compartment received the test item suspension, the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved to be difficult to remove by the rinsing method, the front cover of the holder was opened and the cornea was carefully washed using a gentle stream of incubation medium and eye wipers. The corneae still remained stained orange to red and the test item remained on the surface. The corneae were given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment and opacity was measured (t240).

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae and displays a numerical opacity value. 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 different 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 both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
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 (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 IVIS 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
> 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.

A single testing run composed of at least three corneae should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneae are non-concordant, such that:
• 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
• 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.


Irritation parameter:
in vitro irritation score
Run / experiment:
main experiment
Value:
58.55
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Category 1
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: fulfilled, no category (IVIS 1.25)
- Acceptance criteria met for positive control: filfilled, category 1 (IVIS 104.82)

Results after 240 Minutes Treatment Time

Test Group

Opacity #

value

Permeability ##

 

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

 

0.062

 

0.93

 

 

Negative Control

0

0.33

0.068

0.061

1.02

1.25

No Category

Negative Control

1

 

0.054

 

1.81

 

 

Positive Control

95.67*

 

0.583*

 

104.41

 

 

Positive Control

88.67*

 

0.474*

 

95.77

104.82

Category 1

Positive Control

104.67*

 

0.642*

 

114.29

 

 

Aluminium Orange G

46.67*

 

1.100*

 

63.16

 

 

Aluminium Orange G

37.67*

 

0.976*

 

52.30

58.55

Category 1

Aluminium Orange G

44.67*

 

1.035*

 

60.19

 

 

# Opacity value = Difference (t240 - t0) of Opacity

## Permeability at 490 nm (OD490)

*corrected values

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item is serious eye damaging (EU CLP/UN GHS Category 1).

Executive summary:

This in vitro study was performed to assess the corneal irritation and 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 (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. 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 (physiological saline), neither an increase of opacity nor permeability of the corneae was observed.

The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1).

 

Relative to the negative control, the test item caused an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 58.55.

According to OECD 437 the test item is classified as serious eye damaging (EU CLP/UN GHS Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Classified: H 318 causes serious eye damage

The test item was investigated by means of the BCOP assay using fresh bovine corneae.

Relative to the negative control, the test item caused an increase of the corneal opacity or permeability. The calculated meanin vitroirritancy score was 58.55.

According to OECD 437 the test item is classified as serious eye damaging (EU CLP/UN GHS Category 1).