Registration Dossier

Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UN GHS (2003, last rev. 2017)
Qualifier:
according to guideline
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. QA statement)
Remarks:
Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)
EC Number:
233-357-8
EC Name:
Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)
Cas Number:
10127-27-2
Molecular formula:
C16H11CrN5O8S.Na
IUPAC Name:
sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

In vitro test system

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.

Test animals

Species:
other:

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used

Results and discussion

In vitro

Resultsopen allclose all
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.

Any other information on results incl. tables

 

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

Applicant's summary and conclusion

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.

Categories Display