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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06.01.-20.01.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate
EC Number:
228-810-1
EC Name:
Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate
Cas Number:
6359-85-9
Molecular formula:
C23H21N5O6S2.Na
IUPAC Name:
sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate
impurity 1
Chemical structure
Reference substance name:
Sodium chloride
EC Number:
231-598-3
EC Name:
Sodium chloride
Cas Number:
7647-14-5
Molecular formula:
ClNa
IUPAC Name:
sodium chloride
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Acid Yellow 25- Physical state: yellow solid, powder- Analytical purity: 95 % (w/w)- Impurities (identity and concentrations): 5%(w/w) NaCl (CAS No. 7647-14-5 - Lot/batch No.: 7003/2007- Expiration date of the lot/batch: 2 years/June 2018- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: model EpiDermTM- Tissue: The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 25802 kit C)- Date of initiation of testing: 30.6.2016The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37±1°C
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied: 25 gNEGATIVE CONTROLPBS MatTek 101816ZSA, exp. 18/10/2017 - Amount(s) applied: 25 µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 031617MGKA, exp. 16/03/2018
Duration of treatment / exposure:
60 min.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
85.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Direct MTT reduction - functional check in tubes The test was not performed due to blue colour of the test substance. The next step was performed directly.

Any other information on results incl. tables

Table 1: OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

Treatment

OD570

Avg

SD

Average viability

 

1

2

3

 

 

(% NC)

NC

 

PBS

2.022

1.940

2.050

2.004

0.047

100.0 

%

100.9

96.8

102.3

100.0

2.33

C1

 

389/16

1.882

1.329

1.926

1.712

0.272

85.4 

%

93.9

66.3

96.1

85.4

13.55

PC

 

5% SDS

0.044

0.051

0.050

0.048

0.003

2.4

%

2.2

2.5

2.5

2.4

0.15

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Acid Yellow 25 was 85.4 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model. According to the classification criteria given in chapter 4.5 of this report, the test substance, Acid Yellow 25, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test substance, Acid Yellow 25, was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The test was performed according to the OECD Test GuidelineNo. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

In preliminary experiments neither colour interference with the endpoint nor direct MTT reduction were found.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissuesurface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately
42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental designaverage viability of treated tissues was85.4%,i.e. viability was >50 %.

The effect of the test substancewasnegative inEpiDermTMmodel (tissues were not damaged).

According to the classification criteria given in chapter 4.5.,the test substance,Acid Yellow 25, is considered to have no categoryin regard to skin irritation.