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:
19.04-29.04.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Colour interference test was performed with frozen instead live tissues. As the test substance acts the same way as in MTT test (both post-incubatition media were coloured, the test substance remained in tissues and on walls of inserts after extraction)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate
EC Number:
274-418-9
EC Name:
Trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate
Cas Number:
70210-21-8
Molecular formula:
C26H20ClN7O10S3.3Na
IUPAC Name:
trisodium 7-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]methylamino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2-sulphonate
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): Reactive Orange 5- Physical state: solid, powder- Analytical purity: 90% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Composition of test material, percentage of components:- Lot/batch No.: 8001- Expiration date of the lot/batch: unlisted- 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. 23329)- Date of initiation of testing: 19.04.2016TEMPERATURE 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 (phosphate buffered saline) prepared 25/02/2016, exp. 25/08/2016 and prepared 23/03/2016 exp. 23/09/2016- Amount(s) applied: 25 µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 012616TMB, exp. 26/01/2017
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:
85.6
Negative controls validity:
valid
Positive controls validity:
valid

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 Reactive Orange 5 was 85.6 % of negative control average value, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model (tissues were not damaged).
Executive summary:

The test item, Reactive Orange 5, was assayed for thein vitro skin irritation in human epidermal model EpiDermTM.The test was performed according to the OECD Test Guideline No.439: In VitroSkin 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 (2014).

After pre-incubation of tissues, 25 mg of the test substancewasplaced directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposit ion was 60 minutes. Three tissues were used for the test substance and every control.

After removal of the test substance, tissues were post-incubated for approximately
42 hours due to leave of damage reparation. 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.

The test substance was found to be not direct reducing, so none correction of results for this property must have been done. As the test substace is coloured (orange), steps for determination of appropriate contribution of absorbace to viability of tissues were done. It was found, that no measurable colour passed to final extract.

Average viability of treated tissues was 85.6 %, i.e.viability was >50 %.

The effect of the test substance was negative in EpiDermTMmodel (tissues were not damaged).

According to the classification criteria given in chapter 3.8., the test substance is considered to have no category in regard to skin irritation.