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:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

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:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate
EC Number:
217-699-5
EC Name:
Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate
Cas Number:
1934-21-0
Molecular formula:
C16H12N4O9S2.3Na
IUPAC Name:
trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate
Test material form:
solid: particulate/powder
Details on test material:
No data

In vitro test system

Test system:
other: reconstructed human epidermis (RhE)
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
- Principle of the assay
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential. Evaluation is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean OD value of the negative control tissues. Skin irritation potential of the test substance is predicted if the remaining cell viability is below 50%.

- Test system
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures. Certificate of quality is given in Annex1. The 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.

Test procedure
Direct MTT reduction - functional check in tubes
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional check for this possibility is performed as follows: the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.

- Colour interference
Test chemical has intensive yellow colour.To identify potential interference by coloured test chemical with OD570 absorbance measuring the spectral analysis of the test chemical in water (environment during exposure) and/or isopropanol (extracting solution) should be performed. If the test chemical in water and/or isopropanol absorbs light in the range of 570±30 nm, further colorant controls should be done. In this study the absorbance in isopropanol was detected. A colour may affect the results only if the test chemical is present in the tissues and tracted with MTT at the same time. Due to exclusion of colour influence two frozen tissue replicates, which undergo the entire testing procedure but are incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCkilled) control. The true tissue viability is then calculated as the percent tissue viability obtained with tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with tissues exposed to the interfering test chemical and incubated with medium without MTT.

MTT test
a) Application: The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 25 µL of PBS. The material is spread on the tissue surface. A single testing, composed of three replicate tissues, was run.
b) Procedure:On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations durable for approximately 1 and 18 hours, tissues are topically exposed to the test chemicals for 1 hour (25 minutes at room temperature and the remaining 35 minutes at 37°C, 5% CO2). Three tissues are used per the test substance, for the positive (PC) and negative (NC) controls. Tissues are then thoroughly rinsed with PBS, blotted to remove the test substances, and transferred to fresh medium. After 24±2 hours post-incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18±2 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg•mL-1). After 3 hour of incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/48/3 (VUOS-CETA, 2011).
c) OD570 measuring:OD570 is measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter is used.
d) Viability calculation: Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit.
Amount/concentration applied:
25 mg of the tested substance
Duration of treatment / exposure:
1 and 18 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: irritating effects
Remarks on result:
no indication of irritation
Remarks:
See attcahed table with results

Any other information on results incl. tables

The tested substance is considered to be non irritant for the skin.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the test results, the tested item is to be considered as non-irritant to human skin..
Executive summary:

The results show no irritation on human skin therefore the tested item is to be considered as non-irritant to skin under CLP classification.