Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6.1.2017 - 20.1.2017
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted: 28th July, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No. 761/2009, 23rd July 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium [5-[[4-[(3-chloro-2-hydroxy-5-sulphophenyl)azo]-3-hydroxyphenyl]azoxy]-2-[2-(4-nitro-2-sulphophenyl)vinyl]benzenesulphonato(5-)]cuprate(3-)
EC Number:
274-550-7
EC Name:
Trisodium [5-[[4-[(3-chloro-2-hydroxy-5-sulphophenyl)azo]-3-hydroxyphenyl]azoxy]-2-[2-(4-nitro-2-sulphophenyl)vinyl]benzenesulphonato(5-)]cuprate(3-)
Cas Number:
70304-38-0
Molecular formula:
UVCB
IUPAC Name:
Reaction products of diazotised 3-amino-5-chloro-4-hydroxybenzenesulphonic acid coupled with 3-methoxyaniline and 4,4'-dinitrostilbene-2,2'-disulphonic acid, chelated with copper, sodium salts
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: granular
Details on test material:
Other name:C.I. Direct Brown 103 CAS No.:70304-38-0 EC Number:274-550-7 Mw: 880.55 Batch No.: 7016/2007 Stability/Expiration date: Unlisted

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: tissue for research puposes from accredited institutions
Source strain:
other: Keratinocyte strain 00267
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUEthe reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23388, kit ETEMPERATURE USED FOR TEST SYSTEMculture conditions 37±1°C, 5±1 % CO2, moistened tissueREMOVAL OF TEST MATERIAL AND CONTROLS- Volume and number of washing steps: thoroughly rinsed with PBS- Observable damage in the tissue due to washing: After rinsing, tissues remained uneven coloured brown. The tissue with the lowest OD570 was pierced at rinsing what could have influence on lower intensity of its colouring at MTT test.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 185 min- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter was used.FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATABased on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.NUMBER OF REPLICATE TISSUES: 3NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:1. Direct MTT reduction - functional check in tubes2. Colour interference 3. MTT testPREDICTION MODEL / DECISION CRITERIAOECD Test Guideline No. 439 (1), par. 36:- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2. - The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was placed directly on tissue moistened with 25 µL of PBS. The material was spread on the tissue surface.NC: sterile PBS (phosphate buffered saline) MatTek 092316MHEPC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 111516ZSA
Duration of treatment / exposure:
60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions)
Duration of post-treatment incubation (if applicable):
After 24±2 hours incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18±2 hours (post-treatment incubation).
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
60.9
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After rinsing, tissues remained uneven coloured brown. The tissue with the lowest OD570was pierced at rinsing what could have influence on lower intensity of its colouring at MTT test.The mean OD570 of the NC tissue was 2.004 ±0.047 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.The mean viability of the PC tissues expressed as % of the negative control tissues is 2.4% which meets the acceptance criterion of ≤ 20 %. The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was 12.4 what is < 18 %.All study acceptance criteria were fulfilled.

Any other information on results incl. tables

Direct MTT reduction: functional check in tubes

25 mg ofthe test substance was addedto 1.0 mL of MTT medium. Suspension was incubated for 1 hour at culture conditions. After incubation, the medium was coloured red-brown. The test substance does not reduce MTT directly.

Colour interference

The test substance is soluble in water for injection. OD570 of solution in water for injection was > 3 what is > 0.08.

The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.005 what is < 0.08.

It means that the test substance will go well washed from the tissue and any residue is not dissolved in isopropyl alcohol. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.

MTT test:

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

C3

 

392/16

1.366

1.427

0.871

1.221

0.249

60.9 

%

68.2

71.2

43.5

60.9

12.42

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 Direct Brown 103 was 60.9 % of negative control average value, i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).According to the classification criteria, the test substance, Direct Brown 103, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test item, Direct Brown 103, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In the 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 atop to the previously moistened tissue and it was spread on the entire tissue surface. 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-decribed experimental design, average viability of tissues treated by the test substance was 60.9 %, i.e. viability was > 50 %.

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

According to the classification criteria, the test substance, Direct Brown 103, is considered to have no category in regard to skin irritation.