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Toxicological information

Skin irritation / corrosion

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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:
September 26 to October 03, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm, Model EPI-200-SIT
Version / remarks:
Rev. 26/3/2012, 1-37
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Acid Green 001
IUPAC Name:
Acid Green 001
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal human keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia)
- Tissue batch number(s): 25844 kit A

PREPARATION OF TISSUES
On the day of receipt, EpiDermTM tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubations before treatment was 65 minutes and 19 hours, 4 minutes.

DIRECT MTT REDUCTION
The test was performed as a part of Study No. 447/16/4AC: Acid Green 001 - In vitro Eye Irritation Test (EpiOcularTM Model); VUOS CETA Report No.: 17-473. The test substance does not reduce MTT directly.

COLOUR INTEFERENCE CONTOLS
The test substance was applied on two viable 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 (NSCliving) control. Such treated colorant controls showed minimal OD570, so no correction of result was performed.

TEST ITEM APPLICATION
After pre-incubations, tissues were topically exposed to the test chemicals for 60 ± 1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions).
- Replicates: three tissues were used per the test substance, three for the the positive (PC) and three for the negative (NC) controls.
- Removal of test material and controls: tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.

POST INCUBATION
After 25 hours and 10 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours and 13 minutes.

CELL VIABILITY MEASUREMENTS
Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 176 minutes of MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for 120 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.
- Viability calculation: relative cell viability was 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 was calculated – this value is used for the comparison with limit.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test substance is considered to be non-irritant to skin in accordance 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:
TEST MATERIAL
25 mg of the test substance was dosed directly on the previously moistened tissue (25 μl of PBS per tissue). The substance was spread on the entire tissue surface.
A single experiment, composed of three replicate tissues, was run.
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
90.3
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OHER EFFECTS:
- Visible damage on test system: no colour was eluted to medium during post-incubations and the other steps including extraction. After washing of the test substance off tissues, tissues remained coloured green. Tissues remained coloured green even after extraction with isopropyl alcohol
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD570 of the NC tissue was 1.926 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: the mean viability of the PC tissues expressed as % of the negative control tissues was 2.2 % which meets the acceptance criterion of ≤ 20 %.
- Acceptance criteria met for variability between replicate measurements: the SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.1 % for the positive control, 5.1 % for negative control and 4.9 % for the test substance which is ≤ 18 % in all cases
- Range of historical values if different from the ones specified in the test guideline:

Any other information on results incl. tables

OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities are presented in the table below.

Treatment  OD570 Avg  SD Average
viability (%NC)
1 2 3
NC PBS 2.059 1.822 1.896 1.926 0.0 100.0
% 106.9 94.6 98.5 100.000 5.1
C3 447/16 1.619 1.752 1.848 1.740 0.0 90.3
% 84.1 91.0 96.0 90.341 4.8
C3 CC 447/16 0.010 0.007 0.009 0.0 0.4
% 0.5 0.4 0.467 0.2
PC 5 % SDS 0.039 0.043 0.044 0.042 0.0 2.2
% 2.025 2.233 2.285 2.181 0.1

Applicant's summary and conclusion

Interpretation of results:
other: not classified for skin irritation according to the CLP Regulation (EC) No.1272/2008
Conclusions:
The substance is not a skin irritant.
Executive summary:

The test substance was assayed for its skin irritation potential, in vitro, in the human epidermal model EpiDerm according to the OECD Test Guideline No.439, EU B.46 and Protocol for: In Vitro EpiDermTMSkin Irritation Test for use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT. After pre-incubation of tissues, the test substance was placed directly on tissue for 60 minutes. Three tissues were used for the test substance and for positive and negative controls. Two tissues more were used as colorant control to correction of possible colour interference, which underwent the entire testing procedure except of incubation with MTT medium. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction with isopropyl alcohol followed. 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 design, average viability of treated tissues was 90.3 %,i.e. viability was > 50 %.

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

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