Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2017 - 04 October 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:
Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
EC Number:
239-147-2
EC Name:
Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
Cas Number:
15096-41-0
Molecular formula:
C10H23Cl4Cr2O5
IUPAC Name:
tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The SkinEthic™ RHE-model RHE/S/17, Episkin/SkinEthic Laboratories, Lyon, France.
- Tissue batch number: 17-RHE-086

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper. The inserts were placed in 6-well plates with 2 mL fresh pre-warmed (room temperature) growth medium.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Spectrophotometer (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: not specified
- N. of replicates : 3
- Method of calculation used: see "Any other information on materials and methods"

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the mean tissue viability is > 50 % after incubation period.
- The test substance is considered to be irritating to skin if the mean tissue viability is ≤ 50 % after incubation period.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 µL ± 0.5 µL per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
76.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

 Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

 

Acceptance Criterion

Result

Negative control OD

≥ 0.8 and 3.0

1.930 to 2.127

 

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.2

2.019

Mean viability positive control

< 40 %

1.0 %

SD of group-mean value

18 %

10.0 % (positive control) 4.9 % (negative control)

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.463

2.019

Mean viability positive control

2.98 %

1.0 %

 

Test Item Data Acceptance Criteria:

 

Acceptance Criterion

Result

SD of group-mean value

18 %

7.3 %

 

The study met all acceptance criteria.

The means of the negative controls and the positive control of all performed experiments in the testing laboratory are given in the following table:

Positive Control

Negative Control

Mean Viability [%]

1.45

Mean Absorption [OD570]

2.015

Standard Deviation

0.51

Standard Deviation

0.276

The results obtained after treatment of the reconstructed human epidermis model with the test substance are given in the following table:

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

OD

Viability (%)

OD

Viability (%)

OD

Viability (%)

OD

Viability (%)

Viability (%)

Negative Control

2.127

105.3

1.930

95.6

2.001

99.1

2.019

100.0

4.9

Positive Control

0.021

1.0

0.019

0.9

0.022

1.1

0.021

1.0

10.0

Test Substance

1.510*

71.0

1.587*

82.2

1.540*

77.0

1.546

76.7

7.3

* corrected optical density after true metabolic conversion

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, a cell viaability of 76.7 % was determined. Thus the test substance is not considered as skin irritant.
Executive summary:

In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, the skin irritating potential of the test item was investigated. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either test item, the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. The test item has the ability to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item and three killed untreated tissues were used as negative control. The treatment and MTT assay of the killed tissues was similar to the handling of the living tissues. The obtained OD for a non-specific reduction was subtracted from OD-values obtained after treatment of living tissues with the test item to calculate the cell viability. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test substance, the tissue viability was 76.7 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category). Under the conditions of the present study, the test substance is not considered to possess an irritant potential to skin (UN GHS: No Category).