Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-09 to 2016-09-29
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Batch No.: 86888
- Colour: grey
- Density: 1300-1900 kg/m³
- Purity: > 99.4 %
- Expiry Date: 1 year after the date of delivery
- Storage Conditions: room temperature, dry conditions, container closed tightly

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 16-EKIN-035

SkinEthic Kit:
- EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm²); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 16-EKIN-035):
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 16-MAIN3-056 / 16-MAIN3-060)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 16-ESSC-038)

- Validity controls as provided by the supplier (SkinEthic):
Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 22.5 ± 0.4, CV = 2.0%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n= 14):
Specification ≥ 1.5 mg/mL
Result: 1.8 mg/mL

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C, 5.0 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µL aqua dest.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat No. 14040-91, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium dodecyl sulfat (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3,
Lot No.: 40015277)
- Concentration (if solution): 5% in aqua dest
Duration of treatment / exposure:
15 min ± 0.5 miin
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3 tissues per dose group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
93
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
For detailed results see "Any other information on results" Table 1

Any other information on results incl. tables

Results of the  Pre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSClivingequalled 0%.

Results of the main experiment

Table 1: Results of the test item

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

0.742

0.816

0.676

0.069

0.086

0.086

0.620

0.724

0.736

0.767

0.855

0.689

0.074

0.086

0.081

0.623

0.758

0.784

OD570(Blank-Corrected)

0.699

0.773

0.634

0.027

0.044

0.044

0.578

0.682

0.694

0.724

0.813

0.647

0.031

0.044

0.039

0.581

0.716

0.742

Mean OD570Of The Duplicates (Blank-Corrected)

0.712

0.793

0.641

0.029

0.044

0.041

0.580

0.699

0.718

 Total Mean OD570Of 3 Replicate Tissues (Blank-Corrected)

0.715*

0.038

0.665

SD OD570

0.076

0.008

0.075

Relative Tissue Viabilities [%]

99.5

110.9

89.6

4.1

6.1

5.8

81.0

97.7

100.4

Mean Relative Tissue Viability [%]

100.0

5.3**

93.0

SD Tissue Viability [%]***

10.6

1.1

10.5

CV [% Viability]

10.6

20.6

11.3

* Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is 40%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is  18%.

Table 2: Test Acceptance Criteria

 

Value

Cut off

pass/fail

Mean OD570 nmBlank

0.042

< 0.1

pass

Mean Absolute OD570 nmNK

0.757

0.6 ≤ NK ≤1.5

pass

Mean Relative Viability [%] PC

5.3

≤ 40%

pass

Max. SD of % Viability [%]

10.6

≤ 18%

pass

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

In the present study the skin irritant potential of Chromium diboride (> 99.4 % purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects (93.0 % mean relative tissue viability). As the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%, the test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.