Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010-03-03 to 2010-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Version / remarks:
2007
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 11 December 2009, Vers. 4.
Version / remarks:
2009-12-11
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: solid, odourless, silver-grey powder

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from moisture

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: adult donor
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
not specified
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin TM skin model (purchased from Skinethic Laboratories)
- Tissue lot number: 10-EKIN-007
- Expiration date: 2010-03-08
- Shipping date: 2010-03-01
- Delivery date: 2010-03-02
- Date of initiation of testing: 2010-03-03

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment interval the inserts were removed from the 12-well plate.
- tissues were rinsed with PBS to remove any residual test material.
- excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µg/mL
- Incubation time with MTT: 3 hours
- Extraction of formazan: After the incubation period, the MTT solution was aspirated from the wells and the wells were rinsed with PBS. Tissue samples were cut out of the inserts and transferred into vials. Tissue samples were immersed into extractant solution by pipetting isopropanol into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approx. 70 hours in the refrigerator.
Per each tissue sample 2 × 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate and the optical density was determined with a spectrophotometer. Mean values were calculated from the 2 wells per tissue sample.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm without reference filter

TEST FOR DIRECT MTT REDUCTION
To test for the ability of the test item to directly reduce MTT approximately 15 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: mean optical density of the negative control was 0.857 (historical data range: 0.7 - 1.6)
- Barrier function: IC50 = 1.9 mg/mL (specification ≥ 1.5 mg/mL)
- Morphology: histology scoring = 23.0 (specification ≥ 19.5); well.differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: absence of bacteria, fungus and mycoplasma as well as absence of HIV1 and 2 antibodies, hepatitis C antibodies, and hepatitis B antigen
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (OD test item/ OD negative control) x 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model: for the current test, an irritation potential of a test item according to CLP classification category 2 is predicted if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 15 mg of the test item were applied to each of triplicate tissues.

VEHICLE
- Amount(s) applied (volume or weight with unit): 15 µL of deionised water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL
Duration of treatment / exposure:
15 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 1 hours
Number of replicates:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
90.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour, i.e. the tet item was not persumed to reduce the MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control the absorbance values (0.878, 0.819, and 0.873, respectively) were well above the required acceptability criterion of mean optical density (0.6 ≤ OD ≤ 1.5) for the 15 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 27.4% (acceptability criterion: positive control is ≤ 40 %).
- Acceptance criteria met for variability between replicate measurements: standard deviations between the % variabilities of the test item, the positive and negative controls were below 6% (threshold of the "OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation": 18%).

Any other information on results incl. tables

Results after treatment with vanadium (metal)

 

Dose group

Treatment Interval

Absorbance 570 nm
Tissue 1

Absor-bance 570 nm
Tissue 2

Absorbance 570 nm
Tissue 3

Mean Absorbance of 3 Tissues

Absorbance [%] Tissue 1, 2 + 3

Rel. Standard Deviation

Rel. Absorbance

[% of Negative Control]

Negative Control

15 min

0.878

0.819

0.873

0.857

102.5
95.6
101.9

3.8

100.0

Positive Control

15 min

0.293

0.209

0.203

0.235

34.2
24.4
23.7

5.9

27.4

Vanadium (metal)

15 min

0.752

0.786

0.797

0.778

91.7
93.0
83.6

2.7

90.8

Historical data:

Positive Control

Negative Control

Number of Studies

73

Number of Studies

73

Period

July 2007 - March 2010

Period

July 2007 - March 2010

Mean Viability

16.5 %

Mean OD

1.081

Standard Deviation

11.0%

Standard Deviation

0.262

Range of Viabilities

3% - 36%

Range of ODs

0.7 – 1.6*

* The upper OD value is outside of the range of 0.6 - 1.5 recommended by the OECD guideline. Nevertheless since the OD value is only slightly above the required range, the historical data can still be considered as valid.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Conclusions:
The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.