Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-07-06 to 2016-09-09
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)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin(TM) SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin(TM) test method 15 min - 42 h for the prediction of acute skin irritation of chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name: diytterbium trioxide
- Appearance: white solid
- Further information on test material confidential.
Specific details on test material used for the study:
- Treatment of test material prior to testing: None, the test item was applied as supplied.
- No correction for purity of the test item was applied.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (adult)
Source strain:
other: not applicable
Justification for test system used:
The EPISKIN TM(SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439). Therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM(SM) (0.38 cm2), SkinEthic, France
- Tissue batch number(s): 16-EKIN-027
- Expiry date: 2016-07-11
- Date of initiation of testing: 2016-07-06

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.4-27.4°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (80-100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After 15 min incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: yes, plate reader, not further specified
- Wavelength: 570 nm
- Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test items had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the relative mean viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 50% of the mean variability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

NEGATIVE CONTROL (PBS)
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL (SDS)
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
90.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was 0.021, consequently, Non Specific Colour % was calculated to be 3.0%. This value was below 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was in the recommended range (0.699). Standard deviation of the viability results for negative control samples was 4.1.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 6.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.9.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 2.2.
- The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissue

 Additional control

Optical Density (OD)

 NSC%

     Measured  Blank corrected

       

3.0

 Treated with  1  0.071  0.025
 diytterbium trioxide  2  0.064  0.018
   Mean  ----  0.021

Optical Density (OD) and the calculated relative viability % of the samples

 Substance

Optical Density (OD)       

 Viability

     Measured Blank corrected 

 (%RV)

 Negative Control:  0.718  0.672  96.1
 Phosphate buffered saline  2  0.775  0.729  104.2
   3  0.743  0.697  99.7
   Mean  ---- 0.699   100.0
 Positive Control:  1  0.087  0.041  5.8
 5% (w/v) SDS solution  0.089  0.043  6.1
   0.099  0.053  7.5
   Mean  ----  0.045  6.5
 Test Item:  1  0.673  0.627  89.7
 diytterbium trioxide  2  0.697  0.651  93.1
   3  0.669  0.623 89.1 
   Mean  ----  0.633  90.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test item was determined to be non-irritant to skin. Based on these results, the test item is considered not classified according to the CLP Regulation.