Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-08-10 to 2018-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
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: 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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Yttrium
EC Number:
231-174-8
EC Name:
Yttrium
Cas Number:
7440-65-5
Molecular formula:
Y
IUPAC Name:
yttrium
Test material form:
solid: particulate/powder
Details on test material:
- Name: Yttrium
- Form: solid
- Description: grey powder (dark grey to black)
- Storage conditions: controlled room temperature (15-25°C, ≤ 70 RH%), in the dark (or protected from light)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was applied in its original form, no formulation was required (although it was grounded to fine powder).

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 (SM) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 17-EKIN-032
- Expiry date: 14 August 2017
- Date of initiation of testing: 10 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.2-27.6°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (> 95%) 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 the 15 minutes incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with 2 x ~25 mL 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). Note that the test item was stuck on the surface of the epidermis and could not be removed completely from the surface ot 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

NUMBER OF REPLICATE TISSUES: 3

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

ADDITIONAL CONTROL TISSUES
- For non-specific OD evaluation: 2 additional test item-treated living tissues. The MTT incubation step was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item.
- For MTT-interaction: 2 additional test item-treated killed epidermis and 2 negative control-treated killed epidermis (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed as for the living tissues.
- Because the test item was identified as producing both direct MTT reduction and colour interference, a third set of controls was required (to avoid a potential double correction for colour interference). Killed tissue samples were used for this third set of controls (2 replicates).
- Procedure used to prepare the killed tissues (if applicable): Living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-017, Expiry Date: 1 May 2017) were placed in a 12-well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere for 48 hours (± 1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 28 April 2017 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be non-irritant to skin if the mean relative 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 viability 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 (Phosphate Buffered Saline)
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL (Sodium Dodecyl Sulphate solution)
- 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 (± 1 h)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
101
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
- Colour interference with MTT: Yes. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.020, and Non-Specific Colour % (NSCliving%) was calculated to be 2.7%. This value was below 5%, therefore additional data calculation was not necessary.
- Direct-MTT reduction: Yes. As colour change (purple) was observed after three hours of incubation of the test item in MTT working solution, the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on the observed mean OD (0.022), the calculated NSMTT (Non-Specific MTT reduction) is 2.9%.
- As the test item was shown to be an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.042, and Non-Specific Colour % (NSCkilled%) was calculated as 5.6%. Because the NSCliving% was not used, correction with NSCkilled% 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 (0.751). Standard deviation of the viability results for negative control samples was 3.8%.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 8.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.3%.
- 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 3.1%.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
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 living control tissues

Additional control Optical Density (OD)        NSC (living)% 
     Measured  Blank corrected  
 Treated with 1  0.072  0.025  
 yttrium metal  0.062  0.015 2.7
  mean   -  0.020  

Optical Density (OD) and the calculated Non Specific MTT Reduction (NSMTT) of the additional control tissues (killed epidermis)

 Additional control  Optical Density (OD)        NSMTT  NSMTT %
     Measured  Blank corrected    
 Treated with yttrium metal  1  0.096  0.049  0.026  2.9
   2  0.088  0.041  0.018  
   mean  -  0.045  0.022  
 Treated with PBS  1  0.078  0.031    
   2  0.063  0.016    
   mean  -  0.023    

Optical Density (OD) and the calculated Non Specific Colour % (NSCkilled%) of the additional control tissues (killed epidermis)

 Additional control Optical Density (OD)        NSC (killed)% 
     Measured  Blanc corrected  
 Treated with 1  0.084  0.037  
 yttrium metal  0.093  0.046 5.6
  mean   -  0.042  

Notes (for all 3 tables above): Mean blank value was 0.047. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

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

 Substance  Optical density (OD)           Viability
     Measured  Blank corrected  TODTT   (% RV)
 Negative control:  1  0.819  0.772    102.8
 Phosphate Buffered Saline

 2

 0.765  0.718    95.7
   3  0.809  0.762    101.5
   mean  -  0.751  -  100.0
 Positive control:  1  0.111  0.064    8.5
 5% (w/v) SDS solution  2  0.122  0.075    10.0
   3  0.088  0.041  5.4
   mean  -  0.060  8.0
 Test item:  1  0.852  0.805  0.783  104.3
  yttrium metal  2 0.805  0.758  0.736  98.1
   3  0.824  0.777  0.755 100.6 
   mean  -  0.780  0.758  101.0

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. TODTT: the measured values were corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.022)

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.