Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-05-02 to 2016-08-02
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
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:
other: wet powder
Details on test material:
- Name of test material (as cited in study report): digadolinium oxalate
- CAS nr: 867-64-1
- Physical state: solid
- Appearance: wet white powder
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.

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 EPISKINTM(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): 16-EKIN-018
- Expiry date: May 09, 2016
- Date of initiation of testing: May 04, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25.1-27.2°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 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

NUMBER OF REPLICATE TISSUES: 3

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

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): approximately 20.5 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:
79.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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: no

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 (0.851). Standard deviation of the viability results for negative control samples was 6.6.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 4.8% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.7.
- 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 3.2.
- 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 % of the additional control tissue

 Additional control Optical Density (OD)        NSC% 
     Measured  Blanc corrected  
 Treated with 1  0.052  0.005  
 gadolinium oxalate  0.055  0.008  0.7
  mean   -  0.006  

Notes: 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 (% RV) 
 Negative control:  1  0.952  0.904  106.2
 Phosphate Buffered Saline

 2

 0.905  0.857  100.7
   3  0.839  0.792  93.0
   mean  -  0.851  100.0
 Positive control:  1  0.085  0.038  4.4
 5% (w/v) SDS solution  2  0.085  0.038  4.4
   3  0.095  0.048  5.6
   mean  -  0.041  4.8
 Test item:  1  0.733  0.685  80.5
 gadolinium oxalate  2  0.749  0.702  82.5
   3  0.696  0.648  76.2
   mean  -  0.679  79.7

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.