Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-23 to 2017-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Tungsten zirconium hydroxide oxide
- Physical state: solid
- Appearance: white powder
- Further information on test material confidential
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None; the test item was applied as supplied, 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 EPISKIN (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: EPISKINTM (SM) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 17-EKIN-034
- Expiry date: 28 August 2017
- Date of initiation of testing: August 23, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 24.8-27.6°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (> 95 RH%) 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

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: As the test item appeared to be an MTT-interacting substance, in addition to the normal procedure, two test item treated killed epidermis and two negative control treated killed epidermis were used in the study (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 in case of the killed tissues as in case of the living tissues.
As the test item had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of the test item 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.
To avoid a possible double correction for colour interference, a third set of control for non-specific colour in killed tissues (NSCkilled) was performed.
- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-024, Expiry Date: 19 June 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 16 June 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.
- Number of replicates: 2 for the test item and 2 for the negative control
- Method of calculation used: For test substances detected as able to stain the tissues and interfere with MTT a third set of controls is required before calculation of the “true” viability %.

Non Specific Colour % with killed tissues (NSCkilled%):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test substance treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

In that case the true MTT metabolic conversion (TODTT) will be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV + mean ODCTK]
ODTT: test substance treated viable tissues (incubated with MTT)
ODKT: test substance treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test substance treated viable tissues (not incubated with MTT)
ODCTK: test substance treated killed tissues (not incubated with MTT)

The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100

The mean value of the 3 individual relative viability % for the test substance is calculated as follows:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

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

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 of 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): 10 mg. As the test item was solid, first an appropriate amount (10 µL) of distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis.

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

POSITIVE CONTROL
- 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 per test group (test item, negative control and poitive control)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 / mean of 3 replicates
Value:
86.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
range of values: 82.6 - 91.2
Irritation / corrosion parameter:
other: optical density (TODTT)
Remarks:
corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.014)
Run / experiment:
1 / mean of 3 replicates
Value:
0.657
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of values: 0.624 - 0.690
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 0.007, Non Specific Colour % was calculated as 0.9%. This value was below 5%, therefore additional data calculation was not necessary.
- Direct-MTT reduction: Yes, as colour change (purple precipitate) 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 these observed mean OD (0.014), the calculated NSMTT% was 1.8%. This was considered to be significant, thus correction with NSMTT was made.
- As the test item was showed being 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 0.015, Non Specific Colour % (NSCkilled%) was calculated as 1.9%. 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 (0.756). Standard deviation of the viability results for negative control samples was 6.4%.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 6.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.4%.
- 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 4.3%.
- 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 Control Tissues

 Additional control    OD Measured  OD Blank corrected  NSC% (living)
 Treated with test item  1  0.053  0.006  
   2  0.055  0.008  0.9
   mean  ---  0.007  

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).

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

 Substance     OD Measured  OD Blank corrected  TODTT  Viability (% RV)
 Negative Control:  1  0.850  0.803  ---  106.2
Phosphate buffered saline  2  0.753  0.706  ---  93.4
   3  0.806  0.759  ---  100.4
   mean  ---  0.756  ---  100.0
 Positive Control:  1  0.103  0.056  ---  7.4
 5% (w/v) SDS solution  2  0.095  0.048  ---  6.4
   3  0.082  0.055  ---  4.7
   mean  ---  0.046  ---  6.1
   1  0.717  0.670  0.657  86.9
 Test Item  2  0.685  0.638  0.624  82.6
   3  0.750  0.703  0.690  91.2
   mean  ---  0.670  0.657  86.9

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 0.014)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure to the test item, the mean cell viability was 86.9% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria.
In conclusion, in the in vitro EPISKINTM (SM) model test, the results indicate that the test item is non-irritant to skin.