Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 November 2017 to 15 December 2017
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Reconstructed Human Epidermis (MatTek)
- Tissue batch number(s): 25865
- Delivery date: 12 December 2017
- Date of initiation of testing: 13 December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for the first 35 minutes and room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside.
- Observable damage in the tissue due to washing: None observed
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean 4.37%, range 2.20 to 6.78% for positive control
- Barrier function: 5.13 h (acceptance criteria: ET-50 is 4.77 to 8.72 hours)
- Contamination: No contamination with HIV-1 virus, hepatitis B virus, hepatitis C virus, bacteria, yeast or other fungi
- Reproducibility: Relative standard deviation 21.60% for positive control, 9.40% for negative control

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: The test item passed the MTT- and the colour interference pre-tests.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One experiment performed.

PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: An irritation potential of a test item leading to H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, 7th revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 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): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes (incubated for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2 and moved to a sterile bench at room temperature until the end of treatment)
Duration of post-treatment incubation (if applicable):
42 hours (24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2 after first rinse and another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2 after second rinse)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
99.3
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:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was proven with 10 reference substances, testing performed in March 2014.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, mean OD was 1.830
- Acceptance criteria met for positive control: Yes, mean relative tissue viability was 4.3%
- Acceptance criteria met for variability between replicate measurements: Yes, the highest relative standard deviation was 15.3%
- Range of historical values if different from the ones specified in the test guideline: Negative control mean OD was 1.830 (historical 1.34 - 2.00) and positive control mean OD was 0.079 (historical 0.03 - 0.11)

Any other information on results incl. tables

Table 1. Skin irritation results

 Treatment group  Replicate  OD(570nm) Well 1  OD(570nm) Well 2  OD(570nm) Well 3  Mean OD of 3 Wells  Mean OD of 3 Wells, blank corrected  Mean OD of 3 tissues, blank corrected  Relative viability (%)  Relative standard deviation (%)  Mean relative viability (%)
 Blank  -  0.035  0.035  0.036  0.035  -  -  -  -  -
 Negative control  1  1.989  1.966  1.951  1.969  1.933  1.830  105.6  5.4  100.0
   2  1.863  1.845  1.855  1.854  1.819    99.4    
   3  1.782  1.765  1.772  1.773  1.738    95.0    
 Positive control  1  0.109  0.112  0.113  0.111  0.076  0.079  4.2  9.5  4.3
   2  0.103  0.104  0.116  0.108  0.073    4.0    
   3  0.121  0.118  0.127  0.122  0.087    4.8    
 Test item  1  1.538  1.529  1.538  1.535  1.500  1.818  82.0  15.3  99.3
   2  1.974  1.977  1.964  1.971  1.936    105.8    
   3  2.051  2.044  2.060  2.052  2.016    110.2    

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
After treatment with the test item the mean relative absorbance value decreased irrelevantly to 99.3% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
Executive summary:

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test using the EpiDerm Reconstructed Human Epidermis model. The test item passed the MTT- and the Colour Interference pre-tests. The test item, the negative control (DPBS), and the positive control (5% SLS) were applied to triplicate tissue each. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 2 hours and 45 minutes extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 99.3% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. The acceptability criteria for the negative control, positive control, variablity and historical results were met. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD Guideline 439 (2015) and EU Method B.46 (2008).