Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 14th to 17th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 - 45 °C



In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Human adult donors
Justification for test system used:
The EPISKIN Small ModeITM (EPISKIN-SMTM) is one of the biological systems of the validated alternative method for skin irritation (OECD TG 439).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModeITM (EPISKIN-SMTM) supplied by EPISKIN Laboratoires, 4 Rue Alexander Fleming 69366 Lyon, France.
- Tissue batch number(s): N° 17-EKIN-007
- Delivery date: 14.02.2017
- Date of initiation of testing: 14.02.2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: washing were performed with saline solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 42 hours
- Spectrophotometer: TECAN INFINITE M-200 spectrophotometer.
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0-2 OD

NUMBER OF REPLICATE TISSUES: test item, positive and negative controls were evaluated in triplicate on viable episkin tissues.

INSTRUMENT USED FOR THE TEST:
Analytical bilance XS204 I
CO2 incubator
Microplate Autoreader INFINITE M200
Micropipettes
Refrigerator FRL 360

PREDICTION MODEL / DECISION CRITERIA
- If the mean relative tissue viability (%) of treated tissues exposed to the test substance is > 50 % the test substance is identified as not requiring classification and labeling(NO Category- Non-irritant to skin).
- If the mean relative viability is ≤ 50 % the test substance is identified as potentially requiring classification and labeling as Category 1 (risk phrase H314: causes severe skin burns and eye damage) or Category 2 (risk phrase H315: causes skin irritation).
In absence of information on skin corrosion the test method cannot resolve between UN GHS Categories 1 and 2.

ACCEPTANCE CRITERIA
- Acceptance criteria for negative control: negative control meets the acceptance if the mean OD negative control value of the three tissues is ≥ 0.6 and ≤ 1.5 and if the Standard Deviation value (SD) of the % viability is ≤ 18 %.
- Acceptance criteria for positive control: positive control satisfies acceptance criteria if mean cell viability expressed as percentage compared to Negative Control is ≤ 40 % and Standard Deviation value (SD) is ≤ 18 %.
- Acceptance criteria for test substance: test substance meets the acceptance criteria if the Standard Deviation value (SD) of the cell viability % (expressed as percentage compared to Negative Control) between the 3 tissue replicates is ≤ 18 %.
Control samples:
other: Positive (Sodium Dodecyl Sulfate at 5%) and negative (NaCl 0.9%) controls
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of test item were directly and uniformly applied topically on epidermal surface pre-treated with 10 µL of saline solution.

NEGATIVE CONTROL
- Concentration (if solution):10 µL of saline solution (NaCl 0.9%) were directly and uniformly applied topically on epidermal surface.

POSITIVE CONTROL
- Concentration (if solution):10 µL of Sodium Dodecyl Sulfate at 5% (SDS 5%) were directly and uniformly applied topically on epidermal surface.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Test item, positive and negative controls were evaluated in triplicate on viable episkin tissues.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item mean of three replicates
Value:
ca. 104.16
Vehicle controls validity:
not valid
Remarks:
no vehicle used
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: no direct MTT reduction observed for test item in the qualitative visual inspection.
- Colour interference with MTT: no colour intereference with MTT observed for test item in the qualitative visual inspection.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: negative control meets the acceptance if the mean OD negative control value of the three tissues is ≥ 0.6 and ≤ 1.5 and if the Standard Deviation value (SD) of the % viability is ≤ 18 %.The mean OD negative control value of the three tissues was 0.87 (inside the range of acceptability 0.6 - 1.5) and the Standard Deviation value (SD) of the % viability was 2.87 (≤ 18% as indicated in the accepability criteria).
- Acceptance criteria met for positive control: positive control satisfies acceptance criteria if mean cell viability expressed as percentage compared to negative control is ≤ 40 % and Standard Deviation value (SD) is ≤ 18 %.A cell viability ≤ 40% corresponding to the cut-off value for positive control acceptance criteria was quantified for SDS 5% (26.58%). Also the standard deviation for positive control was inside the range of acceptability 0.86 ≤ 18%.
- Acceptance criteria met for variability between replicate measurements: test substance meets the acceptance criteria if the Standard Deviation value (SD) of the cell viability % (expressed as percentage compared to Negative Control) between the 3 tissue replicates is ≤ 18 %. (i.e. 4.14 % ≤ 18 %).

STATISTICAL ANALYSIS
No specific statistical analysis has been carried out except the standard deviation calculation.

Any other information on results incl. tables

O.D

SAMPLE

BIOLOGICAL

REPLICATE 1

BIOLOGICAL

REPLICATE 2

BIOLOGICAL

REPLICATE 3

NC

0.8164

0.8624

0.8875

0.8759

0.8787

0.8879

SDS 5%

0.2226

0.2262

0.2266

0.2310

0.2419

0.2359

test item

0.8450

0.8818

0.9427

0.9192

0.9096

0.9270

O.D MEAN

SAMPLE

BIOLOGICAL

REPLICATE 1

BIOLOGICAL

REPLICATE 2

BIOLOGICAL

REPLICATE 3

NC

0.8394

0.8817

0.8833

SDS 5%

0.2224

0.2288

0.2389

test item

0.8634

0.9310

0.9183

 

VIABILITY

VIABILITY%

MEAN±SD

 

BIOLOGICAL

REPLICATE 1

BIOLOGICAL

REPLICATE 2

BIOLOGICAL

REPLICATE 3

 

NC

96.69

101.57

101.75

100.00± 2.87

SDS 5%

25.85

26.36

27.52

26.58±0.86

test item

99.46

107.25

105.78

104.16±4.14

Applicant's summary and conclusion

Interpretation of results:
other: not skin irritant according to the CLP Regulation (EC n.1272/2008)
Conclusions:
Non-skin irritant.
Executive summary:

The present study has been conducted  in order to assess the skin irritation of the test item on the reconstructed human epidermis model EPISKIN-SMTMaccording to OECD TG 439 (July 2015).

Skin irritation was assessed at 15 min treatment followed by product washing and a post incubation period of 42h by using the MTT test method to quantify the residual cell viability.

Based on adopted prediction model and to MTT results (104.16 %) the test item was classified as not skin irritant according to UN GHS and according to the CLP Regulation (EC n.1272/2008).