Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-11-2017 to 19-12-2017
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Puyang Ouya Chemical Technology Co., Ltd; 2017073046
- Expiration date of the lot/batch: 31st December 2018
- Purity: 99.87 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and water

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donors
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SK) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media. The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia): Lot No. 25860 kit A. The certificate of analysis is presented in Annex 1

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 minutes at room temperature and the remaining 35 minutes at 37±1°C
- Temperature of post-treatment incubation (if applicable): 42 hours at 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues were then thoroughly rinsed with PBS
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: No deviations

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT (1mg/mL)
- Spectrophotometer: Libra S22
- Wavelength: 570 nm
- Filter: No external filter was used
- Filter bandwidth: Allowed band width is 2-3 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Triplicates for test substance, positive and negative controls.

VIABILITY CALCULATION
Relative cell viability was calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test item was calculated – this value is used for the comparison with limit.

PREDICTION MODEL / DECISION CRITERIA
Evaluation of results and classification
In vitro alternatives that have been validated and accepted may also be used to help in classification decisions making (see Regulation (EC) 1272/2008, 3.2. Skin corrosion/ irritation, 3.2.2. Classification criteria for substances). For further classification, the relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %.
The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439, par. 36:

-In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS * Category 2.
-The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: Direct MTT reduction control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at 37±1°C)
Duration of post-treatment incubation (if applicable):
42 hours at 37±1°C
Number of replicates:
A single experiment, composed of three replicate tissues for each treatment group, was run.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Musk Ketone
Value:
102.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
SD:1.2%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
PBS
Value:
100
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
SD:3%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
5% SDS
Value:
2.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
SD:0.2%
Other effects / acceptance of results:
- Visible damage on test system: No changes in tissue appearance were observed during the experiment.

- Direct-MTT reduction: After approximately 1 hour incubation of the test item with MTT medium at culture conditions, the MTT medium did not change its colour (See Figure 1). The test item did not reduce MTT directly so other steps did not have been performed.

- Colour interference with MTT: The result of measuring of OD570 extract from the test item on the 96-well plate was the following:

OD570 of isopropyl alcohol (average from 2 wells): 0.052
OD570 of the test item (average from 2 wells): 0.043.
Net OD570: 0.043-0.052= -0.009

As the net OD570 for the test item was < 0.08, colour of the test item did not interfere with the endpoint, so next steps did not need to be performed.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 of the negative control tissue was 1.893 which meets the acceptance criteria of ≥ 0.8 and≤ 2.8.

- Acceptance criteria met for positive control: The mean viability of the positive control tissues expressed as % of the negative control tissues was 2.4 % which meets the acceptance criterion of ≤ 20 %.

- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.2 % for the positive control, 3.0 % for negative control and 1.2 % for the test item what is < 18 % in all cases.


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro skin irritation test (EpiDermTM Model), Musk Ketone was not irritating.
Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDerm (17-767), PBS-moistened reconstructed human epidermis tissue was exposed to 25 mg of musk ketone (99.87%) for 60±1 minutes (25 minutes at room temperature and 35 minutes at 37±1°C). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 135 minute isopropyl alcohol extraction period followed. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance musk ketone was 102.4 % ±1.2% of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 2.4 % ±0.2% of negative control average value. According to these results, the test substance is not irritating.

This in vitro skin irritation study in the human epidermal model EpiDerm is acceptable and satisfies the guideline requirement for an OECD 439 study.