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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate Good Laboratory Practice: The Department of Health of the Government ot the United Kingdom, Statement of compliance in accordance with Directive 2004/9/EC, date of inspection 05/07/2016

Test material

Constituent 1
Reference substance name:
dibenzylbenzene, ar-methyl derivative, hydrogenated
EC Number:
943-342-4
Molecular formula:
C21H38
IUPAC Name:
dibenzylbenzene, ar-methyl derivative, hydrogenated
Test material form:
liquid
Specific details on test material used for the study:
- Lot/batch No.of test material: hydrogenius 005
- Expiration date of the lot/batch: > 3 years (unlimited shelf life)
- Storage condition of test material: room temperature in the dark
- Purity: 100%

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, purchased from SkinEthic Laboratories (Lyon, France). The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number: 16-EKIN-040
- Production date: no details mentioned
- Delivery date: 04 October 2016
- Date of initiation of testing: 05 October 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Observable damage in the tissue due to washing: nothing mentioned
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: measured without reference filter
- Linear OD range of spectrophotometer: not mentioned

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period according to:
Relative mean tissue viability is <= 50%: irritant
Relative mean tissue viability is > 50%: non-irritant
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 μL
- Concentration: undiluted, used as supplied

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Test system

Details on study design:
Test for direct MTT reduction by the test material: 10 µL test material + 2 ml 0.3 mg/ml MTT solution incubated for 3 h at 37°C in the dark. Untreated MTT solution was used as control. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint: 10 μL of test item + 90 μL of sterile water, mixing for 15 minutes on a plate shaker, visual assessment of the color was made. A test item may interfere with the MTT endpoint if it is colored.

MAIN TEST
DAY 1-Application of test item and rinsing
Exposure: 15 min
Amount applied: 10 µl
Negative control: 10 µl DPBS
Positive control: 10 µl 5% w/v SDS
Washing: DPBS with Ca++ and Mg++ for 40 seconds
Post-exposure incubation: 42 h

DAY 3-MTT Loading/Formazan Extraction
Plate shaker: 15 min
MTT solution: 2 ml of 0.3 mg/ml per well
Incubation: 3 h
Biopsy of the epidermis was performed. The tissue was then transfered in micro tubes with 500 µl acidified isopropanol and refrigerated at 1-10°C till day 6.

DAY 6-Absorbance/Optical Density at 562 nm with Anthos 2001 microplate reader

DATA EVALUATION
Relative mean viability (%): (mean OD562 test material / mean OD562 negative control) *100

ACCEPTABILITY
Positive control: the relative mean tissue viability should be ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability should be ≤18%.
Negative Control: the mean OD562 negative control should be ≥0.6, and the SD value of the percentage viability ≤18%.
Test Item: the SD calculated from individual percentage tissue viabilities of the three identically treated tissues should be ≤18%.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
106.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. Quantitative correction of results was not necessary and a parallel determination of skin irritation potential on viable and waterkilled tissues was not performed.
- Colour interference with MTT: The solution containing the test item was a very pale green color. This color was attributed to the intrinsic color of the test item itself, it was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD562 for the negative control treated tissues was 0.782 and the standard deviation value of the viability was 12.7%
- Acceptance criteria met for positive control: yes, relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.8%
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation calculated from individual tissue viabilities of test item treated tissues was 12.0%

Any other information on results incl. tables

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD562

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

± SD of

Relative

mean

viability (%)

Negative

Control Item

0.884

0.782

0.099

113.1

100*

12.7

0.775

99.1

0.686

87.8

Positive

Control Item

0.068

0.062

0.006

8.7

7.9

0.8

0.062

7.9

0.056

7.2

Test Item

0.904

0.833

0.094

115.7

106.6

12.0

0.869

111.2

0.727

93.0

 * = the mean viability of the negative control tissues is set as 100%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the tissues exposed to the test material was 106.6%, and hence, the test material is not a skin irritant in this test.
Executive summary:

The skin irritation potential of dibenzylbenzene, ar-methyl derivative, hydrogenated was examined in this in vitro test with the EPISKIN TM reconstructed human epidermis model. The test was performed according to the OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 28 July 2015) and the procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study. Exposure to the test substance lasted 15 minutes and was followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in the human epidermal cultures following topical exposure to the test item, with the MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The optical density was measured at 562 nm and MTT reduction in the test item treated tissues relative to negative control tissues was expressed as percentage viability. The relative mean viability of the test item treated tissues was 106.6% after the 15 -minute exposure period and hence, dibenzylbenzene, ar-methyl derivative, hydrogenated was classified as non-irritant. The study is considered acceptable based on the quality criteria.