Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March, 2016 - 14 March, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Amended by EC No. 640/2012 OJ No. L193, 20 July 2012
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-010
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.4 - 37.2°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow te trazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
Test for Direct MTT Reduction + colour interference:
ISOCYCLOGERANIOL was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 23 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon,
France.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately
69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 25 μL
Duration of treatment / exposure:
15-Minute exposure period and 42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: relative mean viability
Value:
12
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
Direct MTT Reduction:
ISOCYCLOGERANIOL was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that ISOCYCLOGERANIOL did not interact with the MTT endpoint.

Test Item, Positive Control Item and Negative Control Item:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Peomosa compared to the negative control tissues was 12%.

Quality Criteria:
The positive control had a mean cell relative viability compared to the negative control after 15 ± 0.5 minutes exposure of 19%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9% for the negative control, positive control and the test item, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD570 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD570

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.143

1.176

0.096

100

1.102

1.284

Positive Control Item

0.320

0.227

0.081

28

19

0.186

17

0.176

14

Test Item

0.156

0.227

0.081

14

12

0.146

13

0.133

10

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Applicant's summary and conclusion

Interpretation of results:
other: a skin irritant
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 12%. Since the mean relative tissue viability for the substance was below 50%, the substance is considered to be irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 19% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 12%. Since the mean relative tissue viability for Isocyclogeraniol was below 50% after 15 minutes treatment the substance is considered to be irritant and the substance shall be classified as a skin irritant.