Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April, 2016 - 09 May, 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:
(2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

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-015 and 16-EKIN-018
- 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: 35.1 - 37.1°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 tetrazolium 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:
Geraldehyde was checked for possible direct MTT reduction and colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, 10 μL of Geraldehyde was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
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.
Since the test item reacted with the MTT medium in addition to the normal procedure, three killed tissues treated with test item and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 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 70 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.

Test system

Vehicle:
unchanged (no vehicle)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
52
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Irritation / corrosion parameter:
other: relative mean viability (Repeat experiment)
Value:
63
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
Geraldehyde was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that Geraldehyde did interact with the MTT endpoint.
The non-specific reduction of MTT by Geraldehyde was 8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

Test Item, Positive Control Item and Negative Control Item
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Geraldehyde compared to the negative control tissues was 52%. Although the mean relative tissue viability for Geraldehyde was above 50% after 15 ± 0.5 minutes treatment Geraldehyde, the relative tissue viability per skin were 58, 61 and 38%. Therefore, the test was repeated. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Geraldehyde compared to the negative control tissues during the repeat was 63%. The relative tissue viability per skin during the repeat were 63, 69 and 58%. Since the mean relative tissue viability for Geraldehyde was above 50% Geraldehyde is considered to be non-irritant.

Quality Criteria
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 25% and 24% first test and repeat, respectively. The positive control meets the validity criterion meets the validity criterion. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

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

0.907

0.934

0.176

100

0.773

1.122

Positive Control Item

0.220

0.247

0.165

24

25

0.096

12

0.424

38

Test Item

0.618

0.490

0.118

68

52

0.643

83

0.426

38

Repeat:

Item

OD570 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD570

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

0.890

0.832

0.132

100

0.925

0.680

Positive Control Item

0.190

0.199

0.010

21

24

0.210

23

0.195

29

Test Item

0.525

0.526

0.048

59

63

0.574

62

0.479

70

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:
other: Not skin irritating
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
Since the mean relative tissue viability for the substance in two experiments was above 50% the substance is considered to be non-irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in two 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 after 15 ± 0.5 minutes exposure of 25% and 24% first test and repeat, respectively.

The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 52% and 63% first test and repeat, respectively.

Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be non-irritant.