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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 March, 2015 - 03 April, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 439 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
(2013)
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
Test material form:
other: liquid
Details on test material:
- Name of test material in report: IFF 09-0035
- Physical state: Clear liquid
- Storage condition of test material: At room temperature protected from light

Test animals

Species:
other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model
Strain:
other: Not applicable
Details on test animals and environmental conditions:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-013

Test system

Type of coverage:
other: Twenty five μl of the undiluted test substance was added into 12-well plates on top of the skin tissues.
Preparation of test site:
other: The test item was applied topically to the corresponding tissues ensuring uniform covering.
Vehicle:
unchanged (no vehicle)
Controls:
other: Tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control), respectively.
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 animals:
A total of 9 tissues were used: Triplicate tissues were treated with test substance, positive control or negative control.
Details on study design:
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:
IFF 09-0035 was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 507446):
IFF 09-0035 was checked for possible colour interference before the study was started. Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μL of IFF 09-0035 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark.
IFF 09-0035 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 50 μL of IFF 09-0035 was added to 1 mg/mL MTT solution in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently.
In case the test substance reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues must be 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 25 hours at 37°C. Maintenance medium and Assay medium were supplied by SkinEthic Laboratories, Nice, France.
Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
The undiluted test substance 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. In addition three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. 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-well plate prefilled with 2 mL MTT-medium (0.3 mg/mL). 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 68.5 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 substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
7
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure. Reversibility: other: Not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean tissue viability obtained after 15 minutes treatment with FRET 09-0035 compared to the negative control tissues was 7%. Since the mean relative tissue viability for FRET 09-0035 was below 50% it is considered to be irritant.

Any other information on results incl. tables

Direct MTT Reduction

IFF 09-0035 was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model and (project 508001). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that IFF 09-0035 did not interfere with the MTT endpoint.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 7% after a 15-Minute exposure period and 42 hours post-exposure incubation period.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 7% relative to the negative control treated tissues.The positive control acceptance criterion was therefore satisfied.

The mean OD570for the negative control treated tissues was 1.144 and the standard deviationwas 0.047. The negative control acceptance criterion was therefore satisfied. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

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

Item

OD570 of

tissues

Mean OD570

of triplicate

tissues

± SD of

OD570

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.173

1.144

0.047

100

1.090

1.169

Positive Control Item

0.080

0.077

0.003

7

7

0.074

7

0.076

7

Test Item

0.088

0.081

0.013

8

7

0.089

8

0.065

6

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 7%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be irritant.
Executive summary:

The possible skin irritation potential of IFF 09 -0035 was tested through topical application for 15 minutes. The study procedures described in this report were based on the 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. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 7%.

Since the mean relative tissue viability for IFF 09 -0035 was below 50% after 15 minutes treatment the substance is considered to be irritant. The positive control had a mean cell viability of 7% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.