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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:
From August 13 to 18, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 439 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): (E)-1-(2,6,6-TRIMETHYL-1,3-CYCLOHEXADIEN-1-YL)-2-BUTEN-1-ONE
- Physical state: Pale yellow liquid
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Details on test animals and environmental conditions:
EPISKIN™ Reconstructed Human Epidermis Model Kit.
Supplier: SkinEthic Laboratories, Lyon, France.
EpiSkinTM Tissues (0.38cm2) lot number: 14-EKIN-030.
Maintenance Medium lot number: 14-MAIN3-033.
Assay Medium lot number: 14-ESSC-030.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
other: not applicable
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of the test item was applied to the epidermis surface
- Concentration: The test item was used as supplied.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes at room temperature in 12 well plate containing 2 mL of maintenance medium.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 hours
Observation period:
- On Day 3, at the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 hours at 37 °C, 5 % CO2 in air.
- On Day 6, at the end of the formazan extraction period: The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Number of animals:
Triplicate tissues for test item, negative and positive controls
Details on study design:
TEST SITE
- Test item was applied to the epidermis surface

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, each tissue was removed from the well and rinsed using DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

Negative and positive controls (reference substances).
- Negative control: 10 µL of DPBS
- Positive control: 10 µL of 5 % w/v aqueous solution of Sodium Dodecyl Sulfate (SDS)

Results and discussion

In vivo

Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 14.8% after a 15 minute exposure period and 42 hours post exposure incubation period.
Other effects:
None

Any other information on results incl. tables

Direct MTT Reduction:The MTT solution containing the test item remained yellow which indicated that the test item did not directly reduce MTT.

 

Main test:

Table 7.3.1/1: 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

0.976

0.923

0.071

105.7

100*

7.7

0.951

103.0

0.842

91.2

Positive Control Item

0.252

0.289

0.071

27.3

31.3

7.7

0.244

26.4

0.371

40.2

Test Item

0.135

0.137

0.005

14.6

14.8

0.6

0.143

15.5

0.133

14.4

SD=        Standard deviation

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

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 3.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.7%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562 for the negative control treated tissues was 0.923 and the standard deviation value of the percentage viability was 7.7%. The negative control acceptance criterion was therefore satisfied.

 

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 0.6%. The test item acceptance criterion was therefore satisfied.

 

Historical Control Data

 

For the previous 22 experiments conducted from April 2014 to August 2014 using this test method, the mean OD of the positive control was 0.084 ± 0.046 and the mean percentage viability was 9.2 ± 5.3. In this same period the mean OD of the negative control was 0.938 ± 0.102.

Although the positive control parameters for this study were greater than ranges based on the previous 22 experiments the positive control was considered to have reflected the ability to respond to an irritant chemical under the conditions of the test ie the recommended acceptance criterion for the positive control was achieved for this test (≤40% relative to the concurrent negative control). The negative control in this study was comparable to the negative control achieved in the previous 22 experiments conducted with this test method.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test item was classified as irritant according to the annex VI of Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 14.8% after the 15-Minute exposure period and 42 hours post-exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 3.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.7%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.923 and the standard deviation value of the percentage viability was 7.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 0.6%. The test item acceptance criterion was therefore satisfied.

Under the test conditions, test item was classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP). This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.