Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was performed according to OECD guidelines and in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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:
Determination of Skin Irritation potential using the EPISKIN reconstructed human epidermis model
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
-Batch: SC 00010162
-Purity: 100%
-Physical state/appearance: Clear colourless liquid
-Expiry Date: 31 December 2015
- Storage Conditions: Room Temperature

Test animals

Species:
other: N/a: EPISKIN (In vitro): reconstructed human epidermis model
Strain:
other: NA
Details on test animals and environmental conditions:
EPISKIN (In vitro): reconstructed human epidermis model
- Supplier: Skin Ethic Labs, Lyon, France

Test system

Type of coverage:
other: NA
Preparation of test site:
other: NA
Vehicle:
unchanged (no vehicle)
Controls:
other: positive and negative control incubations were included
Amount / concentration applied:
The test substance was used as supplied
Duration of treatment / exposure:
Triplicate tissues were treated with the test substance for an exposure period of 15 minutes. The rinsed tissues were then incubated at 37°C with 5% Carbon Dioxide in air for 42 hours
Observation period:
42 hours
Number of animals:
Tissues were tested in triplicate
Details on study design:
Preparation of Negative and Positive Control Substances, MTT and Acidified Isopropanol:
The negative control substance, PBS, was used as supplied. The positive control substance, SDS, was prepared as a 5% w/v aqueous solution. A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required. A 0.04 N solution of
hydrochloric acid in isopropanol was prepared when required.

Test for Direct MTT Reduction:
As specified, a test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test substance is checked for the ability to directly reduce MTT according to the following procedure: 10 μL of the test substance was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared inassay medium.
The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test substance turns blue, the test substance is presumed to have reduced the MTT and the determination of skin irritation potential would be
performed in parallel onviable and water-killed tissues for quantitative correction of the results.

Pre-incubation (Day 0: Tissue Arrival):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test substance and
each control substance. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test

Application of Test Substance and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test substance for an exposure period of 15 minutes. The test substance was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3μL /cm2) of the test substance was applied to the epidermis surface. Triplicate tissues treated with10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS5% w/v served as the positive controls. To ensure satisfactory contact with the positive control substance the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period,
each tissue was removed from the well using forceps and rinsed using a wash bottle containing 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 substance. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were
incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediatordetermination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2in air. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL ofacidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction offormazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Relative mean viability (%)
Value:
62.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hours. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean viability of the test substance treated tissues was 62.1% after a 15-Minute exposure period and 42 hours post-exposure incubation period.
Other effects:
No other effects reported.

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 17.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.6%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562 for the negative control treated tissues was 0.797 and the standard deviation value of the percentage viability was 7.6%. The negative control acceptance criterion was therefore satisfied.

 

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 1.8%. The test item acceptance criterion was therefore satisfied.

 

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

 

 

Item

 

 

OD562of tissues

 

Mean OD562of triplicate tissues

 

± SD of OD562

 

 

Relative individual tissue viability (%)

 

Relative mean viability (%)

 

± SD of Relative mean

viability (%)

 

 

Negative Control Item

0.734

0.797

0.060

92.1

100*

7.6

0.803

100.8

0.854

107.2

 

Positive Control Item

0.143

0.138

0.005

17.9

17.3

0.6

0.134

16.8

0.138

17.3

Test Item

0.481

0.495

0.014

60.4

62.1

1.8

0.509

63.9

0.494

62.0

 

SD = Standard deviation

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

OD562 = Optical Density

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Isobutyl phenyl acetate does not require classification as a skin irritant based on the results of this assay.
Executive summary:

The potential for the test substance to cause skin irritation was evaluated in the EPISKIN Reconstructed Human Epidermis model. The study was conducted in accordance with OECD guideline 439 and GLP. 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 62.1% after the 15-Minute exposure period and 42 hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under the conditions of the in vitro assay, the test substance was considered to be non-irritating to skin.