Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro in accordance with OECD 439
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 September 2011 - 26 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 439
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : 1,1,3,3-tetramethylbutyl 2-ethylperoxyhexanoate (CAS 22288-43-3)
Description : clear colourless liquid
Batch number : 1009442440
Purity : 90.8%
Date received : 19 July 2011
Expiry date : 01 October 2012
Storage conditions : approximately -20°C in the dark

Test animals

Species:
other: reconstructed human epidermis model
Strain:
other: reconstructed human epidermis model
Details on test animals and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: Not applicable
Controls:
no
Amount / concentration applied:
TEST ITEM

The test item was applied neat.

Amount(s) applied (volume or weight with unit):
Approximately 10 µl of the test item was applied to the epidermis surface.

Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
Area of exposure:
Approximately 10 µl of the test item was applied to the epidermis surface.

% coverage:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.

Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM
Quantitative MTT Assessment (percentage tissue viability):
For the test item the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test item / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant

Mean tissue viability is >50% : Non-Irritant

Results and discussion

In vivo

Results
Irritation parameter:
other: Viability of cells
Basis:
mean
Remarks:
Viability of cells
Time point:
other: Day 6
Reversibility:
other: Not applicable
Remarks on result:
other: See relative mean viability below.
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 105.1% after a 15 Minute exposure period.
Other effects:
No

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 105.1% after a 15-Minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 12.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.678 and the standard deviation value of the percentage viability was 5.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 5.5%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540 of tissues

Mean OD540 of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.718

0.678

0.039

105.9

100*

5.7

0.641

94.5

0.675

99.6

Positive Control Item

0.056

0.084

0.048

8.3

12.4

7.0

0.057

8.4

0.139

20.5

Test Item

0.745

0.712

0.037

109.9

105.1

5.5

0.672

99.1

0.720

106.2


SD=   Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be non-irritant to skin.
Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 -Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

  • OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)
  • Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: 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 540 nm.

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

Results: The relative mean viability of the test item treated tissues was 105.1% after the 15-Minute exposure period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: The test item was considered to be Non-Irritant.