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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:
weight of evidence
Study period:
04 April 2012 - 17 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is carried out based on the OECD No. 439 "In Vitro Skin irritation" and in compliance with OECD principles of Good Laboratory Paractice (GLP).

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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
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
Details on test material:
- Name of test material (as cited in study report): Naphtha
- Substance type: UVCB substance
- Physical state: liquid
- Analytical purity: 100%; UVCB substance
- Lot/batch No.: 11-06053-003
- Expiration date of the lot/batch: 16 August 2013
- Storage condition of test material: room temparature in the dark

Test animals

Species:
human
Strain:
other: epidermal keratinocytes

Test system

Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 microliters
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
Observation period:
Post-exposure incubation period was 42 hours.
Details on study design:
Pre-incubation: Each epidermis unit was transferred into maintenance medium filled wells. The tissues were incubated at 37 deg. C, 5% CO2 in air overnight

Application of the test item: Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 10 microliters were applied to the epidermis surface. Negative control epidermis units were treated with DPBS and positive control epidermis units were treated with 5% SDS (w/v) solution.

Washing: At the end of the 15 min exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca2+ and Mg 2+. The sinsed tissues were transferred to the second column containg 2 ml of maintenance medium. The rinsed tissues were incubated at 37 deg. C, 5% CO2 in air for 42 hours.

Cell viability measurements:Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 min to homogenise the released inflammatory mediators in the maintanance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. The MTT assay is a validated quantitative method which should be used to measure cell viability. The tissue is placed in Formazan solution for 3 hours. The precipitated blue formazan product is then extracted from the tissue using acidic isopropanol, and the concentration of formazan is measured by determining the OD at 570 nm.


SCORING SYSTEM:
Classification of irritation potential is based upon relative mean tissue viability following the 15min exposure period followed by the 42 hour post exposure incubation period.
Criteria for in vitro interpretation Classification
Relative mean tissue viability is ≤ 50% Irritant
Relative mean tissue viability is > 50% Non-Irritant

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: The relative mean viability (%) of the treated tissues
Value:
13.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15min. Max. score: 100.0. Remarks: viability 0-50% is classified as an irritant; viability > 50 % is classified as a non-irritant . (migrated information)

In vivo

Irritant / corrosive response data:
See table 1 in attached background material.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The skin irritation potential of the test item was evaluated using EPISKIN TM reconstructed human epidermis model. Based on the study result the the test item is classified as irritant to the skin.
Executive summary:

The study was regarded reliable without restrictions, since the study is carried out based on the OECD No. 439 "In Vitro Skin irritation" and in compliance with OECD principles of Good Laboratory Paractice (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 period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to micro tubes and stored in freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containg acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the extraction period each tube was mixed and duplicate samples were measured for optical density at 540nm. The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item, and positive control item were calculated. The relative mean viability of the test item treated tissues was 13.3 % after a 15 min exposure period.

This study is used as WoE in the hazard assessment. Based on the study results the classification of this substance is Skin Irrit. 2.