2-Butenedioic acid, 2-methyl-, 1,4-bis(2-ethylhexyl) ester, (2Z)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A non-GLP screening test conducted according to OECD 439.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

non-GLP screening study

Principles of method if other than guideline:

The reconstructed human epidermis model EPISKIN-SM is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. The method is approved by international regulatory agencies as a replacement for the identification of irritants/corrosives in the in vivo rabbit skin assay (OECD 404).
The test is based on quantifying the cytotoxic effects following short term exposure of the stratum corneum of reconstituted human epidermis. The cell viability of the epidermis is assessed immediately after the exposure, based on reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT (thiazolyl blue).

GLP compliance:

no

Remarks:

even if the laboratory has GLP certificate

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

EC Number:

807-715-4

Cas Number:

1354569-12-2

Molecular formula:

C21 H38 O4

IUPAC Name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

Test material form:

other: liquid

Details on test material:

During reception of the test material kit, indicators for pH of the agar medium and transport temperature were checked and found to be in good order. The test kit was kept in their packaging at 37 oC and the sterile assay medium supplied with the kit was stored at 2 - 8 oC until the initiation of the test.

Test animals

Species:

other: EPISKIN-SM human epidermis model

Strain:

other: EPISKIN-SM human epidermis model

Details on test animals or test system and environmental conditions:

Manufacturer: SkinEthic, France, batch n:o 13-EKIN-027, expiry 22.7.2013, manufactured according to ISO 9001. All biological components of the epidermis and the kit culture medium have been tested for viruses, bacteria and mycoplasma. The quality of the final product was assessed at SkinEthic laboratories (attached).

The following indicators for potential false viability were applied:
1. Check-method for possible direct MTT reduction with test substance
2. Check-method to detect the colouring potential of test-substances
3. Additional control(s) for dyes and chemicals able to colour the tissue.

Test system

Type of coverage:

other: topical application of test material to the surface of epidermis

Preparation of test site:

other: 13-day culture period under ISO 9001

Vehicle:

unchanged (no vehicle)

Remarks:

as supplied

Controls:

other: saline buffer as 2 negative controls, one additional test item-treated tissue for non specific OD evaluation

Amount / concentration applied:

50 µL of test item was added to each of the two test and one additional control skin units.
50 µL saline buffer was added to each of the two negative control skin units.

Duration of treatment / exposure:

Application and rinsing (day 0): The plates with the test item treated ad the negative and positive control treated epidermis were incubated for 15 minutes (+- 0.5 min) at room temperature (22.9 - 24.6 oC). After the incubation the EPISKIN-SM units were removed and rinsed with 0.9 % saline buffer solution. The rest of the buffer was removed.
After rinsing the units were placed into the plate wells with fresh pre-warmed maintenance medium (2 mL/well) below them and incubated for 42 hours (+- 1 h) at 37 oC in an incubator with 5 % CO2 protected from light.

Observation period:

2 days (42 hours)

Number of animals:

0

Details on study design:

Pre-incubation (day -1): The maintenance medium was pre-warmed to 37 oC. The appropriate number of wells in an assay plate were filled with pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then inclubated overnight at 37 oC in an incubator with 5 % CO2.

MTT test and formazan reaction (day 2):
All EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The control unit was filled with fresh assay medium. All units were incubated for 3 hours (+- 5 min) at 37 oC in an incubator with 5 % CO2 protected from light.
At the end of incubation with MTT a formazan extraction was undertaken using a biopsy punch into a tube of 500 µL acidified isopropanol. The capped tubes were mixed by a vortex mixer and incubated for abour 2 hours at room temperature protected from light with gentle agitation (150 rpm) for formazan extraction.
Cell viability measurements (day 2): Following the formazan extraction, 2 x 200 µL sample from each tube were placed into the wells of a 96-well plate spectrophotometer and read using wavelengt filter of 540 nm and acified isopropanol solution as blank (6 x 200 µL).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: optical density

Value:

0.601 - 0.678

Remarks on result:

other:

Remarks:

Basis: mean 0.640. Time point: 15 min exposure and 42 h incubation. Max. score: 0.711. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Cell viability (% )

Value:

85 - 95

Remarks on result:

other:

Remarks:

Basis: mean 90 +- 7.07. Time point: 15 min exposure and 42 h incubation. Max. score: 100.0. Remarks: Results converted from measured optical density. (migrated information)

In vivo

Irritant / corrosive response data:

Please see attachment 2 for detailed results.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance was not irritating in an in vivo screening study (EPISkin model).

Executive summary:

The test substance was not irritating in an in vivo screening study (non-GLP EPISkin model, OECD 439). It was rated as reliable with restrictions (Klimisch 2). The screening result is used for classification and labelling.