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

Endpoint summary

Description of key information

Skin corrosion: a negative in vitro EPISKIN model screening test (OECD 431, non-GLP, Klimich 2) used for classification and labelling.
Skin irritation: a negative in vivo screening study (non-GLP EPISkin model, OECD 439, Klimisch 2), and used for classification and labelling.
Eye irritation: a negative in vitro screening test on chicken's eye (OECD 430 but not under GLP, Klimisch 2) and applied for classification and labelling of the substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

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

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.

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).

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)

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)

Irritant / corrosive response data:

Please see attachment 2 for detailed results.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye 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: Non-GLP screening OECD 438 study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Principles of method if other than guideline:

The Enucleated Eye Test with isolated eyes of chickens is a well validated and accepted in vitro test system. It has been recognized as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes, which are obtained from slaughter animals.
This method can provide detailed information about the effects of test items on the cornea, and is useful to compare products, to classify test items for regulatory use when they are severe irritants or corrosive to the eye, and thus to avoid the need to test severe eye irritants in vivo. The test is described in OECD 438 and is approved by international regulatory agencies as a replacement for the identification of corrosives and severe irritants in the in vivo Rabbit eye assay (OECD 405).

GLP compliance:

no

Remarks:

The laboratory has a GLP certicificate

Species:

other: isolated chicken eye

Strain:

other: Ross 308 strain

Details on test animals or tissues and environmental conditions:

Chicken heads collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (v/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3 or 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not have varied by more than ±5% between the -45 and the zero time. Slight changes in thickness (0% to 3%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

Amount / concentration applied:

After the zero reference measurements, the eye (in its retainer) was removed from the chamber, and placed on a layer of tissue with the cornea facing upwards. The eyes were held in horizontal position, while the test item was applied onto the cornea. The test item was applied in a volume of 30 μL onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance and taking care not to damage or touch the cornea.

The positive control eyes were treated with 30 μL Trichloroacetic acid 30 (w/v) %. The negative control eye was treated with 30μL of Saline (Salsol solution, NaCl 0.9% w/v).

Duration of treatment / exposure:

The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item. The time while the eye was out of the chamber was limited to the minimum.

Observation period (in vivo):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within ±5 minutes were considered acceptable. The cornea thickness and cornea opacity were measured at all time points.
Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

5 isolated eyes (1 saline, 2 test item, 2 Triclotoacetic acid TCAA)

Details on study design:

Superfusion chamber apparatus (32 +- 1.5 oC) during the acclimatization and treatment periods.
Examination with a slit lamp microscope.

Irritation parameter:

other: Maximum corneal swelling

Basis:

mean

Time point:

other: 30, 75, 120, 180 and 240 min after rinse

Score:

0

Max. score:

3

Remarks on result:

other: ICE Class I

Irritation parameter:

other: Maximum corneal opacity

Basis:

mean

Time point:

other: 30, 75, 120, 180 and 240 min after rinse

Score:

0.5

Max. score:

1

Remarks on result:

other: ICE Class I

Irritation parameter:

other: Fluorescein retention

Basis:

mean

Time point:

other: 0 and 30 min after rinse

Score:

0.25

Max. score:

0.5

Remarks on result:

other: ICE Class I

Irritant / corrosive response data:

Please see the individual results tables, ICE classification and interpretation criteria in the attachments 1, 2 and 3.

Assessments were made at t=0 (base line), 30, 75, 120, 180 and 240 minutes (cornea thickness and opacity).

Fluorescein retention at t=0 and 30 minutes after the post-treatment rinse.

ICE Classes were determined for mean maximum corneal swelling at up to 75 min and 240 min, mean maximum corneal opacity and mean fluorescein retention.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: OECD 438

Conclusions:

In an in vitro screening test on chicken's eye, the test substance was not ocular corrosive or a severe irritant. The test results indicate that the test item is not irritating at all (ICE classification 3 X I).

Executive summary:

In an in vitro screening test on chicken's eye, the test substance was not ocular corrosive or a severe irritant. The test results indicate that the test item is not irritating at all (ICE classification 3 X I). The test was conducted according to OECD 438 but not under GLP. The results of the screening tests are rated as Klimisch 2 and applied for classification and labelling of the substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The results of the in vitro EPISKIN model screening test (OECD-431, non-GLP, Klimisch 2) indicate that the test item is not corrosive to skin.

The test substance was not irritating in an in vivo screening study (non-GLP EPISkin model, OECD 439, Klimisch 2).

The skin irritation screening study was selected as the key study on skin irritation as the most sensitive endpoint for classification and labelling of the substance.

Justification for selection of skin irritation / corrosion endpoint:
An in vitro screening study (OECD 439 EPISKIN model, non-GLP), rated as Klimisch 2.

Justification for selection of eye irritation endpoint:
An in vitro screening study (OECD 430 Isolated chicken eye, non-GLP), rated as Klimisch 2.

Justification for classification or non-classification

The results of the screening studies do not meet the classification criteria of the CLP Regulation for skin corrosion/irritation.

The results of the screening studies do not meet the classification criteria of the CLP Regulation for serious eye damage/irritation.