2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl-, 2-ethylhexyl ester

Endpoint summary

Description of key information

The skin irritation and sensitization potential of the test material was determined in the human repeated insult patch test (HRIPT) conducted essentially as described by Draize (1955).  In addition, the structurally similar material, undecenyl methoxycrylene (UMC), was tested in vitro in the Episkin Reconstituted Human Epidermis Model for skin irritation.  The eye irritation potential of the test material was measured in vitro in the HET-CAM Test.  In addition, UMC was tested for eye irritation potential in vitro in the SkinEthic Reconstituted Corneal Model.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

other: Method measures cytotoxicity in reconstituted human epidermal cell cultures following topical application

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

between 15 September and 21 September 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The principle of the study was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material 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-diphenyltetrazolium bromide) to a blue formazan salt )within the mitochondria of viable cells) in the test material 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 materials 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. The EPISKIN(TM) model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum coreum.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 19th August 2008, Date of signature 4th March 2009

Species:

other: Human reconstituted epidermis

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Not applicable for in vitro test.

Type of coverage:

other:

Preparation of test site:

other: Not applicable for in vitro test

Vehicle:

other: not applicable for in vitro test

Controls:

other: Not applicable to study.

Amount / concentration applied:

See free text in "Details on study design" section below.

Duration of treatment / exposure:

15 minutes direct exposure. See free text in "Details on study design" section below.

Observation period:

See free text in "Details on study design" section below.

Number of animals:

Not applicable to study

Details on study design:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

Negative and Positive Control Materials:

Dulbecco’s phosphate buffered saline (PBS) with Ca++ and Mg++ was used as the negative control. Sodium dodecyl sulphate (SDS) was used as the positive control.

Preparation of Negative and Positive Control Materials, MTT and Acidified Isopropanol:

The negative control material was used as supplied.

The positive control material was prepared as a 5% w/v aqueous dilution.

A 3 mg/ml MTT stock solution was prepared in PBS. The stock solution was diluted to 0.3 mg/ml with assay medium when required.

A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.

Pre-Test: Assessment of Direct Test Material Reduction of MTT:

MTT dye metabolism, cell viability assay:

The MTT assay, a colourimetric method of determining cell viability, is based on the reduction of the yellow tetrazolium salt (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

Assessment of Direct Test Material Reduction of MTT:

One limitation of the assay is possible interference of the test substance with MTT. A test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test substance is a problem only if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test substance present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by the procedure described as follows:

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, each test substance is checked for the ability to directly reduce MTT according to the following procedure:

10 microliters of the test material was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 deg C, 5% CO2 in air for 3 hours. Untreated MTT solutions were used as a control.

If the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT.

Pre-Incubation (Day 0: tissue arrival):
2ml of maintenance medium, warmed to approx. 37 deg 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 material and each control material. The tissues were incubated at 37 deg C, 5% CO2 in air for at least 24 hours.

Main Test:

Application of the Test Material and Rinsing (Day 1):

2 ml of maintenance medium, warmed to approx. 37 deg C, was pipetted into the second column of 3 wells of the 12-well plate.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 microliters of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 microliters of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). 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 +-0.5 minutes.

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 approx. 40 seconds using a constant soft stream of PBS to gently remove any residual test material. 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 deg C, 5% CO2 in air for approximately 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+-2 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 deg C for possible inflammatory mediator determination.


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 deg C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual Scoring scheme. Following qualitative evaluation of tissue viability a total biopsy of the epidermis was made using the EPISKIN(TM) biopsy punch. 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 microliters of acidified 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 deg C until Day 6 of the experiment, allowing the extraction of formazan 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 coloured solution.

For each tissue, duplicate 200 microliter samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 microliters of acidified isopropanol alone was added to the two wells designated as “blanks.” The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Interpretation of Results:
Quantitative MTT Assessment (percentage tissue viability):

For the test material 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:

Relative mean = (mean OD540 of test material/mean OD540 of negative control) x 100
viability (%)

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 table:

Criteria for in vitro interpretation Classification
Mean tissue viability is <=50% Irritant (I) R38
Mean tissue viability is >50% Non-Irritant (NI)●

● = The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation will be determined for test materials which are found to be borderline non-irritant based upon the MTT cell viability endpoint (mean tissue viability 51-60). This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Quality criteria: The results of the assay are considered acceptable if the following assay acceptance criterion is achieved:

Positive Control:

The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was =<40% relative to the negative control treated tissues, and the Standard Deviation (SD) value of the percentage viability is <20%.

Negative Control:

The assay establishes the acceptance criterion for an acceptable test if the mean OD540
For the negative control treated tissues was =>0.6, and the SD value of the percentage viability is <20%.

Irritation parameter:

other: Qualitative evaluation (see free text)

Basis:

other: Qualitative evaluation (see free text)

Time point:

other: Not applicable

Score:

0

Max. score:

0

Reversibility:

no data

Irritant / corrosive response data:

Direct MTT Reduction:

The MTT solution containing the test material did not turn blue/purple, which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material:

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material were determined and tabulated. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control were determined and tabulated.

The relative mean viability of the test material treated tissues was 105.8% after a 15 minute exposure.

The qualitative evaluation of tissue viability was determined visually and summarized in the table shown in the "Remarks on results including tables and figures" section below.. Following the 15 minute exposure the test material treated tissues appeared blue, which was considered indicative of viable tissue.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was <=40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was <20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was >=0.6 and the SD value of the percentage viability was <=20%. The negative control acceptance criterion was therefore satisfied.

Quantitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative control material	-	-	-
Positive control material	+	+	+
Test material	-	-	-

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be Non-Irritant.

Executive summary:

Introduction. 

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TMreconstituted 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 reconstituted human epidermal cultures following topical exposure to the test material 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 material 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 materials 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.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 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 material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was105.8% after a 15‑minute exposure.

Quality criteria: 

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

Conclusion: 

The test material was considered to be Non-Irritant. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

27 April 2010 to 08 May 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Hillcrest, Belton, Loughborough, UK.

- Age at study initiation: 12 to 20 weeks old.

- Weight at study initiation: At the start of the study the animals were in the weight range of 2.34 to 2.41 kg.

- Housing: The animals were individually housed in suspended cages.

- Diet (e.g. ad libitum): Free access to food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.

- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.

- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C

- Humidity (%): 30 to 70%,

- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour

- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

single application

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2 (male)

Details on study design:

A single rabbit was treated initially. The test material was placed into the conjuctival sac of the right eye. The left eye remained untreated and served as a control. Immediately after administration, an assessment of the initial pain reaction was made according to the six point scale shown in Table 1.

Based on the results from the first animal, a second animal was treated.

Assessment of ocular damage/irritation was made appoximately 1 hour and 24, 48 and 72 hours following treatment according to J.H. Draize (1977). See Table 2.

Irritation parameter:

other: group mean score

Basis:

mean

Time point:

other: 1 hour

Score:

11

Max. score:

110

Irritation parameter:

other: group mean score

Basis:

mean

Time point:

other: 24 hours

Score:

5

Max. score:

110

Irritation parameter:

other: group mean score

Basis:

mean

Time point:

other: 48 hours

Score:

0

Max. score:

110

Irritation parameter:

other: group mean score

Basis:

mean

Time point:

other: 72 hours

Score:

0

Max. score:

110

Irritant / corrosive response data:

See Table 3

Other effects:

The initial pain response value for Animal 69151 was 2.
The initial pain response value for Animal 69181 was 2.

Table 3: Individual Scores

Rabbit Cornea Iris Conjunctivae Total Number Hour A (Opacity) B (Area) (A x B x 5) A A X 5 A (Redness) B (Chemosis) C (Discharge) (A + B + C) x 2 Score 69151 1 0 0 0 0 0 2 (sf) 2 1 10 10 24 0 0 0 0 0 1 (sf) 1 0 4 4 48 0 0 0 0 0 0 (sf) 0 0 0 0 72 0 0 0 0 0 0 (sf) 0 0 0 0 69181 1 0 0 0 0 0 2 (sf) 2 2 12 12 24 0 0 0 0 0 1 (sf) 1 1 6 6 48 0 0 0 0 0 0 (sf) 0 0 0 0 72 0 0 0 0 0 0 (sf) 0 0 0 0

sf = yellow colored staining of the fur around the treated eye

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: other: Kay and Calandra classification system

Conclusions:

The test material produced a maximum group mean score of 11.0 and was classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to the Kay and Calandra classification system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

The test material failed to produce dermal irritation when applied to the skin of 50 human subjects as a 20% solution in corn oil (see Section 7.4.1). The structurally similar material, UMC, was rated "not irritating" when tested in vitro in the Episkin Reconstituted Human Epidermis Test. These results suggest little or no irritant potential for the test material.

Eye Irritation:

The test material failed to produce an irritant response in the HET-CAM test utilizing the hen's egg chorioallantoic membrane. The structurally similar material, UMC, also failed to produce a positive response in vitro when tested in the SkinEthic Reconstituted Corneal Model using human corneal epithelial tissue. The surrogate material, undecenyl methoxycrylene, produced a maximum group mean score for eye irritation of 11.0 based on the Kay and Calandra classification system. This would suggest it would be classified as a minimal irritant. However, with the exception of 1 -hour observations, no corneal, iris, chemosis or discharge scores were in excess of 1 in observations at 24 -, 48- and 72 hours. All minimal effects resolved by 48 hours. These results suggest that the test material has little or no potential for eye irritation.

Justification for selection of skin irritation / corrosion endpoint:
As the substance is a cosmetic ingredient, testing has been conducted on a surrogate material, an in vitro study has been conducted according to appropriate guidelines and according to GLP, as read across has been used to fulfill this endpoint we have assigned a klimisch reliability of 2 to both the in vitro and in vivo studies

Justification for selection of eye irritation endpoint:
As the substance is a cosmetic ingredient, testing has been conducted on a surrogate material, two studies have been conducted, an in vitro study and an in vivo study. Both studies have been conducted according to appropriate guidelines and according to GLP, the in vivo study has been listed as the key study and the in vitro study has been used as supporting data. As read across has been used to fulfill this endpoint we have assigned a klimisch reliability of 2 to both the in vitro and in vivo studies

Justification for classification or non-classification

The subject material would not be classified for skin irritation under either the EU Directive 67/548/EEC or under the EU CLP (Regulation (EC) 1272/2008).

The subject material would not be classified for eye irritation under either the EU Directive 67/548/EEC or under the EU CLP (Regulation (EC) 1272/2008).