Alkenes, C15-18-branched and linear, maleated, hydrolyzed

Endpoint summary

Description of key information

The skin irritation study was carried out according to EU guideline B46. Triplicate EPISKIN reconstituted human epidermal tissues were treated with 10 ul of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes 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 period, each tissue was taken for MTT-loading. After MTT loading , each epidermis was extracted with formazan crystals out of the MTT-loaded tissues and optical density was then measured at 540 nm. Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is not considered to be a skin irritant based on the results of in vitro tests carried out reconstructed human epidermis model (Warren, N., 2009a) 
The study was carried out using an in vitro test that is not internationally validated yet. Triplicate SkinEthic reconstituted corneal epithelial tissues were treated with 30 ul of Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes for an exposure period of 10 minutes. The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes treated SkinEthic reconstituted human corneal epithelial tissues after a 10 minute exposure was 11.7%, and so Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes was considered to be irritant (as the % relative mean tissue viability was < 60%). 
Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes was found to be irritating to eyes under the conditions of this study using SkinEthic reconstituted human corneal epithelial tissues (Warren, N., 2009b). 

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:

2008-11-25 to 2008-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The study was carried out according to EU guideline B46 "In vitro skin irritation: reconstructed human epidermis model test" and complies with GLP. The EPISKIN model is a validated model and suitable to derive classification.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Principles of method if other than guideline:

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. The principle of the assay is based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (witin the mitochondia of viable cells) in the test material treated tissues relative to the negative controls.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Species:

other: In vitro: reconstituted human epidermis model

Strain:

other: EPISKIN reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Triplicate tissues were treated with 10 µl of the test material for an exposure period of 15 minutes at room temperature. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours at 37ºC, 5% CO2 in air. 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 at-14 to -30ºC 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. The tubes were refrigerated at 1 to 10ºC until day 6 to allow for the extraction. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 ul samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm using the Anthos 2001 microplate reader.

Type of coverage:

other: Not relevant

Preparation of test site:

other: Not relevant

Vehicle:

other: Not relevant

Controls:

not required

Amount / concentration applied:

10 µl of the test material was applied to the epidermis surface and triplicate tissues, treated with either 10 ul of PBS ( negative controls) or 10 ul of 5% w/v SDS (positive controls).

Duration of treatment / exposure:

Treatment period of 15 minutes, followed by a post-exposure incubation period of 42 hours.

Observation period:

Not relevant.

Number of animals:

Not relevant.

Details on study design:

The relative mean tissue viability after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control tissues. The relative mean viabilities were calculated as the ratio between the mean optical density at 540 nm of the test material and the mean optical density at 540 nm of the negative control. Classification of irritation potential is based upon the relative tissue viability following the 15 minute exposure period. If the mean tissue viability is ¿ 50%, the test material is classified as Irritant (R38) and if it is >50%, the test material is non-irritating.

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 % viabillity is ¿20%.

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 % viability is ¿20%.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 89.8

Remarks on result:

other:

Remarks:

Basis: other:. Time point: 15 minute exposure followed by a 42 hour post exposure period. Remarks: Non-Irritating.

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues was 89.9% after a 15 minute exposure, and the test material was considered to be non-irritant.

For this in vivo test, if the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT. As the MTT solution did not turn blue/purple, this indicated that the test material did not directly reduce MTT.

The relative mean tissue viability for the positive control treated tissues was less than or equal to 40% relative to the negative control treated tissues and the SD value of the % viability was less than or equal to 20%. The positive control acceptance criterion was therefore satisfied. The mean OD₅₄₀ for the negative control treated tissues was greater than or equal to 0.6 and the SD value of the % viability was less than or equal to 20%. The negative control acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₄₀values and % viabilities for the negative control material, positive control material and test material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability	Relative mean % viability	± SD of % viability
Negative control material	0.770	0.783	0.012	98.3	100*	1.56
	0.794			101.4
	0.784			100.1
Positive control	0.065	0.073	0.020	8.3	9.3	2.61
	0.058			7.4
	0.096			12.3
Test material	0.584	0.703	0.107	74.6	89.8	13.66
	0.792			101.1
	0.733			93.6

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

 

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test material treated tissues was 89.9% after a 15 minute exposure, and the test material was considered to be non-irritant since the mean tissue viability is >50%. On this basis, the test material does not warrant any classification according to Regulation (EC) No 1272/2008.

Executive summary:

Triplicate EPISKIN reconstituted human epidermal tissues were treated with 10 ul of DCI-30.n 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 period, each tissue was taken for MTT-loading. After MTT loading , each epidermis was extracted with formazan crystals out of the MTT-loaded tissues and optical density was then measured at 540 nm.

The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes treated EPISKIN tissues was 89.9% after a 15 minute exposure and therefore Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was considered to be non-irritant since the mean tissue viability is >50%.The results of the negative and positive controls demonstrated the validity of the test. On this basis, the test material does not warrant any classification according to Regulation (EC) No 1272/2008.

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:

2009-01-21 to 2009-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The study was carried out GLP. The SkinEthic test is currently under validation and results are showing that this model is suitable for this type of testing and to derive classification.

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. The experimental design of the study consists of a test for the direct reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test material followed by the main test. For the main test, triplicate SkinEthic tissues were treated with 30 ul of the test material for 10 minutes. Triplicate tissues treated with 30 ul of Solution A serves as the negative control and triplicate tissues treated with 30 ul of 1% w/v Sodium Dodecyl Sulphate served as the positive control. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues. Atfer extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of % viability (MTT conversion relative to negative controls).

GLP compliance:

yes (incl. QA statement)

Species:

other: In vitro: reconstituted human corneal model

Strain:

other: SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France)

Details on test animals or tissues and environmental conditions:

Pre-test
Prior to carrying out the definitive test, on arrival of the tissue, the tissue are prepared and incubated overnight at 37ºC, 5% CO2 in air. Before treatment, the age day 7 tissues were transfered from the arrival plates into the treatment plates containing the maintenance medium.

Main test
Triplicate tissues were treated with 30 µl of the test material for 10 minutes. Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µl of 1% w/v SDS to serve as positive controls. The plates were incubated at 37ºC, 5% CO2 in air durign the exposure time. Further to rinsing with Dulbecco`s Phosphate Buffered Saline (DPBS), the tissues were loaded with MTT and placed in an incubator for approximately three hours at 37ºC, 5% CO2 in air . At the end of the exposure, the tissues were visually examined and the degree of MTT staining evaluated. Further to extraction of MTT, the plate wrapped in aluminium foil were allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, the optical density was measured at 540 nm using the Anthos 2001 microplate reader. Tissues were retained for possible tissue histopathology and stored at room temperature.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

For the main test, triplicate SkinEthic tissues were treated with 30 ul of the test material for 10 minutes. Test materials were applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Duration of treatment / exposure:

The treatment period was 10 minutes.

Observation period (in vivo):

Not relevant.

Number of animals or in vitro replicates:

Not relevant.

Details on study design:

The negative control material, Solution A, was used as supplied and the positive control material, Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile water.

Irritation parameter:

other:

Value:

ca. 11.7

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues after a 10 minute exposure was 11.7%. Therefore as the test was classified according to the following criteria: (1) If the % relative mean tissue viability was greater than or equal to 60% the test material was considered to be non-irritant and (2) If the % relative mean tissue viability was < 60% the test material was considered to be an irritant. According to the above criteria, the test material was considered to be irritant.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 11.7%, and so the test material was considered to be irritant (as the % relative mean tissue viability was < 60%). However, it was considered unnecessary to carry out histopathology on the corneal tissues.

Table 1: Assessmentof eye irritation potential ¿ viability of RHC tissues

Material	Mean tissue viability	Relative mean % viability	± SD of % viability
Negative control material	1.099	1.054	100*
	1.008
Positive control	0.159	0.184	17.5
	0.209
Test material	0.117	0.123	11.7
	0.128

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

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The relative mean viability of the test material treated tissues after a 10 minute exposure was 11.7%, and so the test material was considered to be irritant (as the % relative mean tissue viability was < 60%). Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is classified as Category 2 for serious eye damage with the signal word "Warning" and is assigned the hazard statement H318 "causes serious eye irritation" according to Regulation (EC) No 1272/2008.

Executive summary:

Triplicate SkinEthic reconstituted corneal epithelial tissues were treated with 30 ul of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes for an exposure period of 10 minutes.

The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes treated SkinEthic reconstituted human corneal epithelial tissues after a 10 minute exposure was 11.7%, and so Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was considered to be irritant (as the % relative mean tissue viability was < 60%). Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is classified as Category 2 for serious eye damage with the signal word "Warning" and is assigned the hazard statement H318 "causes serious eye irritation" according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A proprietary study for skin irritation (WArren, N., 2009a) was carried out according to EU guideline B46 "In vitro skin irritation: reconstructed human epidermis model test". Triplicate EPISKIN reconstituted human epidermal tissues were treated with 10 ul of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes 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 period, each tissue was taken for MTT-loading. After MTT loading , each epidermis was extracted with formazan crystals out of the MTT-loaded tissues and optical density was then measured at 540 nm.

The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes treated EPISKIN tissues was 89.9% after a 15 minute exposure and therefore Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was considered to be non-irritant since the mean tissue viability is >50%.The results of the negative and positive controls demonstrated the validity of the test.

A proprietary study for eye irritation (Warren, N., 2009b) was carried out according to SkinEthic reconstituted human corneal epithelial tissues.

Triplicate SkinEthic reconstituted corneal epithelial tissues were treated with 30 ul of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes for an exposure period of 10 minutes.

The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes treated SkinEthic reconstituted human corneal epithelial tissues after a 10 minute exposure was 11.7%, and so Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was considered to be irritant (as the % relative mean tissue viability was < 60%).

Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was found to be irritating to eyes under the conditions of this study.

Effects on eye irritation: irritating

Justification for classification or non-classification

The relative mean viability of DCI-30.n treated EPISKIN tissues was 89.9% after a 15 minute exposure since the mean tissue viability is >50%. The relative mean viability of DCI-30.n treated SkinEthic reconstituted human corneal epithelial tissues after a 10 minute exposure was 11.7%, and so DCI-30.n was considered to be irritant (as the % relative mean tissue viability was < 60%).

The results of the skin and eye irritation studies showed that DCI-30.n is not an irritant to skin but it is an irritant to the eye. On this basis, DCI-30.n is classified as classified as Category 2 for serious eye damage with the signal word "Warning" and is assigned the hazard statement H318 "causes serious eye irritation" according to Regulation (EC) No 1272/2008.