Reaction mass of (6E)-Dec-6-enal and (7E)-Dec-7-enal and (8E)-Dec-8-enal and (8Z)-Dec-8-enal

Endpoint summary

Description of key information

Skin corrosion / irritation
The skin corrosion potential of the test substance, TM 11-212 (FRET 08-0334), was assessed as non-corrosive according to OECD Test Guideline 431 using the EPISKIN™ Reconstructed Human Epidermis Model.
The skin irritation potential of the test substance, TM 11-212 (FRET 08-0334), was assessed according to OECD Test Guideline 439 using EPISKIN™ reconstructed human epidermis model. The relative mean viability of the test item treated tissues was 32.4 % after a 15‑Minute exposure period and 42 hours post‑exposure incubation period and is therefore classified as a Category 2 skin irritant.
Eye irritation
The eye irritation potential of the test substance, TM 11-212 (FRET 08-0334), was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method.  The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.
The eye irritation potential of the test substance, TM 11-212 (FRET 08-0334), was therefore assessed using the SkinEthic reconstructed Human Corneal Epithelium model.  The relative mean viability of the test item treated tissues after a 10-minute exposure period was 83.7 %. The test item was therefore considered to be non-irritant.

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:

The study was conducted between 01 July 2014 and 07 July 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 439 using EPISKIN™ reconstructed human epidermis model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN™ reconstructed human epidermis model

Strain:

other: Not applicable

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours post-exposure incubation period.

Number of animals:

Not applicable

Details on study design:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolizm, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item 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.

Test for Direct MTT Reduction
As specified, a test item 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, the test item is checked for the ability to directly reduce MTT according to the following procedure:

10 µL of the test item 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 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues.

Water-killed tissues were prepared by placing untreated EPISKIN™ tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, the MTT reducing test item was applied to three water killed tissues. In addition, three water killed tissues remained untreated. The untreated water killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.


Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

2 mL of maintenance medium, warmed to approximately 37 °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 item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.


Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL /cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). 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 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. 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 °C, 5% CO2 in air for 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 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 ºC 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 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ 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 µL 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 °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 colored solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:

other: other: Mean

Value:

32.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes exposure. Remarks: Classified as irritant. (migrated information)

Irritant / corrosive response data:

Direct MTT Reduction
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.


Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 32.4% after a 15 Minute exposure period and 42 hours post exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Mean OD₅₆₂Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	±SDof OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.887	1.004	0.101	88.4	100*	10.0
	1.063			105.9
	1.061			105.7
Positive Control Item	0.100	0.087	0.013	10.0	8.7	1.3
	0.088			8.8
	0.074			7.4
Test Item	0.261	0.325	0.058	26.0	32.4	5.8
	0.338			33.7
	0.375			37.4

SD = Standard deviation

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

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.3%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD₅₆₂for the negative control treated tissues was 1.004 and the standard deviation value of the percentage viability was 10.0%. The negative control acceptance criterion was therefore satisfied.

 

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 5.8%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as irritant. The following classification criteria apply:
EU DSD (67/548/EEC) Irritant requires symbol “Xi” risk phrase R38 “Irritating to Skin”.
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.

Executive summary:

The skin irritation potential of the test substance, TM 11-212 (FRET 08-0334), was assessed according to OECD Test Guideline 439 using EPISKIN™ reconstructed human epidermis model. The relative mean viability of the test item treated tissues was 32.4 % after a 15‑Minute exposure period and 42 hours post‑exposure incubation period and is therefore classified as a Category 2 skin irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

The study was conducted between 30 September 2014 and 09 October 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to the Assessment of Ocular Irritation Potential using the SkinEthic reconstructed Human Corneal Epithelium model and in compliance with GLP.

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 item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) 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 direct reduction of MTT (3 [4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.
For the main test, triplicate SkinEthic tissues were treated with the test item for 10 minutes. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues were taken for MTT-loading. After MTT-loading the tissues were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The mean tissue viability for the test item was compared to the negative control and classified.

GLP compliance:

yes (incl. QA statement)

Species:

other: SkinEthic reconstructed Human Corneal Epithelium model

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Human Corneal Epithelium (HCE)
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 07 October 2014
HCE Tissues (0.5cm2) batch number: 14-HCE-040
Maintenance Medium lot number: 14-MIMA-042

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

No data

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Pre-Test – Assessment of Direct Test Item reduction of MTT
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test item is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test item on or in the tissues. To identify this possible interference, the test item was checked for its ability to reduce MTT directly.

30 µL of test item was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced the MTT.


Receipt and Preparation of Tissues
On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5% CO2 in air.


Main Test
Using sterile techniques, 1 mL of maintenance medium at room temperature was dispensed into the appropriate number of wells of 6-well plates designated ‘treatment plates’. Each well was labeled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 day old tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.

Triplicate tissues were treated with 30 µL of the test item for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µL of solution A to serve as negative controls and triplicate tissues were treated with 30 µL of 2% w/v SDS to serve as positive controls. The plates were incubated at 37 °C, 5% CO2 in air during the exposure time.

At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labeled 24-well plate designated ‘holding plate’ containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues were transferred to a pre-labeled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37 °C, 5% CO2 in air.

At the end of the incubation period the inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labeled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminum foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 562nm (OD562) using the Anthos 2001 microplate reader.

Irritation parameter:

other: Relative Mean Viability (%)

Basis:

mean

Time point:

other: 10 minutes

Score:

83.7

Remarks on result:

other: Non-irritant

Irritant / corrosive response data:

Assessment of Direct Test Item Reduction of MTT
The MTT solution containing the test item remained yellow which indicated that the test item did not directly reduce MTT.


Assessment of Eye Irritation Potential
The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 83.7%.

Assay Acceptance Criterion

The relative mean tissue viability for the positive control treated tissueswas 17.8% relative to the negative control treated tissues.

 

The quality criterion required for the acceptance of results in the test was satisfied.

Assessment of Eye Irritation Potential – Viability of HCE Tissues

Item	OD562of Individual Tissue	Mean OD562	Relative Mean Viability (%)
Negative ControlÅ	0.795	0.815	100*
	0.815
	0.834
Positive ControlÅ	0.144	0.145	17.8
	0.148
	0.143
Test Item	0.683	0.682	83.7
	0.671
	0.692

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the study plan followed the test item was considered to be Non-Irritant.
EU DSD (67/548/EEC): Not classified for irritation.
EU CLP (1272/2008/EC)/UN GHS: Not classified for irritation.

Executive summary:

The eye irritation potential of the test substance, TM 11-212 (FRET 08-0334), was assessed using the SkinEthic reconstructed Human Corneal Epithelium model. The relative mean viability of the test item treated tissues after a 10-minute exposure period was 83.7 %. The test item was therefore considered to be non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

Skin corrosion is defined as the production of irreversible damage to the skin, namely visible necrosis through the epidermis and into the dermis following the application of a test substance for up to 4 hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of observations at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia and scars. Skin irritation is defined as the production of reversible damage to the skin following the application of a test substance for up to 4 hours. Several factors are considered in determining the corrosion and irritation potential of substances before in vivo testing is undertaken.

The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. To this end, an in vitro study was performed to assess the potential for skin corrosion using the EPISKIN™in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives (Botham et al., 1995, Barrett et al., 1998 and Fentem et al., 1998). At its 10^thMeeting, held on 31 March 1998 at ECVAM, Ispra, Italy, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the EPISKIN™model as scientifically validated for use as a replacement for the animal test. The EPISKIN™model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.

The EPISKIN™model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

The procedure followed is based on the recommended EpiSkin™ Skin Corrosivity Test protocol INVITTOX No 118. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN™ model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

This study was designed to be compatible with OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

Results showed that the relative mean viabilities of the test item treated tissues were 105.4 % after 3 minutes exposure, 88.6 % after 60 minutes exposure, and 73.8 % after 240 minutes exposure. The test item should not therefore be classified as corrosive.

 

The test item was assessed for skin irritation using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). As described above, the principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric 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 item 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 may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Following a full validation study, the EpiSkin™ reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Test items were applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

This study was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010).

The relative mean viability of the test item treated tissues was 32.4 % after a 15-Minute exposure period and 42 hours post-exposure incubation period. From these results the test item was classified as an irritant.

 

 

Eye Irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay of vision following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application. Eye irritation means the production of changes in the eye following application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

The classification system for substances involves a tired testing and evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage.

 The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and bioitem function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The In Vitro Irritancy Score for the test item was 11.7 and therefore no prediction of eye irritation can be made. The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

As the BCOP study did not permit the conclusion that the test item does not require classification for eye irritation a further in vitro study was performed.

 

The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) 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 SkinEthic HCE model consists of transformed human corneal epithelial cells of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye (Nguyen et al., 2003). The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum free and chemically defined medium.

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.

Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test item treated tissues (quantitative measurement of tissue viability) relative to the negative control.

 

Relative mean tissue viability of ≤ 60 % results in a prediction of ocular irritancy. After a 10 minute exposure, the relative mean viability of the test item treated tissues was 83.7 % and the test substance is therefore considered not classified for eye irritation.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted on the target substance in an appropriate test species and according to internationally recognised guidelines.

Justification for selection of eye irritation endpoint:
The study was conducted on the target substance in an appropriate test species.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin Corrosion/Irritation

Using a quantitative MTT assessment, the classification of corrosivity potential is based on relative viabilities for each exposure time. At 3 minutes treatment time, a relative mean tissue viability (percentage of negative control) of < 35 indicates corrosivity. Likewise at 3/60 minutes and 60/240 minutes treatment time, a relative mean tissue viability (percentage of negative control) of ≥ 35 / <35 also indicates corrosivity. A relative mean tissue viability of ≥ 35 at 240 minutes treatment however is an indication that the substance is non-corrosive. The relative mean viability of the test substance, at 3, 60 and 240 minutes was 105.4 %, 88.6 % and 73.8% respectively and therefore the test substance is classified as non-corrosive to the skin.

Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period and a 42-hour post-exposure incubation period. The test item is considered an irritant if the relative mean tissue viability is ≤ 50 % and a non-irritant if the relative mean tissue viability is >50%. The relative mean viability of the tissues treated with the test item was 32.4 % and the test substance is therefore classified as a skin irritant.

 

Eye Irritation

The classification for substances involved a tired testing and evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage.

A study was performed to assess the ocular irritancy potential of the test substance to isolate bovine cornea, using an in vitro irritancy score. If the test item induced an in vitro irritancy score≥55, it is defined as an ocular corrosive or severe irritant and will be labelled EU CLP/UN GHS Category 1. If the irritancy score is≤3 then no classification to UN GHS or EU CLP is required. If a score of > 3 and≤55 is achieved then no prediction of eye irritation can be made. The in vitro irritancy score for the test substance was 11.7. The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

As the BCOP study did not permit the conclusion that the test substance does not require classification for eye irritation, a further study was performed using the SkinEthic reconstructed Human Corneal Epithelium model using a treatment period of 10 minutes.

In this test, a substance is classified as an irritant if the relative mean tissue viability ≤ 60 % and is considered non-irritant if the relative mean tissue viability > 60 %. The relative mean viability of the tissues treated with the test item was 83.7 % after a 10 minute exposure period. The test substance is therefore classified as non-irritant to the eye.