Fatty acids, tall-oil, compds. with ethanolamine

Endpoint summary

Description of key information

Skin corrosion in vitro: Relative mean viability of tissues after exposure to test item were 93.8 % at 3 minutes and 89.5 % at 60 minutes (OECD 431 and EU Method B.40 bis).

Skin irritation in vitro: Relative mean viability of the test item treated tissues was 84.7% after the 15-minute exposure period and 42 -hour post exposure incubation period (OECD 439 and EU Method B.46).

Eye irritation in vitro: The in vitro irritancy score (IVIS) was determined to be in the range > 3 to ≤ 55 for the test item (OECD 437 and EU Method B.47). Corrected viability of tissues was determined to be ≤ 60 % (OECD 492).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2016 to 29 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

results of the freeze-killed tissues not used for quantitative correction of results or for reporting purposes with no impact on integrity of study (see below)

Qualifier:

according to guideline

Guideline:

other: EU Method B40 bis (In Vitro Skin Corrosion Human Skin Model Test)

Deviations:

yes

Remarks:

results of the freeze-killed tissues not used for quantitative correction of results or for reporting purposes with no impact on integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: neonatal

Vehicle:

unchanged (no vehicle)

Details on test system:

PURPOSE OF THE TEST
- The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes.
- Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
- This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS AND MTT
- The negative control item was used as supplied.
- The positive control item was used as supplied.
- A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to use.

EPIDERM RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: MatTek
- Date received: 27 July 2016
- EpiDerm tissues (0.63 cm2) lot number: 23347
- Assay medium lot number: 072116ZSA
- Upon receipt of the Epiderm tissues, the sealed 24-well plate was stored in a refrigerator until use.

MTT DYE METABOLISM, 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 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, 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 is still a sufficient amount 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.
- As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
- Test item (0.05 mL) was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT.
- 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 corrosion potential was performed in parallel on viable and freeze- killed tissues. This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
- Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERM tissues in an empty 12-well plate and storing in a freezer (−14 to −30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
- Test item (0.05 mL) was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the colour was then made.

PRE-INCUBATION
- The assay medium was pre-warmed before use in the main test. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods.
- EpiDerm tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

APPLICATION OF TEST ITEM AND RINSING
- Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre-incubation of the EpiDerm tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure.
- For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. Test item (50 μL) and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
- When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure.
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours.
- Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
- After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

ABSORBANCE / OPTICAL DENSITY MEASUREMENTS
- After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution.
- Aliquots (3 x 200 µL) of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate.
- Isopropanol (200 µL) alone was added to the three wells designated as blanks.
- Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.
- The relative mean viabilities were calculated using the equation relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100.

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) Negative control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
(ii) Positive control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
(iii) Coefficient of variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

MAJOR COMPUTERISED SYSTEMS
- Delta building monitoring system

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL

Duration of treatment / exposure:

3 minutes or 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

93.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative viability (% of negative control)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

89.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative viability (% of negative control)

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred.
- It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item was a yellow colour.
- This colour was attributed to the intrinsic colour of the test item and was considered not to have the potential to cause colour interference.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD562 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 1.716 for the 3-Minute exposure period and 1.735 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

RELATIVE MEAN VIABILITIES FOR EACH TREATMENT GROUP

Exposure period	Percentage viability negative control*	Percentage viability positive control	Percentage viability test item
3 minute	100	5.2	93.8
60 minutes	100	5.4	89.5

* Mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Remarks:

see Appendix 2 (attached)

Conclusions:

The relative mean viability of the test item treated tissues were reported as 93.8 % (3 minute exposure period) and 89.5% (60 minute exposure period).

Executive summary:

GUIDELINE

The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item. The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 5.2 % (positive control and 93.8 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 5.4 % (positive control) and 89.5 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues were reported as 93.8 % (3 minute exposure period) and 89.5% (60 minute exposure period).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 September 2016 to 19 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

results of the water-killed tissues not used for quantitative correction of results or for reporting purposes with no impact on integrity of study (see below)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

results of the water-killed tissues not used for quantitative correction of results or for reporting purposes with no impact on integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

other: adult

Vehicle:

unchanged (no vehicle)

Details on test system:

PURPOSE OF THE TEST
- The purpose of this test was to evaluate the skin irritation potential of the test item 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). 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.
- 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 of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Following a full validation study the EpiSkinTM 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.
- The procedure followed is based on the recommended EpiSkiN SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.
- Test items are 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.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS AND MTT
- The negative control item, DPBS, was used as supplied.
- The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
- A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
- A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

EPISKIN RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: SkinEthic Laboratories, Lyon, France
- Date received: 13 September 2016
- EpiSkin Tissues (0.38cm2) lot number: 16-EKIN-037
- Maintenance Medium lot number: 16-MAIN3-064
- Assay Medium lot number: 16-ESSC-040

MTT SALT METABOLISM 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 by using killed tissues to act as controls.

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 was checked for the ability to directly reduce MTT.
- Test item (10 μL) 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 or purple, 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 item like viable tissues.
- Water-killed tissues were prepared by placing untreated EPISKINTM 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.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
- Test item (10 Μl) was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

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, temperature indicator colour and agar medium colour were all found to be satisfactory.
- Maintenance medium (2 mL), warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 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.

APPLICATION OF TEST ITEM AND RINSING (DAY 1)
- Maintenance medium (2 mL), 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 center). After a 7-Minute 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 (re-spreading is not required for the negative control or test item). The plates 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-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
- MTT (2 mL of a 0.3 mg/mL solution), freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. 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 EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 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 coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 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.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- For the test item the relative mean tissue viabilities obtained after the 15-minute exposure period followed by the 42-hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3).
- Relative mean viabilities were calculated using the equation relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100.

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria
are achieved:
(i) Positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
(ii) Negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
(iii) Test item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL of test item (26.3 μL/cm2)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test

Value:

84.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: relative mean viability of tissues after 15-minute exposure and 42-hour post-exposure incubation

Other effects / acceptance of results:

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.
- 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.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item was a pale yellow colour. This colour was attributed to the intrinsic colour of the test item and was not considered capable of causing interference with the MTT endpoint. It was therefore unnecessary to run colour correction tissues.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1 (attached). The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1.
- The relative mean viability of the test item treated tissues was 84.7% after a 15-minute exposure period and 42-hour post-exposure incubation period.
- It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

QUALITY CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 11.7% relative to the negative control treated tissues and the standard deviation value of the viability was 2.7%. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 0.772 and the standard deviation value of the viability was 2.0%. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.6%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Remarks:

see Appendix 2 (attached)

Conclusions:

The relative mean viability of the test item treated tissues was 84.7% after the 15-minute exposure period and 42-hour post exposure incubation period.

Executive summary:

GUIDELINE

The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a postexposure

incubation period of 42 hours. The principle of the assay was 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 study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46.in vitroskin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period each tissue was rinsed before incubating for 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 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

The relative mean viability of the test item treated tissues was 84.7% after the 15-minute exposure period and 42-hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

CONCLUSION

The relative mean viability of the test item treated tissues was 84.7% after the 15-minute exposure period and 42 -hour post exposure incubation period.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF BOVINE EYES
- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Excised eyes were transported to the test facility over ice packs on the same day of slaughter.
- Corneas were refrigerated on arrival and used within 24 hours of receipt.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three

Details on study design:

PREPARATION OF CORNEAS
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
- The medium from both chambers of each holder was replaced with fresh complete EMEM.
- A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (see Annex 1, attached). The average opacity for all corneas was calculated.
- Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.
- At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
- The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes.
- After incubation, the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated.
- The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL).
- The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
- After incubation the medium in the posterior chamber of each holder was decanted and retained.
- Medium representing each cornea (360 μL) was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
- The corneas were retained after testing for possible conduct of histopathology.
- Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface.
- The cassette was immersed in 10% neutral buffered formalin.

DATA EVALUATION
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
- The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading.
- These values were then corrected by subtracting the average change in opacity observed for the negative control
corneas.
- The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
- The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea.
- The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

VISUAL OBSERVATION
- The condition of the cornea was visually assessed post treatment and post incubation.

CRITERIA FOR AN ACCEPTABLE TEST
- Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2014 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.
- Sodium chloride solution (0.9 % w/v) was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 2.9 and for permeability ≤ 0.103.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.

Irritation parameter:

in vitro irritation score

Value:

10.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL OPACITY AND PERMEABILITY MEASUREMENTS
- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (attached).

CORNEAL EPITHELIUM CONDITION
- The condition of each cornea is given in Appendix 2 (attached)
- The corneas treated with the test item were clear post treatment and post incubation.
- The corneas treated with the negative control item were clear post treatment and post incubation.
- The corneas treated with the positive control item were cloudy post treatment and post incubation.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 10.8 for the test item, 2.7 for the negative control and 39.7 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity of ≤ 2.9 and permeability ≤ 0.103. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

study cannot be used for classification

Conclusions:

In vitro irritancy scores were reported as 10.8 for the test item, 2.7 for the negative control and 39.7 for the positive control.

Executive summary:

GUIDELINE

The study was performed in accordance with OECD 437 (updated 26 July 2013) and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require 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 biochemical 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.

 

METHODS

The undiluted test item was applied to the excised eyes of adult cattle 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).

 

RESULTS

In vitro irritancy scores were reported as 10.8 for the test item, 2.7 for the negative control and 39.7 for the positive control.

 

CONCLUSION

The in vitro irritancy score (IVIS) was determined to be in the range > 3 to ≤ 55 for the test item and, in accordance with OECD 437 (26 July 2013), the result was inconclusive with respect to GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2016 to 01 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

EpiOcular assay medium not warmed during cell culture and assessment of coloured or staining materials was not applicable because the test item did not dye water or isopropanol

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

PURPOSE OF STUDY
- Eye irritation is generally defined as "the production of reversible changes in the eye". The potential for chemical induced eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It is usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a pre-validation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the EpiOcular human cornea model and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
- The EpiOcular TM Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
- The test consists of a topical exposure of a human reconstructed cornea model to the neat test item followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

CELL CULTURE
- EpiOcular kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular tissues (surface 0.6 cm2) were cultured on specially prepared cell culture inserts (MILLICELL , 10 mm Ø).
- EpiOcular tissues were shipped at 2 to 8 °C on medium-supplemented agarose gels in a 24- well plate. On the day EpiOcular tissues were received (29 November 2016), the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. Aliquots (1.0 mL) were placed into the appropriate wells of pre-labelled 6-well plates.
- Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70 % isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50 % of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular tissues was incubated at standard culture conditions overnight (about 17 hours).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL test item

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Two

Details on study design:

TEST ITEM PREPARATION
- Test item (50 µL) was dispensed directly onto duplicate EpiOcular tissue for 30 minutes.
- The test item was spread to cover the upper surface of the tissue.

EPIOCULAR KIT COMPONENTS
- Lot number: 23754
- Sealed 24-well plate: Contains 12/24 inserts with EpiOcular tissues on agarose
- Serum-free test medium: DMEM-Medium
- Positive control: Methyl Acetate (CAS#79-20-9)
- 12-well plate: Holding plate
- 24-well plates: For MIT viability assay
- 6-well plates: For storing inserts, or for topically applying test agents
- Ca++Mg++-Free D-PBS: Fo Dulbecco's Phosphate Buffered Saline

MTT-100 ASSAY KIT COMPONENTS
- 1 vial (2 mL): MIT concentrate
- 1 vial (8 mL): MIT diluent (supplemented DMEM) for diluting MIT concentrate prior to use in the MTT assay
- 1 bottle (60 mL): Extractant solution (lsopropanol) for extraction of formazan crystals

MTT SOLUTION
- On the day of the experiment, an MTT solution of 1 mg/mL in DMEM was prepared.

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST ITEM
- Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 µL of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
- Since the test item proved to reduce MTT, an additional test using freeze-killed tissue controls (killed controls = KC) had to be performed to determine a correction factor for calculating the true viability in the main experiment.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
- Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and is extracted together with MTT. Therefore, each test item must be checked for its colorant properties.
- Since the test item was intrinsically coloured (orange), it had to be tested for its ability to dye water or isopropanol and to absorb significantly light at the wavelength used for the MTT determination. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 mL of isopropanol, incubated in a glass tube, and placed on an orbital plate shaker for 2 to 3 hours at room temperature. Since the test item did not dye water or isopropanol, it was not necessary to measure the OD of an aliquot, and an additional test on viable tissues (without MTT addition) did not have to be performed.

EXPERIMENTAL PROCEDURE
- After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free­ DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
- Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular tissues. The tissues were incubated at standard culture conditions for 30 minutes.
- At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilised a different set of three beakers.
- The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps.
- The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
- Since it was not possible to remove the visible test item completely, this was noted in the study file. No further rinsing should have been done according to the test kit supplier's recommendation.
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
- At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
MTT ASSAY
- After post-treatment incubation of 120 minutes the MTT assay was performed.
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
- Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2 to 8 °C in the dark and then shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
- The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax Molecular Devices, 85737 Ismaning, Germany; Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

KILLED CONTROLS FOR ASSESSMENT OF RESIDUAL TEST ITEM REDUCTION OF MTT
- Since the test item reduced MTT a functional check on killed controls (KC) was performed in the definitive assay to show that the test material was not binding to the tissue and leading to a false MTT reduction signal.
- To determine whether residual test item was acting to directly reduce the MTT, two freeze­ killed tissues per test item were used. Freeze-killed tissues were prepared by placing untreated EpiOcular constructs (in a 24-well plate) in the -20 °C freezer overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once frozen, the tissue can be stored indefinitely in the freezer.
- Two killed tissues were treated with the test item in the normal fashion. All assay procedures were performed as for the viable tissue. Two freeze-killed tissues treated with sterile deionised water (negative control) were tested in parallel since a small amount of MTT reduction is expected from the residual NADH and associated enzymes within the killed tissue.

DATA RECORDING
- The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item and the controls.

DATA EVAUATION
- The mean OD value of the blank control wells (OD Blk) for each experiment was calculated.
- The mean OD Blk was subtracted from each mean OD value of the same experiment (blank corrected values).
- The mean value of the two aliquots for each tissue (= corrected test item OD) was calculated.
- The mean value of the two relating tissues for each control and test item (= corrected mean OD) was calculated. For further calculations only the corrected mean negative control OD value was needed and in case of the freeze-killed tissues the negative control OD value (OD Nc_Kc).
- The corrected OD value of the negative control corresponds to 100% viability (corrected negative control OD = Negative Control OD – OD Blk = 100% Viability).

CALCULATIONS FOR VIABILITY TESTS ONLY
- The percentage viability of each of the two relating tissues was calculated relative to the negative control (100 % control) for each control and test item using the equation viability [%] = 100 x corrected test item OD / corrected mean negative control OD.
- The difference in viability between duplicate tissues was calculated. The test is considered invalid if the difference is > 20 %.
- The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model.

CALCULATIONS FOR VIABILITY PLUS KILLED CONTROL TESTS
- The OD values of the freeze-killed tissue experiment were determined and blank control corrected = corrected OD test item KC.
- The mean negative control freeze-killed tissue value (OD Nc_Kc) was substracted from the test item-treated freeze-killed tissue to determine the individual freeze-killed tissue values for the test item (OD Kc). The equation used was OD killed control = corrected OD test item Kc – OD Nc_Kc. The net OD killed control represented the amount of reduced MTT due to direct reduction by test item residues.
- The freeze-killed tissue OD for the two relating tissues was converted to a percentage value relative to the viability scale (freeze-killed tissue “viability”) by comparison to the corrected viability negative control OD of the relating experiment in which the substance viability test was performed. The equation used was killed control viability [%] = 100 x corrected test item OD / corrected mean negative control OD.
- The difference in viability of the duplicate killed tissues was calculated. The freeze-killed tissue test is considered invalid if the difference is > 20 %.
- The mean viability for the freeze-killed tissue was calculated and this mean freeze-killed tissue “viability” value (KC “viability”) was subtracted from the relating mean viability of the test item (TI viability) to determine the freeze-killed tissue corrected viability (KC corrected viability). The equation used was KC corrected viability = TI viability – KC viability.
- The test item was classified regarding the freeze-killed tissue corrected viability according to the prediction model.

PREDICTION MODEL
- If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant.
- If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

ACCEPTABILITY OF ASSAY
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5.
2) The mean relative viability of the positive control is below 50 % of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control. This applies also to the freeze-killed (single items and negative control), which are calculated as percent values related to the viability of the negative control.
- The OD was determined in a microplate reader (Versamax Molecular Devices; SoftMax Pro Enterprise version 4.7.1) at 570 nm (OD570) without reference filter.

Irritation parameter:

other: percentage cell viability

Remarks:

true viability

Value:

41.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability reported as 50.2 % before correction factor taken into account

Other effects / acceptance of results:

RESULTS
- Results after treatment for 30 minutes with test item and the controls are shown in the attached table.
- Historical data for positive and negative controls are presented in Annex 1 (attached).
- The optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water did not lead to a change in colour. An additional test with viable tissues (without MTT addition) did not have to be performed to determine a correction factor for calculating the true viability in the main experiment.
- Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed blue colour. An additional test with freeze-killed tissues had to be performed to determine a correction factor for calculating the true viability in the main experiment.
- The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 50.2% (threshold for irritancy ≤ 60%) without taking the correction factor into consideration. Taking correction factors into account, the true viability was 41.2%, consequently the test item was irritant to eye.

ACCEPTANCE CRITERIA
- The negative control OD was > 0.8 and < 2.5 (values between 1.617 and 1.690).
- The mean relative viability of the positive control was below 50 % of the negative control viability (17.9%).
- The difference of viability between the two relating tissues of a single item was < 20% (values between 0.1% and 8.2%) in the same run (for positive and negative control tissues and tissues of single test item and additional test).

DISCUSSION

- The in vitro study was performed to assess the eye irritation potential of test item by means of the Human Cornea Model Test.

- An additional test with freeze-killed tissues was performed, since the test item proved to be a MTT reducer.

- Tissues of the EpiOcular human cornea model were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

- Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 17.9 %, thus the validity of the test system is ensured.

- Relevant irritating effects were observed following 30 minutes incubation with test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 50.2 % compared with the value of the negative control even without taking the correction factor derived from the additional test with freeze-killed tissues into account (threshold for irritancy ≤ 60%). Considering the correction factor, the true viability value was reduced to 41.2 %.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the experimental conditions reported, the test item possesses an eye irritating potential (corrected viability of tissues ≤ 60 %).

Executive summary:

GUIDELINE

An in vitro study was conducted to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The investigation was performed in compliance with the OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July 2015) and the MatTek Corporation Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular Model (29 June 2015).

METHODS

The test item proved to be an MTT reducer in the MTT pre-test but it did not dye water or isopropanol in the colour interference pre-test. Therefore, an additional test with freeze-killed tissues had to be performed to determine a correction factor for calculating the true viability in the main experiment. An aliquot (50 µL) of test item, negative control (deionised water) or positive control (methyl acetate) was applied to duplicate tissues for 30 minutes.

RESULTS

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % compared with the negative control value in the relative absorbance thus ensuring the viability of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the corrected viability of the tissues decreased below 60 % (41.2 %).

CONCLUSION

Under the experimental conditions reported, the test item possesses an eye irritating potential (corrected viability of tissues ≤ 60 %).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion in vitro

 

A key study was performed to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item. The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B40 bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 5.2 % (positive control and 93.8 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 5.4 % (positive control) and 89.5 % (test item). Relative mean viability of tissues after exposure to test item were 93.8 % at 3 minutes and 89.5 % at 60 minutes.

Skin irritation in vitro

 

A key study was performed to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed 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 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 study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46 in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period each tissue was rinsed before incubating for 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 theappropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 84.7% after the 15-minute exposure period and 42-hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

Eye irritation in vitro

Bovine Corneal Opacity and Permeability (BCOP) test

 

A key study was performed in accordance with OECD 437 (updated 26 July 2013) and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require 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 biochemical 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 undiluted test item was applied to the excised eyes of adult cattle 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).

 

In vitro irritancy scores were reported as 10.8 for the test item, 2.7 for the negative control and 39.7 for the positive control.

 

The in vitro irritancy score (IVIS) was determined to be in the range > 3 to ≤ 55 for the test item and, in accordance with OECD 437 (26 July 2013), the result was inconclusive with respect to GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments.

Human Cornea Model Test

 

A second key study was conducted in vitro to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The investigation was performed in compliance with the OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July 2015) and the MatTek Corporation Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular Model (29 June 2015).

 

The test item proved to be an MTT reducer in the MTT pre-test but it did not dye water or isopropanol in the colour interference pre-test. Therefore, an additional test with freeze-killed tissues had to be performed to determine a correction factor for calculating the true viability in the main experiment. An aliquot (50 µL) of test item, negative control (deionised water) or positive control (methyl acetate) was applied to duplicate tissues for 30 minutes.

After treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % compared with the negative control value in the relative absorbance thus ensuring the viability of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the corrected viability of the tissues decreased below 60 % (41.2 %).

 

Under the experimental conditions reported, the test item possesses an eye irritating potential (corrected viability of tissues ≤ 60 %).

Justification for classification or non-classification

Skin

Investigation of skin corrosivity potential in vitro using OECD 431 showed that the test item was not corrosive (relative mean tissue viability ≥ 50 % after 3-minute exposure and ≥ 15 % after 60-minute exposure). Results of an in vitro test (OECD 439) showed that the test item was not irritating to skin (relative mean tissue viability > 50 % after 15-minute exposure). Classification for skin irritation/corrosion is therefore not required under the terms of Regulation (EC) No 1272/2008.

 

Eye

The BCOP assay conducted in accordance with OECD 437 demonstrated that classification of the test item for Category 1 ocular effects was not required (in vitro irritancy score (IVIS) < 55). However, corrected viability of tissues was reported as ≤ 60 % using the Human Cornea Model Test (OECD 492) indicating that the substance cannot be considered non-hazardous. Classification as an eye irritant (Category 2) is therefore appropriate under the terms of Regulation (EC) No. 1272/2008.