2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Endpoint summary

Description of key information

Skin corrosion in vitro: The test item was determined to be non-corrosive to skin becausecell viability was determined to be 93.6 % after 3 minutes and 47.0 % after 60 minutes (OECD 431; EpiDerm Human Skin Model).

 

Skin corrosion in vivo: The test item was determined to be non-corrosive to skin(OECD 404, EU Method B.4, OPPTS 870.2500 and relevant Japanese methods).

 

Skin irritation in vitro: The test item was determined to be non-irritant to skin because the relative mean viability of the test item treated tissues was determined to be 54.6 % after the 15-minute exposure period and 42-hours post-exposure incubation (OECD 439; EPISKIN reconstructed human epidermis model).

 

Skin irritation in vivo: The test item was determined to be non-irritant to skin (OECD 404, EU Method B.4, OPPTS 870.2500 and relevant Japanese methods).

Eye damage/irritation in vitro: The in vitro irritancy score (IVIS) was determined to be 10.2 using the BCOP assay and no prediction of eye irritation could be made because the IVIS value was in the range > 3 to ≤ 55 (OECD 437 and EU Method B.47). However, the test item was determined to be non-irritant to the eye because test item-treated tissue viability corrected for direct MTT reduction was determined to be 93.4 % relative to negative control treated tissue in the Human Cornea Model (OECD 492).

 

Eye damage/irritation in vivo: Under the conditions of the study, the test substance was determined to be mildly irritating to the eye according to a Kay and Calandra classification scheme. However, the reported individual mean scores for corneal opacity, iritis, conjunctival redness and chemosis do not cause the test item to be classified as causing serious eye damage or eye irritation under classification rules applicable in the EU (OECD 405, EU Method B.5,OPPTS 870.2400 and relevant Japanese guidelines).

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:

02 July 2019 to 04 July 2019

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)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

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 In Vitro Life Sciences Laboratories
- Date received: 02 July 2019
- EpiDerm tissues (0.63 cm2) lot number: 30804
- Assay medium lot number: 062719MSC
- 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/purple 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 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.

TEST FOR DIRECT MTT REDUCTION
- 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 (50 µL) 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 turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.
- The MTT solution containing the test item turned a brown color as opposed to blue/purple. Therefore, even though the solution did not blue/purple, the darkening to brown indicated that the test item may have the potential to directly reduce MTT and corrective procedures should be undertaken to avoid a false negative result. 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 or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
- Test item (50 μL) 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.

MAIN TEST PRE-INCUBATION
- The assay medium was brought to room temperature before use.
- An aliquot (0.9 mL) of this assay medium was pipetted into the appropriate wells of two pre-labelled 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. 50 μL of the test item 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 Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, 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 tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and 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 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.
- Servicing, calibration, pass band width and linearity range of the microplate reader is given in Annex 1 (attached).

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 OD570 of test item / mean OD570 of negative control) x 100.
- It was determined that the test item may have the potential to directly reduce MTT and therefore freeze-killed tissues were employed, the results of the MTT assay were corrected using the equation true viability = mean OD tvt – (OD tkt – OD ukt) where OD = optical density at 570 nm; tvt = treated viable tissues, tkt = treated killed tissues; ukt = untreated killed tissues.
- If direct reduction by the test item is greater than 30 % of the negative control value, additional steps must be taken into account or the test item may be considered incompatible with this test system.
- If direct reduction by the test item is less than 30 % of the negative control value, the mean OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) Negative control: The absolute OD570 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 OD570 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 8.0 N 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.
- Labtech LT-4500 microplate reader and LT-com analysis software.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL of test item

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicate tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

93.6

Vehicle controls validity:

not examined

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:

6-minute exposure

Value:

47

Vehicle controls validity:

not examined

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
- The MTT solution containing the test item turned a brown colour as opposed to blue/purple. Therefore, even though the solution did not blue/purple, the darkening to brown indicated that corrective procedures should be undertaken to avoid a false negative result. Therefore, an additional procedure using freeze-killed tissues was performed.
- The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

INTERFERENCE BY THE TEST ITEM RELATIVE TO THE CORRESPONDING NEGATIVE CONTROL
- 3 minutes exposure: Mean of test item treated killed tissues (tkt) = 0.144 OD570; Mean of untreated killed tissues (ukt) = 0.148 OD570. The direct reduction by the test item relative to the negative control value: (0.144 (tkt) – 0.148 (ukt)) / 2.269 (mean of negative control) = 0.0% (negative value treated as 0.0% to avoid artificially inflating the results).
- 60 minutes exposure: Mean of test item treated killed tissues (tkt) = 0.223 OD570; Mean of untreated killed tissues (ukt) = 0.135 OD570. The direct reduction by the test item relative to the negative control value: (0.223 (tkt) – 0.135 (ukt)) / 2.362 (mean of negative control) = 3.7 %.
- The interference by the test item relative to the corresponding negative control was 0.0 % after 3 minutes exposure and 3.7 % after 60 minutes exposure. Therefore, direct reduction was < 30 % relative to the negative control and considered acceptable.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item did not become coloured.
- This observation was taken to indicate the test item did not have the potential to cause colour interference.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD570 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 OD570 for the negative control treated tissues was 2.269 for the 3-minute exposure period and 2.362 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 2.5 % 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	3.3	93.6
60 minute	100	2.5	47.0

* Mean viability of the negative control tissues is setat 100%

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test item treated tissues was 93.6 % after 3 minutes exposure and 47.0 % after 60 minutes exposure.

Executive summary:

GUIDELINE

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 (29 July 2016) and Method B.40bis 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. Negative and positive control groups were treated for each exposure period. It was determined that the test item may have the potential 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 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 of 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-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data were 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), 3.3 % (positive control) and 93.6 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 2.5 % (positive control) and 47.0 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues was 93.6 % after 3 minutes exposure and 47.0 % after 60 minutes exposure.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2019 to 02 September 2019

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)

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

with no impact on integrity or validity of study results (see below)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

yes

Remarks:

with no impact on integrity or validity of study results (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 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 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 was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.
- 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 (ECVAM, 2009) 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 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.
- The study was repeated due to a quality control failure affecting the positive control group in the initial run. The repeated run was successful. Therefore, the study report was based on the repeated run and the main test results from the initial run were not used for reporting purposes.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS AND MTT
- The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.
- The positive control item, Sodium dodecyl sulphate (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: EpiSkin Laboratories, Lyon, France
- Date received: 27 August 2019
- EpiSkin Tissues (0.38cm2) lot number: 19-EKIN-035
- Maintenance Medium lot number: 19-MAIN3-038
- Assay Medium lot number: 19-ESSC-036

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
- 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.
- 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 turned blue or purple, the test item was 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 MTT solution containing the test item turned a brown color as opposed to blue/purple. Even though the solution did not blue/purple, the darkening to brown indicated that the test item may have the potential to directly reduce MTT and corrective procedures should be undertaken.
- 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 prior to the study 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 a minimum of 48 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-labeled micro tubes and stored in a freezer at -35 to -10 °C 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 EPISKINT 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 2 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-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
- Servicing, calibration, pass band width and linearity range of the microplate reader is given in Annex 2 (attached).

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 OD570 of test item / mean OD570 of negative control) x 100.

ACCEPTANCE 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 OD570 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.
- Labtech LT-4500 microplate reader and LT-com analysis software.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL of test item

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test

Value:

56.4

Vehicle controls validity:

not examined

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
- The MTT solution containing the test item turned a brown color as opposed to blue/purple. Even though the solution did not turn blue/purple, the darkening to brown indicated that corrective procedures should be undertaken to avoid the possibility of a false negative result.
- An additional procedure was performed using water-killed tissues. However, the results obtained showed that no 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 colourless.
- It was therefore unnecessary to run colour correction tissues.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- The individual and mean OD570 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 56.4 % 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.

ACCEPTANCE CRITERIA
- The test was repeated due to a failure to meet the assay acceptance criteria.
- The relative mean tissue viability for the positive control treated tissues was 5.4 % 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 OD570 for the negative control treated tissues was 0.738 and the standard deviation value of the viability was 2.6 %. 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 7.3 %. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

Relative to the concurrently treated negative control, the viability of the test item treated tissues was 56.4 %.

Executive summary:

GUIDELINE

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. 640/2012, of 06 July 2012, 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. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to have the possibility to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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-labeled 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 570 nm. Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

The results obtained showed that no interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the water-killed for quantitative correction of results or for reporting purposes. The relative mean viability of the test item treated tissues was 56.4 % after the 15-minute exposure period 42-hour post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

CONCLUSION

Relative to the concurrently treated negative control, the viability of the test item treated tissues was 56.4 %.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2019 to 26 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 12-Nousan-8147

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: New Zealand albino

Details on test animals or test system and environmental conditions:

ANIMAL INFORMATION
- Sex: Female, nulliparous and non-pregnant.
- Age: Young adult (14-5 weeks)
- Bodyweight: 2193-2662 grains at start of study.
- Source: Received from Robinson Services, Inc on 27 November 2019 and 11 December 2019.

HUSBANDRY
- Housing: The animals were singly housed in suspended stainless steel caging, which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.
- Animal Room Temperature was 19 to 23 °C and relative humidity was 38 to 69 %.
- Animal Room Air Changes: 12 per hour. Airflow measurements were evaluated regularly and the records kept on file at Product Safety Labs.
- Photoperiod: 12-hour light/dark cycle.
- Acclimation Period: 5 or 13 days.
- Food: Certified RSI 5025 High Fiber Rabbit Diet (Rowe Nutrition, LLC). A designated amount of diet (approximately 150 grams/day) and Alfalfa Timothy Hay Cubes (Standlee Premium Western Forage) were available to each rabbit.
- Water: Filtered tap water was supplied ad libitum.
- Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of the study. Analyses of the food and water were conducted regularly and the records kept on file at Product Safety Labs.

IDENTIFICATION
- Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
- Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal. This number, together with a sequential animal number assigned to study 51953, constituted unique identification. Only the sequential animal number was presented in the report.

PREPARATION AND SELECTION OF ANIMALS
- On the day before application, a group of animals was prepared by clipping the dorsal area of the trunk. On the day of dosing, but prior to application, the animals were examined for health and the skin checked for any abnormalities. Three healthy, naive animals (not previously tested) without pre-existing skin irritation were selected for test.
- Initially, only one rabbit was placed on test. Since no significant dermal irritation was observed at the dose site on this animal, the test was completed with two additional rabbits.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

- Exposure time of 3 minutes, 1 hour and 4 hours in the first animal
- Exposure time of 4 hours in two additional animals

Observation period:

10 days

Number of animals:

Three

Details on study design:

PREPARATION OF TEST SUBSTANCE
- The test substance was applied as received and mixed well prior to use.
- The pH was determined for the test substance prior to the application and was within a pH range of 2 and 11.5, therefore testing proceeded. The procedure used and the results were retained in the raw data.

APPLICATION OF TEST SUBSTANCE
- Initial testing was conducted on one rabbit to determine the irritation/corrosion potential of the test substance.
- Three dose sites, each approximately 6 cm2, were delineated on this rabbit.
- Test substance (0.5 mL) was applied to the skin of each dose site and covered with a 1 x 1 inch, 4-ply gauze pad. The pads and entire trunk of the animal were then wrapped with semi-occlusive 3-inch Micropore tape to avoid dislocation of the patches.
- An Elizabethan collar was placed on the rabbit after removal of the 3-minute patch and it was returned to its designated cage.
- The pads were removed at the appropriate intervals (3 minutes, 1 hour, and 4 hours).
- All dose sites were evaluated for corrosion 30 to 60 minutes after patch removal.
- Subsequent evaluations were performed approximately 24, 48, 72 hours and/or 7 and 10 days after removal of the four-hour patch.
- Since there was no corrosion observed at any of the dose sites for this animal, two additional rabbits (each with one patch only for a 4-hour exposure period), were selected for further testing and treated in the same manner as described above.
- After the 3-minute and 1-hour exposure periods, the pads were removed and the dose sites were
cleansed with a 3 % soap solution followed by tap water and a clean paper towel to remove any
residual test substance.
- After the 4-hour exposure period, the pads and collars were removed and the dose sites were
cleansed with a 3 % soap solution followed by tap water and a clean paper towel to remove any
residual test substance.

EVALUATION OF DOSE SITES
- Individual dose sites were scored according to the Draize scoring system (Draize et al., 1944: see
Table 6, below) at approximately 30 to 60 minutes, 24, 48, and 72 hours and/or 7 and 10 days after patch removal in addition to evaluation for corrosion at these intervals for the first animal only.
- The classification of irritancy was obtained by adding the average erythema and edema scores for the 30 to 60 minute. 24, 48, and 72-hour scoring intervals and dividing by the number of evaluation intervals.
- The resulting Primary Dermal Irritation Index (PDII) was classified as: PDII = 0 (non-irritating); PDII > 0 to 2.0 (slightly irritating); PDII = 2.1 to 5.0 (moderately irritating); PDII > 5.0 (severely irritating).

IN-LIFE OBSERVATIONS
- The animals were observed for signs of gross toxicity and behavioural changes at least once daily during the test period.

BODY WEIGHTS
- Individual weights of animals were recorded shortly before application of the test substance (initial) and at the completion of testing (terminal).

STUDY TERMINATION
- Once testing was complete, the animals were released for euthanasia and humanely euthanised.

STATISTICAL ANALYSIS
- Statistical analysis was limited to the calculation of the mean irritation scores.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3501 (female)

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: animal 3502 (female)

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: animal 3503 (female)

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: animal 3501 (female)

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: animal 3502 (female)

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: animal 3503 (female)

Irritant / corrosive response data:

RESULTS
- Individual body weights are presented in Table 1 (attached).
- Individual in-life observations are presented in Table 2 (attached).
- Individual skin corrosion and irritation scores are presented in Tables 3 and 4 (attached).
- A summary of primary skin irritation scores used for calculation of Primary Dermal Irritation Index is presented in Table 5 (attached).
- The Draize Primary Skin Irritation Scoring System is presented in Table 6 (above).
- All animals appeared active and healthy and gained body weight during the study.
- Apart from the dermal irritation noted below, there were no other signs of gross toxicity, adverse clinical effects, or abnormal behaviour.
- No visible necrosis (corrosion) of the skin tissue was observed at the 3-minute, 1-hour and 4-hour dose sites during the study.
- Three minute exposure site: Within 30 to 60 minutes of patch removal, very slight erythema and very slight edema was noted at the 3-minute exposure site, which resolved by 24 hours.
- One hour exposure site: Within 30 to 60 minutes of patch removal, very slight erytherna was noted at the 1-hour exposure site, which resolved by 48 hours.
- Four hour exposure site: Within 24 hours of patch removal, all three treated sites exhibited well-defined erythema and/or very slight edema. The overall incidence and severity of irritation decreased gradually with time. Due to desquamation noted at the dose sites on Day 7, scoring continued until Day 10 (study termination).
- The Primary Dermal Irritation Index for the test item was determined to be 1.7.

Interpretation of results:

GHS criteria not met

Conclusions:

The Primary Dermal Irritation Index (PDII) calculated for the test substance was 1.7 after an exposure time of 4 hours. However, the reported individual mean scores for erythema and edema do not cause the test item to be classified as corrosive or irritating to skin under classification rules applicable in the EU.

Executive summary:

GUIDELINE

A primary skin irritation test was conducted with rabbits to determine the potential for the test item to produce irritation after a single topical application. The study was performed in accordance with US EPA Health Effects Test Guidelines, OPPTS 870.2500 (1998), OECD Guidelines for Testing of Chemicals, Test No. 404 (2015), JMAFF 12-Nousan-8147 (2000) and the Official Journal of the European Union, Methods for the Determination of Toxicity and Other Health Effects, Part B.4 (Dermal Irritation/Corrosion), Council Regulation (EC) No. 440/2008.

 

METHODS

Initially, one rabbit was tested to determine the irritation/corrosion potential of the test substance. Three dose sites, each approximately 6 cm2, were delineated on this animal. The neat test substance (0.5 mL) was applied to the skin of each dose site. The pads and entire trunk of the animal were then wrapped with semi-occlusive 3-inch Micropore tape to avoid dislocation of the patches. The patches were removed at the appropriate intervals (3 minutes, 1 hour, and 4 hours). All dose sites were evaluated for skin irritation according to the Draize method of scoring and for corrosion 30 to 60 minutes after the appropriate patch removal. Subsequent evaluations were performed approximately 24, 48 and 72 hours after removal of the four-hour patch. Since there was no corrosion observed at any of the dose sites for this animal, two additional rabbits were selected for testing and had the test substance applied for four hours only. All dose sites were evaluated for skin irritation according to the Draize scoring system at 30 to 60 minutes plus 24, 48, 72 hours and at 7 and 10 days after patch removal.

 

RESULTS

No visible necrosis (corrosion) of the skin tissue was observed at the 3-minute, 1-hour and 4-hour

dose sites during the study. Within 30 to 60 minutes of patch removal, very slight erythema and very slight edema was noted at the 3-minute exposure site, which resolved by 24 hours. Within 30-60 minutes of patch removal, very slight erythema was noted at the I-hour exposure site, which resolved by 48 hours. Within 24 hours of patch removal, all three treated sites exhibited well-defined erythema and/or very slight edema. The overall incidence and severity of irritation decreased gradually with time. Due to desquamation noted at the dose sites on Day 7, scoring continued until Day 10 (study termination).

 

CONCLUSION

The Primary Dermal Irritation Index (PDII) calculated for the test substance was 1.7 after an exposure time of 4 hours. However, the reported individual mean scores for erythema and edema do not cause the test item to be classified as corrosive or irritating to skin under classification rules applicable in the EU.

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:

16 July 2019

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)

Version / remarks:

09 October 2017

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)

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).
- Eyes were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL test item

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three replicates

Details on study design:

PURPOSE OF THE TEST
- The Bovine Corneal Opacity and Permeability (BCOP) test is designed to identify test items that can induce serious eye damage (UN GHS Category 1) and to identify test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) using the corneas of eyes isolated from cattle. It is not recommended for the identification of test chemicals that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test chemicals that should be classified as mildly irritating to eyes (UN GHS Category 2B). The 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, corneal effects produced by the test item are assessed by quantitative measurements of changes in corneal opacity and permeability. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements are used to calculate an In Vitro Irritancy Score (IVIS), which is used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of a test item.
- The test method is based on the ICCVAM (2010). ICCVAM Recommended BCOP Test Method Protocol and the ICCVAM (2010). ICCVAM Test Method Evaluation Report which was initially developed from the INVITTOX protocol, 124. The INVITTOX protocol, 124 was based on the BCOP test method first reported by Gautheron et al (1992) and has been superseded by INVITTOX (2009) Protocol 127. The ocular irritancy potential of a test item is measured by its ability to induce opacity and increase permeability in an isolated bovine cornea.
- The effects are measured by decreased light transmission through the cornea (opacity), increased passage of sodium fluorescein dye through the cornea (permeability) and evaluation of fixed and sectioned cornea at the light microscopic level, if applicable.
- The opacity and permeability assessments of the cornea following exposure to a test item are considered individually, and also combined, to derive an In Vitro Irritancy Score, which is used to classify the irritancy level of the test item. Histological evaluation can be useful for identifying injury to tissue layers that do not result in significant opacity or increased permeability of the cornea or when a more complete characterization of corneal damage is needed. Collection of histological data will be conducted in accordance with current OECD guidance on the collection of eye tissues for histological evaluations and collection of data (OECD, 2018).

REFERENCE ITEM PREPARATION
- The negative control item, sodium chloride 0.9% w/v, was used as supplied.
- The positive control item, ethanol, was used as supplied.

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 65 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 2, 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 ºC 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.
- Media (360 μL) representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate.
- The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 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 * 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 during the previous 12 months for the testing facility.
- 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 calculated from the previous 12 months data for the testing facility. The applicable data range is presented in Appendix 3 (attached).

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
- Labtech LT-4500 microplate reader and LT-com software.

Irritation parameter:

in vitro irritation score

Run / experiment:

Main Test

Value:

10.2

Vehicle controls validity:

not examined

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 had cloudy patches 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 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- Assay acceptance criteria are given in Appendix 3 (attached).
- The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity and permeability values below the established upper limits. 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 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control. Under the experimental conditions reported, no prediction of eye irritation could be made.

Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008.

 

METHODS

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).

 

RESULTS

In Vitro Irritancy Scores were reported as 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control.

CONCLUSION

Under the experimental conditions reported, no prediction of eye irritation could be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 October 2019 to 24 October 2019

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)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

EPIOCULAR HUMAN CORNEAL MODEL (0.6 cm2)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Date received: 22 October 2019
- Epiocular tissues lot number: 30631
- Assay medium lot number: 102119ISA

MTT SOLUTION
- MTT concentrate and MTT diluent were supplied as an MTT test kit (MTT-100).
- An MTT solution was prepared when required.
- The MTT concentrate was diluted in MTT diluent (2 mL of concentrate to 8 mL diluent) to produce a 1.0 mg/mL MTT solution and used within 1 hour.

MISCELLANEOUS ASSAY REAGENTS
- DPBS (without Ca++ Mg++) Lot Number 2026759
- Isopropanol (MTT) extractant) Lot Number 1672280

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 replicates

Details on study design:

PURPOSE OF STUDY
- The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.
- The potential for chemically induced serious eye damage/eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. According to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), test chemicals inducing serious eye damage are classified as Category 1, while those inducing eye irritation are classified as Category 2. Chemicals that do not meet the requirements for classification as UN GHS Category 1 or 2 are referred to as UN GHS No Category. The EpiOcular EIT is an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category). The results of the test cannot be used in isolation to differentiate between eye irritation/reversible effects to the eye (UN GHS Category 2) and serious eye damage/irreversible effects to the eye (UN GHS Category 1).
- 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. The EpiOcular tissue mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular tissues are cultured on specially prepared cell culture inserts with a porous synthetic membrane through which nutrients can pass to the cells.
- The method directly measures cytotoxicity resulting from penetration of the chemical, applied topically, through the cornea and production of cell and tissue damage following chemical exposure. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison to negative controls is used to predict the eye hazard potential of the test chemical.
- The method is based on 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; 02 October 2017.
- The EpiOcular EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST ITEM
- A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. Therefore, it was necessary to assess this ability of the test item to directly reduce MTT prior to conducting the assay. This property of the test item is only a problem, if at the time of the MTT test (after the test item has been rinsed off) 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 by the procedure described.
- Test item (50 μL) was added to 1 mL of MTT solution and incubated at 37 °C, 5 % CO2 for three hours. A control (50 μL sterile water in MTT solution) was run concurrently. If the MTT solution turned blue/purple, the test item was presumed to have directly reduced the MTT.
- The MTT solution containing the test item turned a brown color as opposed to blue/purple. Therefore, even though the solution did not turn blue/purple (indicative of a direct MTT reducer), the darkening to brown indicated that the test item may have the potential to directly reduce MTT and corrective procedures should be undertaken to avoid a false negative result. 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 eye irritation potential was performed in parallel on viable and freeze-killed tissues.
- The following step employs freeze-killed tissues that possess negligible metabolic activity but absorb and bind the test item similar to viable tissues.
- Freeze-killed tissues were prepared in-house (outside of the confines of the study) by placing untreated EpiOcular tissues in a freezer (-35 to -10 °C) overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once killed, the tissues may be stored indefinitely in the freezer. Freeze-killed tissues were thawed for approximately 60 minutes at 37 ± 2 °C, 5 ± 1 % CO2 in air before use.
- Each MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues remained untreated (the untreated controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay was performed on the frozen tissues in parallel to the viable tissues.
- If the interference by the test item was ≤ 60 % of the negative control value, the net OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- Data were corrected using the equation True viability = Viability of treated tissue – direct MTT interference from test item = OD tvt – (mean OD tkt – mean OD ukt) where OD = optical Density at 570 nm; tvt = treated viable tissues; tkt = treated killed tissues; ukt = untreated killed tissues.
- If the interference by the test item was greater than 60 % of the negative control value the test item may be considered incompatible with this test system.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- Coloured test items or those which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colorant properties.
- Test items which absorb light and appear red, yellow, green or blue should be considered as intrinsic colorants. A test item which appears black may absorb light and should be considered as a colorant. Blue, purple and black test items may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue/purple MTT product. Such test items should also be tested on killed controls because it may not be possible to assess their potential to directly reduce MTT.
- For non-coloured test items additional tests have to be performed to assess if they become colorants after contact with water or isopropanol. As the test item was a light yellow color as opposed to a strong yellow color it was treated as a non-colored substance. For this purpose 50 μL of the test item was added to 1.0 mL of water in a 6-well plate and the mixture is incubated in the dark at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air for at least 1 hour. Furthermore, 50 μL of the liquid test item was added to 2 mL of isopropanol, the same amount as used for MTT extraction, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature.

PREPARATION AND PRE-INCUBATION OF EPIOCULAR TISSUES
- Upon receipt of the EpiOcular tissues, the sealed 24-well plate and the assay medium were placed into the refrigerator (2 to 10 °C) until the equilibration step. The vial containing the MTT concentrate was placed in the freezer (-35 to -10 °C) and the MTT diluent placed in the refrigerator (2 to 10 °C). The positive control, Methyl Acetate, was stored at room temperature, in the dark.
- On the day of receipt the equilibration step (15 minutes at room temperature in the 24-well shipping container) was started. An appropriate volume of EpiOcular Assay medium was warmed to approximately 37 °C and 1 mL of the medium aliquoted 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 ethanol-soaked tissue paper. The sterile gauze was removed and each tissue 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 container 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 1 hour in Assay Medium. After 1 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 (16 to 24 hours).

APPLICATION OF TEST ITEM AND RINSING
- After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5 % CO2 for 30 ± 2 minutes.
- Test item (50 μL) was applied atop duplicate cultures for an exposure period of 30 ±2 minutes at 37 °C, 5 % CO2 followed by rinsing, a post-treatment immersion and a post-treatment incubation (described below). Negative and positive controls (50 μL) were similarly applied.
- At the end of the test item exposure period, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS at room temperature. Three clean beakers (glass or plastic with minimum 150 mL capacity), containing a minimum of 100 mL each of Ca++ Mg++ free DPBS were used per test item or control with each test item or control item utilizing a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. 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 paper towel 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 inserts were 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 paper. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent paper (to break the surface tension).
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labelled 12-well plate for a 12 ± 2 minutes immersion incubation (post‐treatment immersion) 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‐treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 120 ± 15 minutes at 37 °C, 5 % CO2 (post-treatment incubation).

MTT ASSAY
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were placed into the 24-well plate containing 0.3 mL of 1.0 mg/mL MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated at 37 °C, 5 % CO2 in air for 3 hours.
- Each insert was removed from the 24‐well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and transferred to a pre-labelled 24‐well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol flowed into the insert on the tissue surface. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) or a standard plate sealer and stored overnight at 2 to 10 °C in the dark. At the end of the extraction period, each tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.

ABSORBANCE / OPTICAL DENSITY MEASUREMENTS
- At the end of the extraction period, using a pipette fitted with a 1000 μL tip, the extraction solution was forced vigorously up and down to thoroughly mix. The tissues and empty inserts were discarded.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. 200 μL of isopropanol alone was added to eight wells designated as ‘blanks’. All wells were examined and any air bubbles were removed. The absorbance at 570 nm (OD570) of each well was measured using the LabTech LT-4500 microplate reader and LT-com analysis software. Servicing, calibration, pass band width and linearity range of the microplate reader is given in Annex 2 (attached).
- The plate reader LT-com analysis software was set to correct for blanks and calculate the mean OD570 values of the duplicate wells representing each tissue. The mean OD570 values of the duplicate tissues were manually calculated.

DATA EVALUATION
- The relative mean tissue viabilities were compared to the mean of the negative control (n=2) treated tissues.
- The relative mean tissue viabilities were calculated using the equation Relative mean tissue viability = (mean OD570 of test item / mean OD570 of negative control) * 100

ACCEPTABILITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) The negative control OD is > 0.8 and < 2.8.
(ii) The mean relative viability of the positive control is below 50 % of the negative control viability for either the 30 minutes or 6 hours exposures.
(iii) 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 killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
- Labtech LT-4500 microplate reader and LT-com analysis software.

Irritation parameter:

in vitro irritation score

Run / experiment:

Main Test

Value:

93.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: results expressed as relative mean viability after correction for direct MTT reduction

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The MTT solution containing the test item turned a brown colour as opposed to blue/purple. Therefore, even though the solution did not turn blue/purple (indicative of a direct MTT reducer), the darkening to brown indicated that the test item may have the potential to directly reduce MTT and corrective procedures should be undertaken to avoid a false negative result. Therefore, an additional procedure using freeze-killed tissues was performed. The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

ASSESSMENT OF COLOUR INTERFERENCE WITH MTT ENDPOINT
- As the test item was a light yellow colour, as opposed to a strong yellow colour, it was treated as a non-coloured substance.
The water and isopropanol solutions were colourless. It was therefore unnecessary to run color correction tissues.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- The individual and mean OD570 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 (attached).
- The relative mean viability of the test item treated tissues, corrected for direct MTT reduction, was 93.4% after a 30-Minute exposure period and 2-Hour post-exposure incubation period.
ACCEPTANCE CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 42.6 % relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
- The mean OD570 for the negative control treated tissues was 2.200. The negative control acceptance criterion was therefore satisfied.
- The difference in viability between the two relating tissues in each treatment group was < 20 %. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test item treated tissues, corrected for direct MTT reduction, was 93.4%. The criteria required for acceptance of results in the test were satisfied. Under the experimental conditions reported, the test item was classified as non-irritant.

Executive summary:

GUIDELINE

The purpose of the study is to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

METHODS

Duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120 minutes. It was determined that the test item may have the potential to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). 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, corrected for direct MTT reduction, was 93.4%. The criteria required for acceptance of results in the test were satisfied.

 

CONCLUSION

Under the experimental conditions reported, the test item was classified as non-irritant.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2019 to 20 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 12-Nousan-8147

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: New Zealand

Remarks:

albino

Details on test animals or tissues and environmental conditions:

ANIMALS
- Source: Received from Robinson Services Inc. on 27 November 2019 and 11 December 2019.
- Number of animals: 3
- Sex: Female. All animals assigned to test were nulliparous and non-pregnant.
- Age: Young adult (l4-16 weeks) at experimental start.
- Body Weight: 2355 to 2847 g at experimental start.

HUSBANDRY
- Housing: The animals were singly housed in suspended stainless steel caging, which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.
- Animal Room Temperature was 19 to 23 °C and relative humidity was 38 to 46 %.
- Animal Room Air Changes: 12 per hour. Airflow measurements were evaluated regularly and the records kept on file at Product Safety Labs.
- Photoperiod: 12-hour light/dark cycle.
- Acclimation Period: 6 or 20 days.
- Food: Certified RSI 5025 High Fiber Rabbit Diet (Rowe Nutrition, LLC). A designated amount of diet (approximately 150 grams/day) and Alfalfa Timothy Hay Cubes (Standlee Premium Western Forage) were available to each rabbit.
- Water: Filtered tap water was supplied ad libitum.
- Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of the study. Analyses of the food and water were conducted regularly and the records kept on file at Product Safety Labs.

IDENTIFICATION
- Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
- Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal. This number, together with a sequential animal number assigned to study 51952, constituted unique identification. Only the sequential animal number was presented in the report.

PREPARATION AND SELECTION OF ANIMALS
- Prior to test initiation, both eyes of a group of animals were examined using a white light source and a fluorescein dye procedure. One drop of ophthalmic fluorescein sodium dye was instilled into both eyes of each rabbit. The eyes were rinsed with physiological saline (0.9% NaCI) after instillation of the fluorescein and then evaluated for corneal damage using an ultraviolet light source. Prior to test substance instillation, the eyes were re-examined and scored for abnormalities according to the “Scale for Scoring Ocular Lesions” (Draize et al., 1944; see Table 4, attached). Three healthy, naive animals (not previously tested) without pre-existing ocular irritation were selected for test.
- A systemic analgesic (buprenorphine SR) was administered to relieve potential discomfort associated with eye irritation which provides therapeutic relief for periods of up to 76 hours. Prior to test substance instillation, 0.1 mg/kg of body weight of the analgesic was administered to the animals and at appropriate intervals to maintain therapeutic blood levels.

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated left eye served as control

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

Single instillation

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three animals

Details on study design:

PREPARATION OF TEST SUBSTANCE
- The test substance was applied as received and mixed well prior to use.
- The pH was determined for the test substance prior to the application and was within a pH range of 2 and 11.5, therefore testing proceeded. The procedure used and the results were retained in the raw data.

INSTILLATION
- Prior to instillation, 1-2 drops of ocular anaesthetic (tetracaine hydrochloride ophthalmic solution LSP, 0.5%) were placed into both the treated and control eye of each animal.
- Test substance (0.1 mL) was then instilled into the conjunctival sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were gently held together for about one second before releasing to minimise loss of the test substance.
- The other eye of each rabbit remained untreated with the test substance and served as a control.
- The rabbits were then returned to their designated cages.

OCULAR SCORING
- Ocular irritation was evaluated using a white light source in accordance with the Draize method of scoring (Draize et al., 1944; see Table 4, attached) at 1, 24, 48, and 72 hours post-instillation.
- The fluorescein dye evaluation procedure described previously was used in the treated eye at 24 hours and as needed at subsequent scoring intervals to evaluate the extent of corneal damage or to verify reversal of effects.
- Individual scores were recorded for each animal.
- In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted. The average score for all rabbits at each scoring period was calculated to aid in data interpretation.

CLASSIFICATION OF EYE SCORES
- The time interval with the highest mean score (Maximum Mean Total Score - MMTS) for all rabbits was used to classify the test substance by the system of Kay and Calandra (Kay & Calandra, 1962) described in Table 5 (attached).

IN-LIFE OBSERVATIONS
- The animals were observed for signs of gross toxicity and behavioural changes at least once daily during the test period.

BODY WEIGHTS
- Individual weights of animals were recorded shortly before instillation of the test substance (initial) and at the completion of testing (terminal).

STUDY TERMINATION
- Once testing was complete, the animals were released for euthanasia and humanely euthanised.

STATISTICAL ANALYSIS
- Statistical analysis was limited to the calculation of the mean irritation scores.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: animal 3401 (female)

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: animal 3402 (female)

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3403 (female)

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: animal 3401 (female)

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: animal 3402 (female)

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: animal 3403 (female)

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3401 (female)

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3402 (female)

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3403 (female)

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3401 (female)

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3402 (female)

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: animal 3403 (female)

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, the test substance was determined to be mildly irritating to the eye according to a Kay and Calandra classification scheme. However, the reported individual mean scores for corneal opacity, iritis, conjunctival redness and chemosis do not cause the test item to be classified as causing serious eye damage or eye irritation under classification rules applicable in the EU.

Executive summary:

GUIDELINE

The study was performed in accordance with US EPA Health Effects Test Guidelines, OPPTS 870.2400 (1998), OECD Guidelines for the Testing of Chemicals, Test No. 405 (2017), JMAFF 12-Nousan-8147 (2000), the Official Journal of the European Union, Methods for the Determination of Toxicity and Other Health Effects, Part B.5 (Acute Eye Irritation/Corrosion), Commission Regulation (EU) No. 2017/735 amending Regulation (EC) No. 440/2008.

 

METHODS

A primary eye irritation test was conducted with rabbits to determine the potential for the test item to produce irritation from a single instillation via the ocular route. Test substance (0.1 mL) was instilled into the right eye of three healthy rabbits. The left eye remained untreated and served as a control. Ocular irritation was evaluated by the Draize method of scoring.

 

RESULTS

Within one hour after test substance instillation, all three treated eyes exhibited positive conjunctivitis. By 24 hours, corneal opacity developed in one treated eye. There was no iritis observed in any treated eye during the study. The overall incidence and severity of irritation decreased with time. All animals were free of ocular irritation by study termination at 72 hours.

 

CONCLUSION

Under the conditions of the study, the test substance was determined to be mildly irritating to the eye according to a Kay and Calandra classification scheme. However, the reported individual mean scores for corneal opacity, iritis, conjunctival redness and chemosis do not cause the test item to be classified as causing serious eye damage or eye irritation under classification rules applicable in the EU.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion in vitro

A key 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 (29 July 2016) and Method B.40bis 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. Negative and positive control groups were treated for each exposure period. It was determined that the test item may have the potential 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 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 of 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-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data were 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), 3.3 % (positive control) and 93.6 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 2.5 % (positive control) and 47.0 % (test item).

 

Skin irritation in vitro

A key 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. 640/2012, of 06 July 2012, 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. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to have the possibility to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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-labeled 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 570 nm. Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The results obtained showed that no interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the water-killed for quantitative correction of results or for reporting purposes. The relative mean viability of the test item treated tissues was 56.4 % after the 15-minute exposure period 42-hour post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

Skin corrosion / irritation in vivo

A key primary skin irritation test was conducted with rabbits to determine the potential for the test item to produce irritation after a single topical application. The study was performed in accordance with US EPA Health Effects Test Guidelines, OPPTS 870.2500 (1998), OECD Guidelines for Testing of Chemicals, Test No. 404 (2015), JMAFF 12-Nousan-8147 (2000) and the Official Journal of the European Union, Methods for the Determination of Toxicity and Other Health Effects, Part B.4 (Dermal Irritation/Corrosion), Council Regulation (EC) No. 440/2008.

 

Initially, one rabbit was tested to determine the irritation/corrosion potential of the test substance. Three dose sites, each approximately 6 cm2, were delineated on this animal. The neat test substance (0.5 mL) was applied to the skin of each dose site. The pads and entire trunk of the animal were then wrapped with semi-occlusive 3 inch Micropore tape to avoid dislocation of the patches. The patches were removed at the appropriate intervals (3 minutes, 1 hour, and 4 hours). All dose sites were evaluated for skin irritation according to the Draize method of scoring and for corrosion 30 to 60 minutes after the appropriate patch removal. Subsequent evaluations were performed approximately 24, 48 and 72 hours after removal of the four-hour patch. Since there was no corrosion observed at any of the dose sites for this animal, two additional rabbits were selected for testing and had the test substance applied for four hours only. All dose sites were evaluated for skin irritation according to the Draize scoring system at 30 to 60 minutes plus 24, 48, 72 hours and at 7 and 10 days after patch removal.

 

No visible necrosis (corrosion) of the skin tissue was observed at the 3-minute, 1-hour and 4-hour dose sites during the study. Within 30 to 60 minutes of patch removal, very slight erythema and very slight edema was noted at the 3-minute exposure site, which resolved by 24 hours. Within 30 to 60 minutes of patch removal, very slight erythema was noted at the I-hour exposure site, which resolved by 48 hours. Within 24 hours of patch removal, all three treated sites exhibited well-defined erythema and/or very slight edema. The overall incidence and severity of irritation decreased gradually with time. Due to desquamation noted at the dose sites on Day 7, scoring continued until Day 10 (study termination).

 

The Primary Dermal Irritation Index (PDII) calculated for the test substance was 1.7 after an exposure time of 4 hours. However, the reported individual mean scores for erythema and edema do not cause the test item to be classified as corrosive or irritating to skin under classification rules applicable in the EU.

 

Eye damage/irritation in vitro

 

BCOP assay

 

A key study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008.

 

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).

 

In Vitro Irritancy Scores were reported as 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control.

 

Human Cornea Model

 

The purpose of the key study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

 

Duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120 minutes. It was determined that the test item may have the potential to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). 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, corrected for direct MTT reduction, was 93.4%. The criteria required for acceptance of results in the test were satisfied.

 

Eye damage/irritation in vivo

 

The key study was performed in accordance with US EPA Health Effects Test Guidelines, OPPTS 870.2400 (1998), OECD Guidelines for the Testing of Chemicals, Test No. 405 (2017), JMAFF 12-Nousan-8147 (2000), the Official Journal of the European Union. Methods for the Determination of Toxicity and Other Health Effects, Part B.5 (Acute Eye Irritation/Corrosion), Commission Regulation (EU) No. 2017/735 amending Regulation (EC) No. 440/2008.

 

A primary eye irritation test was conducted with rabbits to determine the potential for the test item to produce irritation from a single instillation via the ocular route. Test substance (0.1 mL) was instilled into the right eye of three healthy rabbits. The left eye remained untreated and served as a control. Ocular irritation was evaluated by the Draize method of scoring.

 

Within one hour after test substance instillation, all three treated eyes exhibited positive conjunctivitis. By 24 hours, corneal opacity developed in one treated eye. There was no iritis observed in any treated eye during the study. The overall incidence and severity of irritation decreased with time. All animals were free of ocular irritation by study termination at 72 hours.

 

Under the conditions of the study, the test substance was determined to be mildly irritating to the eye according to a Kay and Calandra classification scheme. However, the reported individual mean scores for corneal opacity, iritis, conjunctival redness and chemosis do not cause the test item to be classified as causing serious eye damage or eye irritation under classification rules applicable in the EU.

Justification for classification or non-classification

Skin corrosion: The test item was considered to be non-corrosive to skin because cell viability was determined by OECD 431 to be≥50 % after 3 minutes and≥15 % after 60 minutes using the EpiDerm Human Skin Model. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 and subsequent amendments is therefore not required.These data were confirmed via testing of skin irritation in vivo becauseno visible necrosis of skin was observed after exposure of one rabbit for 3 minutes, 1 hour and 4 hours.

 

Skin irritation: The test item was determined to be non-irritant to skin using OECD 439 because percentage tissue viability after exposure and post-treatment incubation was found to be>50 % using the EPISKIN reconstructed human epidermis model. Classification for skin irritation in accordance with Regulation (EC) No. 1272/2008 and subsequent amendments is therefore not required.These data were confirmed via testing of skin irritation in vivobecause mean scores for erythema and edema after an exposure period of 4 hours were < 2.3 from gradings at 24, 48 and 72 hours in all threerabbits. Furthermore, any reactions were observed to fully resolve during the 10-day observation period.

Eye damage/irritation: The In Vitro Irritancy Score for the test item was found to lie in the range > 3 to ≤ 55 using the BCOP assay and, in accordance with OECD 437, no prediction of eye irritation could be made. However, test item-treated tissue viability corrected for direct MTT reduction was determined to be > 60 % relative to negative control treated tissue using the Human Cornea Model (OECD 492; 18 June 2019). Classification as an eye irritant (category 2) is therefore not required under the terms of Regulation (EC) No. 1272/2008 and subsequent amendments.These data were confirmed via testing of eye damage/irritation in vivo because mean scores from gradings at 24, 48 and 72 hours were < 1 (corneal opacity), < 1 (iritis), < 2 (conjunctival redness) and < 2 (chemosis) in all three rabbits. Furthermore, any reactions were observed to fully resolve within 72 hours observation period.