Registration Dossier

Administrative data

Description of key information

Skin corrosion in vitro: The research material was determined to be non-corrosive to skin becausecell viability was determined to be 94.3 % after 3 minutes and 104.6 % after 60 minutes (OECD 431; EpiDerm Human Skin Model).

 

Skin irritation in vitro: The research material was determined to be non-irritant to skin because the corrected relative mean viability of the test item treated tissues was determined to be 107.9 % after the 15-minute exposure period and 42-hours post-exposure incubation (OECD 439; EPISKIN reconstructed human epidermis model).

 

Skin irritation in vivo: The research material was determined to be non-irritant to skin becausescores of zero were reported for erythema and edema in both animals at 24, 48 and 72 hours(OECD 404 and EU Method B.4).

 

Eye damage/irritation in vitro: The research material was determined to be non-irritantbecause the IVIS value was 0.6 (OECD 437 and EU Method B.47; BCOP assay).

 

Eye damage/irritation in vivo: The research material was determined to be non-irritant to the eye becausemean scores for both animals at 24/48/72 hours were reported as 0.0 for corneal opacity, 0.0 for iritis, 0.7 for conjunctival redness and 0.3 for chemosis and the effects that were seen fully reversed(OECD 405 and EU Method B.5).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 September 2016 to 03 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: neonatal
Vehicle:
unchanged (no vehicle)
Details on test system:
PURPOSE OF THE TEST
- The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes.
- Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
- This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

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

EPIDERM RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: MatTek
- Date received: 01 September 2016
- EpiDerm tissues (0.63 cm2) lot number: 23352
- Assay medium lot number: 082516ZSC
- Upon receipt of the Epiderm tissues, the sealed 24-well plate was stored in a refrigerator until use.

MTT DYE METABOLISM, CELL VIABILITY ASSAY
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
- As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below.
- Test item (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.

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 (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.

PRE-INCUBATION
- The assay medium was pre-warmed before use in the main test.
- Assay medium (0.9 mL) 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. Test item (50 µL) and 50 μL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. Sterile water (25 μL) was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60-minute exposure period.
- When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
- After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

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

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

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) Negative control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
(ii) Positive control: Potassium hydroxide 8.0 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
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Two
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
97.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean relative viability (% of negative control)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-minute exposure
Value:
95.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean relative viability (% of negative control)
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item did not become coloured.
- This 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 OD562 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 1.557 for the 3-Minute exposure period and 1.503 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 4.1 % 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

4.1

97.7

60 minutes

100

4.1

95.2

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

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the test item treated tissues was 97.7 % after 3 minutes exposure and 95.2 % 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 (28 July 2015) 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). The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and cytotoxicity is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

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. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 4.1 % (positive control) and 97.7 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.1 % (positive control) and 95.2 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues was 97.7 % after 3 minutes exposure and 95.2 % after 60 minutes exposure.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 to 03 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: adult
Vehicle:
unchanged (no vehicle)
Details on test system:
PURPOSE OF THE TEST
- The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 1 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The actual post-exposure incubation period was 43.75 hours and was recorded as a deviation in the study file. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period may also be determined for
test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.
- The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Following a full validation study, the 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.
- The procedure followed was based on the recommended EpiSkin SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.
- Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

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

EPISKIN RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: SkinEthic Laboratories, Lyon, France
- Date received: 27 September 2016
- EpiSkin Tissues (0.38cm2) lot number: 16-EKIN-039
- Maintenance Medium lot number: 16-MAIN3-066
- Assay Medium lot number: 16-ESSC-042

MTT SALT METABOLISM CELL VIABILITY ASSAY
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

TEST FOR DIRECT MTT REDUCTION
- As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item 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.

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 centre). 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. 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 homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °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 EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the
extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE / OPTICAL DENSITY MEASUREMENTS (DAY 6)
- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

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

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

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL
Duration of treatment / exposure:
4 hours
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test
Value:
107.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: relative mean viability of tissues after 15-minute exposure and 42-hour post-exposure incubation
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

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 OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1 (attached). The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1.
- The relative mean viability of the test item treated tissues was 107.9 % after a 15-minute exposure period and 42-hour post-exposure incubation period.
- It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

QUALITY CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 8.1 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.4 %. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 0.922 and the standard deviation value of the viability was 3.7 %. 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 11.0 %. 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 107.9 %.
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. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINT reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

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. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

The relative mean viability of the test item treated tissues was 107.9 % 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 107.9 %.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 November 2016 to 04 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW
Details on test animals and environmental conditions:
ANIMAL INFORMATION
- Two New Zealand White (Hsdlf:NZW) were supplied by Envigo RMS (UK) Limited, Leicestershire, UK.
- At the start of the study the animals weighed 4.32 or 4.49 kg and were 12 to 52 weeks old.
- After an acclimatisation period of at least 5 days each animal was given a number unique within the study. The number was written with black indelible marker pen on the inner surface of the ear and on the cage label.

ANIMAL CARE AND HUSBANDRY
- The animals were individually housed in suspended cages.
- Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon UK) was allowed throughout the study.
- The diet and drinking water were not considered to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70 % respectively.
- Lighting was controlled by a timer switch to give 12 hours continuous light and 12 hours darkness.
- The rate of air exchange was at least 15 changes per hour.
- Animals were provided with environmental enrichment items which were not considered to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
Two
Details on study design:
MEASUREMENT OF pH
- The pH of the test item was determined prior to commencement of the study and the findings are shown in the table below.

TEST SYSTEM
- On the day before the test, two rabbits were clipped free of fur on the dorsal/flank area using veterinary clippers.
- Only animals where gross observation showed healthy intact epidermis were selected for the study.
- On the day of the investigation a suitable test site was selected on the back of each rabbit.
- Test item (0.5 mL) was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch.
- The patch was secured in position with a strip of surgical adhesive tape.
- To prevent animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset.
- Animals were returned to their cages for the duration of the exposure period.
- Four hours after application the corset and patches were removed from each animal and any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water.
- Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for primary irritancy and scored according to the scale shown in the table below.
- Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

DATA EVALUATION
- Mean scores erythema/edema scores following grading at 24, 48 and 72 hours after patch removal are presented in Appendix 1 (attached).
- If the study director judges that irreversible alteration of the dermal tissue has taken place in any rabbit (including ulceration and clear necrosis or signs of scar tissue), the test item is considered to be corrosive to skin. Grading according to Draize may therefore not be applicable.

MAJOR COMPUTERISED SYSTEMS
- Delta Controls ORCAview
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: 75624 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: 75624 female
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: 75626 female
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: 75626 female
Irritant / corrosive response data:
SKIN REACTIONS
- The individual scores for erythema/eschar and edema are given in Appendix 1 (attached).
- No evidence of skin irritation was noted during the study.
Other effects:
BODY WEIGHT
- Individual body weights and body weight change are given in Appendix 2 (attached).
- Both animals showed body weight loss during the study.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item produced individual mean scores of 0.0 for erythema and 0.0 for edema.
Executive summary:

GUIDELINE

The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit in compliance with the OECD Guideline for the Testing of Chemicals No 404 “Acute Dermal Irritation/Corrosion” (adopted 28 July 2015) and Method B.4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No 440/2008.

 

METHODS

Two animals were clipped free of fur on the dorsal/flank area using veterinary clippers and 0.5 mL of test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in place with a strip of surgical adhesive tape and the trunk of each rabbit was wrapped in an elasticated corset. Four hours after application, the corset and patches were removed from each animal and any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water. Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for primary irritancy and scored.

 

RESULTS

A single 4-hour semi-occluded application of test item to the intact skin of two rabbits produced no evidence of skin irritation.

 

CONCLUSION

The test item produced individual mean scores of 0.0 for erythema and 0.0 for edema.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF BOVINE EYES
- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Excised eyes were transported to the test facility over ice packs on the same day of slaughter.
- Corneas were prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
REFERENCE ITEM PREPARATION
- The negative control item, 0.9% w/v sodium chloride solution, 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 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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

TREATMENT OF CORNEAS
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC 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 (5 mg/mL).
- The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

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

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

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

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

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

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Score = mean opacity value + (15 * 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.

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

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
Irritation parameter:
in vitro irritation score
Run / experiment:
Main test
Value:
0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
CORNEAL OPACITY AND PERMEABILITY MEASUREMENTS
- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (attached).

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

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 0.6 for the test item, 0.5 for the negative control and 48.8 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was above the range of 29.6 to 52.0. The positive control acceptance criterion was therefore not satisfied. This was reported as a deviation.
- The negative control gave opacity of ≤ 2.9 and permeability ≤ 0.103. The negative control acceptance criteria were therefore satisfied.
Interpretation of results:
GHS criteria not met
Conclusions:
In Vitro Irritancy Scores were reported as 0.6 for the test item, 0.5 for the negative control and 48.8 for the positive control.
Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

METHODS

The undiluted test item was applied for 10 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 anIn Vitro Irritancy Score (IVIS).

 

RESULTS

In Vitro Irritancy Scores were reported as 0.6 for the test item, 0.5 for the negative control and 48.8 for the positive control.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 2016 to 02 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW
Details on test animals or tissues and environmental conditions:
ANIMAL INFORMATION
- Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK.
- At the start of the study the animals weighed 2.85 or 3.41 kg and were 12 to 52 weeks old.
- After an acclimatisation period of at least 5 days each animal was given a number unique within the study which was written with black indelible marker pen on the inner surface of the ear and on the cage label.

ANIMAL CARE AND HUSBANDRY
- Animals were individually housed in suspended cages.
- Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Diet and drinking water were not considered to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70 % respectively.
- Rate of air exchange was at least 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
- Animals were provided with environmental enrichment items that were not considered to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated left eye
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
Single dose to non-irrigated left eye
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Two
Details on study design:
MEASUREMENT OF pH
- The pH of the test item was determined prior to commencement of the study and the findings are shown in the table below.

TEST SYSTEM
- Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard opthalmoscope.
- Only animals free of ocular damage were used.
- Initially, a single rabbit was treated.
- A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to the test item application to provide a therapeutic level of systemic analgesia.
- Five minutes prior to test item application, a pre-dose anaesthesia of ocular anaesthetic (two drops of 0.5 % proxymetacaine hydrochloride) was applied to each eye.
- Test item (0.1 mL) was placed into the conjunctival sac of the right eye by gently pulling the lower lid away from the eyeball.
- To prevent loss of the test item, the upper and lower eyelids were held together for about one second immediately after treatment and then released.
- The left eye remained untreated and was used for control purposes.
- An assessment of the initial pain reaction was made immediately after administration using the scoring system described in Annex 2 (attached).
- Eight hours after test item application, a subcutaneous injection of post-dose analgesia (buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg) was administered to provide a continued therapeutic level of systemic analgesia.
- The treated animal was checked for signs of pain and suffering approximately 12 hours later and no further analgesia was required.
- After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
- Assessment of ocular damage/irritation was made at approximately 1 hour plus 24, 48 and 72 following treatment according to the numerical evaluation of Draize, 1977 (see Annex 3, attached).
- Any other ocular effects were also noted.
- Examination of the eye was facilitated by the use of the light source from a standard opthalmoscope.
- If present, any clinical signs of toxicity were also recorded.
- Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

DATA EVALUATION
- The mean scores following grading at 24, 48 and 72 hours after instillation of the test item were calculated for corneal opacity, iridial inflammation, conjunctival redness and chemosis (see Appendix 2, attached).
- Interpretation according to a modified version of the system described by Kay and Calandra, 1962 (see Annex 5, attached) is presented in Annex 4 (attached).
- If evidence of irreversible ocular damage was noted, the test item would be considered to be corrosive to the eye.

MAJOR COMPUTERISED SYSTEMS
- Delta Controls ORCAview
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: 75622 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: 75634 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: 75622 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: 75634 female
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: 75622 female
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
3
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: 75634 female
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: 75622 female
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: 75634 female
Irritant / corrosive response data:
OCULAR REACTIONS
- Individual and individual total scores for ocular irritation are given in Appendix 1 (attached).
- Mean scores for corneal opacity, iridial inflammation, conjunctival redness and chemosis are given in Appendix 2 (attached).
- No corneal effects were noted during the study.
- Iridial inflammation was noted in one treated eye one hour after treatment.
- Moderate conjunctival irritation was noted in both treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour and 48-hour observations.
- Both treated eyes appeared normal at the 72-hour observation.
Other effects:
BODY WEIGHT
- Individual body weights and body weight change are given in Appendix 3 (attached).
- Both animals showed body weight loss during the study.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item produced individual mean scores in both animals of 0.0 for corneal opacity, 0.0 for iritis, 0.7 for conjunctival redness and 0.3 for chemosis.
Executive summary:

GUIDELINE

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit in compliance with the OECD Guideline for the Testing of Chemicals No 405 “Acute Eye Irritation/Corrosion” (adopted 02 October 2012) and Method B.5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No 440/2008.

 

METHODS

The test involved a single application of the test item to the non-irrigated eye of two rabbits. Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to the test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre-dose anaesthesia of ocular anaesthetic (two drops of 0.5 % proxymetacaine hydrochloride) was applied to each eye. Test item (0.1 mL) was placed into the conjunctival sac of the right eye by gently pulling the lower lid away from the eyeball. To prevent loss of the test item, the upper and lower eyelids were held together for about one second immediately after treatment and then released. The left eye remained untreated and was used for control purposes. An assessment of the initial pain reaction was made immediately after administration. Eight hours after test item application, a subcutaneous injection of post-dose analgesia (buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg) was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required. After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated. Assessment of ocular damage/irritation was made at approximately 1 hour plus 24, 48 and 72 following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard opthalmoscope. If present, any clinical signs of toxicity were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

 

RESULTS

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72-hour observation.

 

CONCLUSION

The test item produced individual mean scores in both animals of 0.0 for corneal opacity, 0.0 for iritis, 0.7 for conjunctival redness and 0.3 for chemosis.

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 (28 July 2015) 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). The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and cytotoxicity is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

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. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 4.1 % (positive control) and 97.7 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.1 % (positive control) and 95.2 % (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. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINT reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

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. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 107.9 % 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 irritation in vivo

A key study was performed to assess the irritancy potential of the research material to the skin of the New Zealand White rabbit in compliance with the OECD Guideline for the Testing of Chemicals No 404 “Acute Dermal Irritation/Corrosion” (adopted 28 July 2015) and Method B.4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No 440/2008.

Two animals were clipped free of fur on the dorsal/flank area using veterinary clippers and 0.5 mL of test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in place with a strip of surgical adhesive tape and the trunk of each rabbit was wrapped in an elasticated corset. Four hours after application, the corset and patches were removed from each animal and any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water. Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for primary irritancy and scored.

 

A single 4-hour semi-occluded application of test item to the intact skin of two rabbits produced no evidence of skin irritation. The test item produced individual mean scores of 0.0 for erythema and 0.0 for edema.

Eye damage/irritation in vitro

A key study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The undiluted research material was applied for 10 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 0.6 for the test item, 0.5 for the negative control and 48.8 for the positive control.

Eye damage/irritation in vivo

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit in compliance with the OECD Guideline for the Testing of Chemicals No 405 “Acute Eye Irritation/Corrosion” (adopted 02 October 2012) and Method B.5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No 440/2008.

The test involved a single application of the research material to the non-irrigated eye of two rabbits. Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to the test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre-dose anaesthesia of ocular anaesthetic (two drops of 0.5 % proxymetacaine hydrochloride) was applied to each eye. Test item (0.1 mL) was placed into the conjunctival sac of the right eye by gently pulling the lower lid away from the eyeball. To prevent loss of the test item, the upper and lower eyelids were held together for about one second immediately after treatment and then released. The left eye remained untreated and was used for control purposes. An assessment of the initial pain reaction was made immediately after administration. Eight hours after test item application, a subcutaneous injection of post-dose analgesia (buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg) was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required. After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated. Assessment of ocular damage/irritation was made at approximately 1 hour plus 24, 48 and 72 following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard opthalmoscope. If present, any clinical signs of toxicity were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72-hour observation. The test item produced individual mean scores in both animals of 0.0 for corneal opacity, 0.0 for iritis, 0.7 for conjunctival redness and 0.3 for chemosis.

Justification for classification or non-classification

Skin corrosion: The research material was considered to be non-corrosive to skin because cell viability was determined by OECD 431 (28 July 2015) 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 is therefore not required.

 

Skin irritation: The research material was determined by OECD 439 (28 July 2015) to be non-irritant to skin because percentage tissue viability after exposure and post-treatment incubation was found to be > 50 % using the EPISKIN reconstructed human epidermis model. These data were confirmed in vivo where scores of zero were reported for erythema and edema in both animals. Classification for skin irritation in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

Eye damage/irritation: The In Vitro Irritancy Score was determined to be3 using the BCOP assay and, in accordance with OECD 437 (26 July 2013), the research material is not classified for eye damage/irritation. These data were confirmed in vivo where mean scores for both animals at 24/48/72 hours were reported as <1 for corneal opacity, < 1 for iritis, < 2 for conjunctival redness and < 2 for chemosis and the effects that were seen fully reversed. Classification for eye damage/irritation is therefore not required under the terms of Regulation (EC) No. 1272/2008.