[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin corrosion in vitro: The test item was determined to be non-corrosive to skin (Epiderm human skin model; OECD 431).

 

Skin irritation in vitro: The test item was determined to be non-irritant to skin (EPISKIN reconstructed human epidermis model; OECD 439).

 

Skin irritation in vivo: The test item was determined to be non-irritant to rabbit skin (OECD 404 and EU Method B.4).

 

Eye damage/irritation in vitro: The test item was determined to be non-irritant (BCOP assay; OECD 437).

 

Eye damage/irritation in vivo: The test item was determined to be non-irritant to the rabbit eye (OECD 405 and EU Method B.5).

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:

13 July 2016 to 15 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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.
- MTT solution (1.0 mg/mL) was prepared from a MatTek MTT-100 kit immediately prior to use.

EPIDERM RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: MatTek
- Date received: 12 July 2016
- EpiDerm Tissues (0.63 cm2) lot number: 23345
- Assay medium lot number: 080716ZSB
- Upon receipt of the EpiDerm tissues, the sealed 24-well plate was stored in a refrigerator until use.

PRE-TEST PROCEDURE - MTT DYE METABOLISM CELL VIABILITY ASSAY
- The MTT assay is a colorimetric method of determining cell viability and is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan dye by mitrochondrial 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.

PRE-TEST PROCEDURE - TEST FOR DIRECT MTT REDUCTION
- To identify possible interference, the test item was checked for ability to directly reduce MTT.
- Test material (50 µL) was added to 1 mL of a freshly prepared 1.0 mg/L MTT solution.
- The solution was incubated in the dark at 37 °C for 60 minutes in an atmosphere of 5 % CO2 in air.
- Untreated MTT solution was tested concurrently 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.

PRE-TEST PROCEDURE - 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 for 60 minutes in an atmosphere of 5 % CO2 in air. A visual inspection of the colour was then made.

MAIN TEST – PRE-INCUBATION
- The assay medium was pre-warmed before use.
- Assay medium (0.9 mL) was pipetted into the appropriate wells of two pre-labelled 6-well plates for 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.

MAIN TEST – 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 after chemical exposure and MTT loading.
- Another 24-well plate was prepared for the MTT loading.
- Pre-warmed assay medium (300 µL) was dispensed into each well of the holding plate and the plate was placed into the incubator until required.
- MTT medium (300 µL) was dispensed into each well of the MTT loading plate and the plate was 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 whilst 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. Tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue was subjected to an equal exposure time. Test item (50 µL) and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60-minute exposure period.
- When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure period was so short the tissues were dosed at regular intervals to ensure that each tissue received equal exposure 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. The tissues were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for three hours.
- The same rinsing and MTT loading procedure was repeated after the 60-minute exposure period was complete.
- After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. The MTT extractant (isopropanol, 2 mL) was used to completely immerse each insert and the plate was covered with plate sealer to prevent isopropanol evaporation. The plates were allowed to stand overnight at room temperature to allow evaporation 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 of the extract (3 x 200 µL) were transferred to the appropriate wells of a pre-labelled 96-well plate.
- Isopropanol alone (200 µL) was added to the three wells designated as blanks.
- Absorbance at 562 nm (OD562) was measured for each well using an Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3-minute and 60-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.
- 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
- Results of the assay were considered acceptable if the assay acceptance criteria for negative control, positive control and coefficient of variation were achieved.
- 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 storage 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.
- Positive control: Potassium hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criteria if mean relative tissue viability of the 60-minute positive control is < 15 %.
- Coefficient of variation: In the range 20 to 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 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:

98.7

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:

103.2

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 result was taken to indicate that 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 result 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).
- Relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- Mean OD562 for the negative control treated tissues was 1.773 for the 3-minute exposure period and mean OD562 for the negative control treated tissues was 1.727 for the 60-minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Relative mean tissue viability for the positive control treated tissues was 4.8 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criteria 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 minutes	100*	5.2	98.7
60 minutes	100*	4.8	103.2

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

Interpretation of results:

GHS criteria not met

Remarks:

see classification criteria given in Appendix 2 (attached)

Conclusions:

Relative mean viability of cells after exposure to test item was 98.7 % at 3 minutes and 103.2 % at 60 minutes.

Executive summary:

GUIDELINE

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

 

METHODS

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. 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 MTT-loading. After MTT loading, each tissue was placed in 2 mL isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm. Data were presented in the form of percentage viability (MTT reduction in the test item-treated tissues relative to negative control tissues).

 

RESULTS

For the 3-minute exposure period, relative mean cell viabilities were 100 % (negative control), 5.2 % (positive control) and 98.7 % (test item). For the 60-minute exposure period, relative mean viabilities were 100 % (negative control), 4.8 % (positive control) and 103.2 % (test item). The quality criteria for acceptance of results in the test were satisfied.

 

CONCLUSION

Relative mean viability of cells after exposure to test item was 98.7 % at 3 minutes and 103.2 % at 60 minutes.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2016 to 01 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 a single donor

Source strain:

other: adult

Vehicle:

unchanged (no vehicle)

Details on test system:

PURPOSE OF TEST
- The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction
assay.
- Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.
- The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.
- The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

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.
- MTT stock solution (3 mg/mL) was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
- A solution of hydrochloric acid (0.04 N) in isopropanol was prepared when required.

EPISKIN RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: SkinEthic Laboratories, Lyon, France
- Date received: 26 July 2016
- EpiSkin tissues (0.38 cm2) lot number: 16-EKIN-030
- Maintenance medium lot number: 16-MAIN3-049
- Assay medium lot number: 16-ESSC-031

PRE-TEST PROCEDURE - MTT SALT METABOLISM CELL VIABILITY ASSAY
- The MTT assay is a colorimetric method of determining cell viability 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.

PRE-TEST PROCEDURE - TEST FOR DIRECT MTT REDUCTION
- To identify possible interference, the test item was checked for ability to directly reduce MTT.
- Test material (10 µL) was added to 2 mL of a freshly prepared 0.3 mg/L MTT solution freshly prepared in assay medium.
- The solution was incubated in the dark at 37 °C for 3 hours in an atmosphere of 5 % CO2 in air.
- Untreated MTT solution was tested concurrently 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. Determination of skin irritation potential would then be performed in parallel on viable and water-killed tissues for quantitative correction of results.

PRE-TEST PROCEDURE – ASSESSMENT OF COLOUR INTERFERENCE WITH 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-TEST PROCEDURE – 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 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.

MAIN TEST – 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.
- Test item (10 μL; 26.3 μL/cm2) 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 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.

MAIN TEST – 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.
- Maintenance medium (1.6 mL) from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
- MTT (2 mL of a 0.3 mg/mL solution), freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper.
- The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air.
- At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry.
- A total biopsy of the epidermis was made using the 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 from the MTT-loaded tissues.

MAIN TEST – 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.
- Acidified isopropanol alone (200 μL) was added to the two wells designated as ‘blanks’.
- The optical density (OD562) was measured (quantitative viability analysis) at 562 nm (without a reference filter) using an Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- Relative mean tissue viabilities obtained after the 15-Minute exposure to the test item 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
- Results of the assay were considered acceptable if assay acceptance criteria for positive control, negative control and test item were achieved.
- 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%.
- 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%.
- 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 Management System

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL of test item (26.3 μL/cm2)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test

Value:

96.5

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 MTT ENDPOINT
- The solution containing the test item was colourless.
- It was therefore unnecessary to run colour correction tissues.

TEST ITEM, POSITIVE CONTROL 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 (attached).
- The relative mean viability of the test item treated tissues was 96.5% 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 9.3% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 0.873 and the standard deviation value of the viability was 2.1%. 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 2.4%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Remarks:

see classification criteria given in Appendix 2 (attached)

Conclusions:

The relative mean viability of the test item treated tissues was 96.5% after the 15-minute exposure period and 42-Hours post-exposure incubation period.

Executive summary:

GUIDELINE

The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt

(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46.in vitroskin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 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 were 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 96.5% after the 15-minute exposure period and 42-Hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

CONCLUSION

The relative mean viability of the test item treated tissues was 96.5% after the 15-minute exposure period and 42-Hours post-exposure incubation period.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2016 to 23 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or test system 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 3.09 or 3.78 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:

Four hours

Observation period:

72 hours

Number of animals:

Two

Details on study design:

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).
- Calculation of the primary irritation index and grading of irritancy potential using the Draize scheme is is shown in Annex 2 (attached).
- If the study director judges that irreversible alteration of dermal tissue (such as ulceration or clear necrosis) has taken place in any rabbit, the test item is considered to be corrosive to rabbit skin.

MAJOR COMPUTERISED SYSTEMS
- Delta Controls ORCAview

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: 75551 male

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: 75552 male

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: 75551 male

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: 75552 male

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 expected gain in body weight 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

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

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

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 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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

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

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

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

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

DATA EVALUATION
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
OPACITY MEASUREMENT
- The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading.
- These values were then corrected by subtracting the average change in opacity observed for the negative control
corneas.
- The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

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

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

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

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

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.

Irritation parameter:

in vitro irritation score

Value:

0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

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

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 0.1 for the test item, 1.2 for the negative control and 41.3 for the positive control.

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

Interpretation of results:

GHS criteria not met

Conclusions:

In vitro irritancy scores were reported as 0.1 for the test item, 1.2 for the negative control and 41.3 for the positive control.

Executive summary:

GUIDELINE

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

 

METHODS

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

 

RESULTS

In vitro irritancy scores were reported as 0.1 for the test item, 1.2 for the negative control and 41.3 for the positive control.

 

CONCLUSION

The in vitro irritancy score (IVIS) was determined to be ≤ 3 for the test item and, in accordance with OECD 437 (26 July 2013), no classification is required under the terms of GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 October 2016 to 15 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 3.99 or 4.16 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 night 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:

24 hours

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

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: 75539 male

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: 75539 male

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: 75539 male

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: 75539 male

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: 75591 female

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: 75591 female

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: 75591 female

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean score

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

other: 75591 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 or iridial effects were noted during the study.
- One hour after treatment, moderate conjunctival irritation was noted in both treated eyes.
- Minimal conjunctival irritation was noted at the 24 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 expected body weight gain during the study.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced individual mean scores 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 moderate conjunctival irritation. Both treated eyes appeared normal at the 72-hour observation.

 

CONCLUSION

The test item produced individual mean scores 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 adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion in vitro

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

 

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

 

For the 3-minute exposure period, relative mean cell viabilities were 100 % (negative control), 5.2 % (positive control) and 98.7 % (test item). For the 60-minute exposure period, relative mean viabilities were 100 % (negative control), 4.8 % (positive control) and 103.2 % (test item). The quality criteria for acceptance of results in the test were satisfied.

 

Skin irritation in vitro

The purpose of the key study 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. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt

(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 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-labeled 96-well plate. The optical density was measured at 562 nm. Data were 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 96.5% after the 15-minute exposure period and 42-Hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 96.5% after the 15-minute exposure period and 42-Hours post-exposure incubation period.

Skin irritation in vivo

The key 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.

 

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 animals 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 irritation in vitro

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

 

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

 

In vitro irritancy scores were reported as 0.1 for the test item, 1.2 for the negative control and 41.3 for the positive control.

 

The in vitro irritancy score (IVIS) was determined to be ≤ 3 for the test item and, in accordance with OECD 437 (26 July 2013), no classification is required under the terms of GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments.

Eye irritation in vivo

The key 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 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.

 

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72-hour observation. The test item produced individual mean scores 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: Investigation of in vitro skin corrosivity potential in accordance with OECD 431 showed that the test item was not corrosive (relative mean tissue viability ≥ 50 % after 3-minute exposure and ≥ 15 % after 60-minute exposure). Results of an in vitro test (OECD 439) showed that the test item was not irritating to skin (relative mean tissue viability > 50 % after 15-minute exposure) and an in vivo investigation showed that the test item produced individual mean scores of 0.0 for erythema and 0.0 for edema. Classification for skin irritation/corrosion is therefore not required under the terms of Regulation (EC) No 1272/2008.

Eye: Investigation of in vitro eye irritation potential in accordance with OECD 437 showed that the test item was non-irritant (IVIS ≤ 3) and, when applied to the non-irrigated right eyes of two rabbits, the test item produced individual mean scores (24/48/72 h) of 0.0 for corneal opacity, 0.0 for iritis, 0.7 for conjunctival redness and 0.3 for chemosis. Classification for eye effects is therefore not required under the terms of Regulation (EC) No 1272/2008.