2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Endpoint summary

Description of key information

The test substance was not corrosive or irritating to the skin in a GLP compliant OECD 431 and 439 study.

The test substance was not irritating to the eyes in a GLP compliant OECD 437 study.

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 bis ( In vitro skin corrosion: human skin model test)

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST FOR DIRECT MTT REDUCTION
- 25 mg of the test item 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 and 5% CO2 for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
- If the MTT solution containing the test item turns purple/blue relative to the control, the test item is considered to be able to directly reduce 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.
- The test item (25 mg) was added to sterile water (300 μL). The solution was incubated in the dark at 37 °C and 5% CO2 for 60 minutes. A visual assessment of the colour was then made.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis
- Tissue batch number: 23328

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

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

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 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 before treatment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Tissues were blotted and transferred to the 24-well plate prepared for MTT loading.
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- 2 mL of MTT extract (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.
- extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks.
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562nm

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 treated with sterile distilled water.
- The relative mean viabilities were calculated by dividing the mean OD562 of test item by the mean OD562 of negative control, and multiplying this by 100.

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- 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.
- An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute exposure for the positive control is < 15%.
- In the range 20 and 100% viability, the coefficient of variation between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg
- Other: 25 μL of sterile water was added for wetting of the test item to increase tissue surface contact.

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test: 3 minute exposure

Value:

101.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test: 1 hour exposure

Value:

97.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after one hour of incubation with the test item in MTT working solution, thus the test item did not react with MTT. Therefore, additional controls and data calculations were not necessary to account for the MTT-reducing potential of the test item.
- Colour interference with MTT: The solution containing the test item was a pale yellow colour. This colour was attributed to the intrinsic colour of the test item and was considered not to have the potential to cause colour interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.112 for the 3-minute exposure period and 2.105 for the 60-minute exposure period. The negative control acceptance criteria as per the OECD guidelines was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.7% relative to the negative control following the 60-mnute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: 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.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro test according to OECD 431 and EU method B.40 bis, the test item was not found to be corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model in accordance with OECD 431 and GLP, after treatment periods of 3 and 60 minutes. The corrosion potential of a test item is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to predict the corrosivity potential of the test item. Duplicate tissues were treated with the test item (25 mg) 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 μL 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 is evaluated in the form of percentage cell viability (MTT reduction in the test item treated tissues relative to negative control tissues). The quality criteria required for acceptance of results in the test were satisfied. The percent cell viability for the test item were 101.6% and 97.7% after a 3 minute and 60 minute exposure, respectively, therefore under the conditions of this assay the test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST FOR DIRECT MTT REDUCTION
- 10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
- If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and additional controls 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.
- 10 mg of test item 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.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ reconstructed human epidermis model kit
- Tissue batch number(s): 16-EKIN-018

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 Satisfactory
- 2 mL of maintenance medium, 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)
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT solution: 0.3 mg/mL
- Incubation time: 3 hours
- MTT loading/formazan extraction (day 3): 2 mL of MTT 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. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
- Absorbance/optical density measurements (day 6): At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’.
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)

NUMBER OF REPLICATE TISSUES: 3

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).
- The relative mean viabilities were calculated by dividing the mean OD562 of test item by the mean OD562 of negative control, and multiplying this by 100.

PREDICTION MODEL
- The test substance is considered to be irritant to skin (Hazard - Code H315; Category 1 or 2) if the relative mean tissue viability after 15 minutes exposure is lower than or equal to 50%.
- The test substance is considered to be non-irritant to skin (Hazard - Code H315; Category 2; Statement “Causes Skin Irritation”) if the relative mean tissue viability after 15 minutes exposure is greater than 50%.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg (26.3 mg/cm2)
- Other: The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis.

NEGATIVE CONTROL
- Amount applied: 10 μL of DPBS

POSITIVE CONTROL
- Amount applied: 10 μL of SDS 5% w/v
- Other: To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value irritation test: 15 min exposure

Value:

127.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple. Therefore the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to use additional colour correction tissue controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.730 and the standard deviation value of the viability was 5.0%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 4.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 4.6%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro test according to OECD 439 and EU method B.46, the test item was not found to be irritant to the skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model in accordance with OECD 439 and GLP. 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 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 10 mg of 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. Tissues were concurrently treated with 5% w/v SDS (positive control) or with DPBS (negative control). At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from 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 as percentage tissue viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of test item treated tissues was 127.3% compared to the negative control. This is above the 50% threshold therefore the test item was considered to be non-irritant to the skin under the conditions of this assay. The quality criteria required for acceptance of results in the test were met therefore the study is considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Species:

cattle

Strain:

other: bovine

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Characteristics of donor animals: Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Storage, temperature and transport conditions of ocular tissue: 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). They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% w/v solution in 0.9% w/v sodium chloride solution.

VEHICLE
- Amount applied: 0.75 mL - Concentration (if solution):
- Concentration: 0.9% w/v sodium chloride solution

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
- 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. 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 vehicle control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
3

VEHICLE CONTROL USED
sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

APPLICATION DOSE AND EXPOSURE TIME
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation (20% w/v solution) 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 240 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others: 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), which was calculated as follows: IVIS = mean opacity value + (15 x mean permeability OD492 value)

DATA EVALUATION
- 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.
- 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.
- The condition of the cornea was visually assessed post treatment.

DECISION CRITERIA: according to OECD Guideline 437

CRITERIA FOR AN ACCEPTABLE TEST
For an acceptable test the following positive and negative control criterion should be achieved:
- 20% w/v Imidazole 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 66.9 to 101.4.
- 0.9% w/v sodium chloride solution 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 solids the negative control limit for opacity should be ≤4.1 and for permeability ≤0.105.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean in vitro irritancy score

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

deviation: see 'acceptence of results'

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

mean opacity

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

deviation: see 'acceptence of results'

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

mean permeability

Value:

0.005

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

deviation: see 'acceptence of results'

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was 110.7, and therefore not within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore not satisfied. However, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. This deviation was considered to have not affected the integrity or validity of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

In this BCOP test according to OECD 437 and EU Method B.47, the test item was not found to be irritant to the eye.

Executive summary:

The purpose of this GLP compliant test according to OECD 437 was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and 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 test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

750 µL of the test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Vehicle only control and positive control (20% w/v imidazole in 0.9% w/v sodium chloride solution) items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS 2.8 and 110.7 for the vehicle control and positive control, respectively. Acceptance criteria for the negative control were met. The IVIS of the positive control was higher than the criteria range set for an acceptable test. However, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. This deviation was considered to have not affected the integrity or validity of the study. The IVIS of the test item was 1.0, indicating that the requirements for classification as UN GHS/ EU CLP Category 1 or 2 are not met. In conclusion, classification for eye irritation is not required for the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

In vitro:

Skin irritation

The skin irritation potential of the test item was evaluated in a GLP compliant OECD 439 study using the EPISKIN™ reconstructed human epidermis model. Triplicate tissues were treated with 10 mg of 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 cell viability was measured by enzymatic reduction of tetrazolium salt. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of test item treated tissues was 127.3% compared to the negative control. This is above the 50% threshold therefore the test item was considered to be non-irritant to the skin under the conditions of this assay (Warren, 2017a).

Skin corrosion

The corrosion potential of a test item was evaluated in a GLP compliant OECD 431 study using the EpiDerm™ Human Skin Model. Duplicate tissues were treated with the test item (25 mg) for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before cell viability was measured by enzymatic reduction of tetrazolium salt. The quality criteria required for acceptance of results in the test were satisfied. The percent cell viability for the test item were 101.6% and 97.7% after a 3 minute and 60 minute exposure, respectively, therefore under the conditions of this assay the test item was considered to be non-corrosive to the skin (Warren, 2017b).

In vivo:

Rats were dermally exposed to the undiluted test substance under occlusive conditions. Five applications gave a total dose of 0.6 g.

Slight local irritation was observed (Moses, 1979).

Eye irritation:

In vitro:

A BCOP test method was performed according to OECD 437 and GLP to establish if the test item can induce serious eye damage or if it does not require classification for eye irritation or serious eye damage. 750 µL of the test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS of the test item was 1.0, indicating that the requirements for classification as UN GHS/ EU CLP Category 1 or 2 are not met. In conclusion, classification for eye irritation is not required for the test item (Henzell, 2017).

In vivo:

The eyes of New Zealand white rabbits were exposed to the undiluted test substance. Animals were observed for 7 days. Instillation into the eye caused moderate initial pain and slight irritation. The group mean scores after 24, 48 and 72 hours were 0 (out of 80) for the cornea, 0 (out of 10) for the iris, and 0.67 (out of 20) for the conjunctiva (fully reversible within 48 hours) (Moses, 1979).

Justification for classification or non-classification

Based on the available data on skin irritation / corrosion and eye irritation, the substance is not classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.