Reaction products of Dihydro-3-(tetrapropenyl)furan-2,5 dione with propane-1,2,diol

Endpoint summary

Description of key information

Skin Corrosion

Under the conditions of this study, the test material was determined to be non-corrosive to the skin.

Skin Irritation

Under the conditions of this study, the test material was determined to be a skin irritant.

Eye Irritation

Under the conditions of the study, the test material was found to cause serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2016 to 22 February 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)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was heated in a water bath to 37 °C, taken up into a sufficient number of syringes and allowed to cool to room temperature prior to dosing.

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes.

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

SOURCE ANIMAL
Model: EPISKIN™ 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.
- Supplier: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ reconstructed human epidermis model from SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 16-EKIN-007
- Delivery date: 16 February 2016
- 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 Satisfactory: Yes
Temperature Indicator Colour Satisfactory: Yes
Agar Medium Colour Satisfactory: Yes
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.
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):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with approximately 10 μL of the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. An infinite dose of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature.
- Temperature of post-treatment incubation: The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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 material. 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.
- Observable damage in the tissue due to washing: None specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
- MTT concentration: 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates.
- Incubation time: The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5 % CO2 in air.
At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
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: The optical density was measured (quantitative viability analysis) using the Anthos 2001 microplate reader.
- Wavelength: 562 nm
- Filter: Without a reference filter

NUMBER OF REPLICATE TISSUES: Triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
Approximately 10 mg of 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 the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. Approximately 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.

QUANTITATIVE MTT ASSESSMENT 9PERCENTAGE 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 in the following way:

Relative mean viability (%) = (mean OD562 of test item / mean OD 562 of negative control) x 100

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be an irritant to skin if the viability after 15 minutes exposure is less than, or equal to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 μL

NEGATIVE CONTROL
- Amount applied: 10 μL

POSITIVE CONTROL
- Amount applied: 10 μL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate tissues were treated with the test material and with the positive and negative controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three runs/experiments

Value:

9.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS
- Direct-MTT reduction: The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test material was a very pale yellow colour. This colour was attributed to the intrinsic color of the test item itself and was not considered to affect the MTT endpoint. It was therefore unnecessary to run colour correction tissues.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD562 for the negative control treated tissues was 0.908 and the standard deviation value of the viability was 3.5 %. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.5 %. 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 material treated tissues was 1.7 %. The test material acceptance criterion was therefore satisfied.

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Material

Material	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative Individual tissue viability (%)	Relative Mean viability (%)	± SD of Relative Mean viability (%)
Negative Control Material	0.943	0.908	0.031	103.9	100*	3.5
	0.882			97.1
	0.889			99.0
Positive Control Material	0.056	0.052	0.005	6.2	5.7	0.5
	0.053			5.8
	0.047			5.2
Test Material	0.070	0.085	0.016	7.7	9.4	1.7
	0.085			9.4
	0.101			11.1

OD = Optical Density

SD = Standard deviation

∗ = The mean viability of the negative control tissues is set at 100 %

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this study, the test material was determined to be a skin irritant.

Executive summary:

A study was performed in vitro to assess the irritancy potential of the test material. The study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material 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.

The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.5 %. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.908 and the standard deviation value of the viability was 3.5 %. 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 1.7 %. The test item acceptance criterion was therefore satisfied.

The relative mean viability of the test material treated tissues was 9.4 % which is below the threshold of 50 %, therefore under the conditions of this study the test material was determined to irritating to the skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 January 2016 to 22 January 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: Method B.40bis of Commission Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed human epidermis

Cell source:

other: EPISKIN™ reconstructed human epidermis

Source strain:

other: Not applicable

Details on animal used as source of test system:

SOURCE ANIMAL
Model: Three-dimensional reconstructed human epidermis.
- Supplier: MatTek
- Batch number: 23308
- Upon receipt of the EPISKIN™ tissues, the sealed 24-well plate was stored in a refrigerator until use.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ reconstructed human epidermis
- Tissue batch number: 23308
- Delivery date: Received 19 January 2016
- Date of initiation of testing: 21 January 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C, then at room temperature over-night.
PRE-INCUBATION
Pre-Incubation: The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted.
- Observable damage in the tissue due to washing: None specified.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 50 μL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.
- Incubation time: The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes.
After extraction, each tissue was pierced with a pipette fitted with a 1 000 μL tip and the 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

NUMBER OF REPLICATE TISSUES: Duplicate tissues

ASSESSMENT OF MTT DIRECT INTERFERENCE
- Preparation of MTT: A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to usage.
- MTT Dye Metabolism, Cell Viability Assay: The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
- Test for Direct MTT Reduction: As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT.
- A test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT.
- 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. 50 μL of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

INTERPRETATION OF RESULTS
Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x 100

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is < 50 %, or if the viability after 3 minutes exposure is ≥ 50 % and the viability after 1 hour exposure is < 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL, used as supplied.

POSITIVE CONTROL
- Amount applied: 50 μL, used as supplied.

Duration of treatment / exposure:

3 minute and 60 minute exposure periods.

Duration of post-treatment incubation (if applicable):

3 Hours

Number of replicates:

Duplicate tissues were treated with the test material, negative and positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

68.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure period

Value:

65.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test material did not turn blue. This was taken to indicate the test material did not reduce MTT.
- Colour interference with MTT: The solution containing the test material was yellow. This colour was attributed to the intrinsic colour of the test material and was taken to indicate that the test material did not 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 1.854 for the 3 Minute exposure period and 1.908 for the 60 Minute exposure period. 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 2.6 % relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20-100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Material

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.946	1.854	0.130	7.0	100*
		1.762
	60 Minutes	1.888	1.908	0.028	1.5
		1.927
Positive Control	3 Minutes	0.101	0.087	0.020	N/A	4.7
		0.073
	60 Minutes	0.053	0.050	0.004	N/A	2.6
		0.047
Test Item	3 Minutes	1.233	1.277	0.062	4.9	68.9
		1.321
	60 Minutes	1.249	1.246	0.004	0.3	65.3
		1.243

OD = Optical density

* = The mean % viability of the negative control tissue is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test material was determined to be non-corrosive to the skin.

Executive summary:

A study was performed in vitro to assess the corrosive potential of the test material in accordance with the standardised guidelines OECD 431 and EU Method B.40bis under GLP conditions.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.

After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

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

The quality criteria required for acceptance of results in the test were satisfied.

The relative mean viabilities for each treatment group were as follows:

The mean viability of the negative control was set at 100 %.

The mean percentage viability of the test material after a 3 minute exposure period was 68.9. After 60 minutes exposure the test material viability was 65.3.

The mean percentage viability of the positive control after a 3 minute exposure period was 4.7 and after 60 minutes exposure was 2.6.

The test material was therefore considered to be non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

Study period:

04 March 2016 to 08 March 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

Strain:

not specified

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

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was used as supplied.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount 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 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 material and three corneas to the positive control material.

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.

NUMBER OF REPLICATES
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.

NEGATIVE CONTROL USED
0.9% w/v sodium chloride solution.

POSITIVE CONTROL USED
Ethanol, used as supplied.

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 or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 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.

- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.
- Corneal permeability: 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 ºC for 90 minutes.
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of medium representing each cornea 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.
- Others: Each cornea was visually observed. The condition of the cornea was visually assessed post treatment and post incubation.
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). Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score. The following formula was used to determine the In Vitro Irritancy Score:

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.

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.

DECISION CRITERIA:
≤ 3: No category. Not requiring classification to UN GHS or EU CLP.
> 3, ≤ 55: No prediction of eye irritation can be made.
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

Irritation parameter:

in vitro irritation score

Run / experiment:

Treatment with 0.75 mL of the test item

Value:

66.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤ 2.9 and permeability ≤ 0.103. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: 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.

In Vitro Irritancy Scores

Treatment	In Vitro Irritancy Score
Test Material	66.2
Negative Control	0.4
Positive Control	47.4

Table 2: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number		Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	2	2	3	1		0.007
	2	2	2	2	0		0.003
	4	2	2	2	0		0.007
					0.3*		0.006**		0.4
Positive Control	3	3	37	36	33	32.7	1.312	1.306
	5	2	28	30	28	27.7	1.063	1.057
	6	3	35	35	32	31.7	0.983	0.977
						30.7+		1.114+	47.4
Test Item	7	2	46	65	63	62.7	0.281	0.275
	8	5	40	66	61	60.7	0.700	0.694
	9	3	26	58	55	54.7	0.418	0.412
						59.3+		0.461+	66.2

OD = Optical density            

* = Mean of the post-incubation pre-treatment values         

** = Mean permeability                    

+ = Mean corrected value

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the conditions of the study, the test material was found to cause serious eye damage.

Executive summary:

A study was performed in vitro to assess the eye irritancy potential of the test material in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions.

The Bovine Corneal Opacity and Permeability (BCOP) test method was used to assess the potential of the test material to cause eye irritancy. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The corneas treated with the test item or the positive control item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation.

The positive control gave an IVIS of 47.4. The negative control had an IVIS of 0.4. The test material scored 66.2.

Under the conditions of the study, the test material was found to cause serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The following studies were assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

 

Skin Corrosion

A study was performed in vitro to assess the corrosive potential of the test material in accordance with the standardised guidelines OECD 431 and EU Method B.40bis under GLP conditions.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.

After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

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

The quality criteria required for acceptance of results in the test were satisfied.

The relative mean viabilities for each treatment group were as follows:

The mean viability of the negative control was set at 100 %.

The mean percentage viability of the test material after a 3 minute exposure period was 68.9. After 60 minutes exposure the test material viability was 65.3.

The mean percentage viability of the positive control after a 3 minute exposure period was 4.7 and after 60 minutes exposure was 2.6.

The test material was therefore considered to be non-corrosive to the skin.

 

Skin Irritation

A study was performed in vitro to assess the irritancy potential of the test material. The study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material 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.

The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.5 %. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.908 and the standard deviation value of the viability was 3.5 %. 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 1.7 %. The test item acceptance criterion was therefore satisfied.

The relative mean viability of the test material treated tissues was 9.4 % which is below the threshold of 50 %, therefore under the conditions of this study the test material was determined to irritating to the skin

Eye Irritation

The Bovine Corneal Opacity and Permeability (BCOP) test method was used to assess the potential of the test material to cause eye irritancy. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The corneas treated with the test item or the positive control item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation.

The positive control gave an IVIS of 47.4. The negative control had an IVIS of 0.4. The test material scored 66.2.

Under the conditions of the study, the test material was found to cause serious eye damage.

Justification for classification or non-classification

Skin

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material requires classification for skin irritation. The test material is assigned to Category 2 and requires the hazard statement “causes skin irritation”.

Eye

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material requires classification for eye irritation. The test material is assigned to Category 1 and requires the hazard statement “Causes serious damage to eyes”.