Dipraseodymium trioxide

Endpoint summary

Description of key information

Skin Corrosion

Under the conditions of this study, the test material is considered to be non corrosive to the skin.

Skin Irritation

Under the conditions of this study, the test material was found not to require classification as a skin irritant.

Eye Irritation

The In Vitro Irritancy Score for the test material was 6.3 and as a result, under the conditions of this study, no prediction of eye irritation can be made.

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:

24 January 2017 to 26 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: PR2O3-4/16
- Expiration date of the lot/batch: 21 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark over SiGel

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit, supplied by MatTek
- Tissue lot number(s): 23389
- Delivery date: 24 January 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

TEST FOR DIRECT MTT REDUCTION
A test material 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 material was checked for the ability to directly reduce MTT: 25 mg of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37°C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 25 mg of test material was added to 300 µL of sterile water. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. A visual assessment of the colour was then made.

MAIN TEST

PRE-INCUBATION
The assay medium was 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.

TREATMENT
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 minute and 60 minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 minute exposure period was returned to the incubator, while the other was being dosed for the 60 minute exposure. For the 60 minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test material and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test material to increase tissue surface contact. The plate was returned to the incubator (37°C, 5% CO2) for the 60 minute exposure period. When dosing for the 60 minute exposure period was complete, the same procedure was repeated for the 3 minute exposure period.

REMOVAL OF TEST MATERIAL AND CONTROLS
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The plate was incubated (37°C, 5% CO2) for 3 hours. After the 3 hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.
- 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. 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. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

NUMBER OF REPLICATE TISSUES: 2

DATA EVALUATION
- 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.
- Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x 100
Classification of corrosivity potential is based on relative viabilities for both exposure times according to the following:
- Relative mean tissue viability (% of negative control):
- 3 min < 50: H314 Category 1A
- 3 min ≥ 50, 1 hour < 15: H314 Category 1B or 1C
- 3 min ≥ 50, 1 hour ≥ 15: Not classified for corrosivity

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.
- Potassium hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
- 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(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2 per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

111.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

100.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The solution containing the test material was a pale green colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore considered unnecessary to run colour correction tissues.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 1.613 for the 3 minute exposure period and 1.729 for the 60 minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 3.8% relative to the negative control following the 60 minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Table 1: Mean OD562 Values and Viabilities for the Negative Control, Positive Control 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	1.661	1.613	0.068	4.2	100*
		1.565
	60 Minutes	1.757	1.729	0.040	2.3
		1.701
Positive Control	3 Minutes	0.068	0.074	0.008	n/a	4.6
		0.079
	60 Minutes	0.069	0.065	0.006	n/a	3.8
		0.061
Test Material	3 Minutes	1.828	1.800	0.040	2.2	111.6
		1.771
	60 Minutes	1.701	1.736	0.049	2.8	100.4
		1.770

 

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

n/a = Not applicable

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

Coefficient of Variation = (standard deviation / mean OD562 of duplicate tissues) x 100

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is considered to be non corrosive to the skin.

Executive summary:

The potential of the test material to cause skin corrosion was determined in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions. The corrosivity potential of the test material was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

During the study, duplicate tissues were treated with discs of the test material 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 material 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).

The quality criteria required for acceptance of results in the test were satisfied. The 3 minute exposure time gave a relative mean viability of 111.6% and the 60 minute exposure time was 100.4%.

Under the conditions of this study, the test material is 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:

key study

Study period:

07 February 2017 to 20 February 2017

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: PR203-4/16
- Expiration date of the lot/batch: 21 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic Laboratories, Lyon, France.
- Tissue lot number: 17-EKIN-007
- Delivery date: 14 February 2017

TEST FOR DIRECT MTT REDUCTION
A test material 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 material is checked for the ability to directly reduce MTT: 10 mg of the test material 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 material turns blue/purple, the test material 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 material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 10 mg of test material was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

PRE-INCUBATION (DAY 0)
- Before removal from the transport plate each tissue was inspected for: any air bubbles between the agarose gel and the insert, whether tissues were satisfactory, whether the temperature indicator colour was satisfactory and whether the agar medium colour was 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 material and each control material. The tissues were incubated at 37°C, 5% CO2 in air overnight.

MAIN TEST

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: 37°C

APPLICATION (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 the test material for an exposure period of 15 minutes. The test material 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 material and the epidermis. Approximately 10 mg (26.3 mg/cm²) of the test material was then applied to the epidermal 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 material 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 material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
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 Ca²+ and Mg²+. 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.

MTT LOADING /FORMAZAN EXTRACTION (DAY 3)
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination.
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. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

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

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
- Quantitative MTT Assessment (Percentage Tissue Viability): For the test material the relative mean tissue viabilities obtained after the 15-minute exposure period followed by the 42-hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3).
- Relative mean viability (%) = (mean OD562 of test material/ mean OD562 of negative control) x 100
- Classification of irritation potential is based upon relative mean tissue viability. If relative mean tissue viability is ≤ 50%: irritant (H315 Category 2), if relative mean tissue viability is > 50%: non-irritant (not classified for irritation).

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

NEGATIVE CONTROL
- Amount applied : 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

100.6

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:

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.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test material was a pale green colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.

TEST MATERIAL TREATMENT
- The individual and mean OD562 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1.
- The relative mean viability of the test material treated tissues was 100.6% 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 14.3% relative to the negative control treated tissues and the standard deviation value of the viability was 7.4%. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 0.784 and the standard deviation value of the viability was 11.9%. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 5.4%. The test material acceptance criterion was therefore satisfied.

Table 1: Mean OD562 Values and Viabilities for the Negative Control, Positive Control and Test Material

Treatment	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	0.678	0.784	0.094	86.5	100*	11.9
	0.817			104.2
	0.856			109.2
Positive control	0.132	0.112	0.058	16.8	14.3	7.4
	0.158			20.2
	0.047			6.0
Test material	0.819	0.789	0.042	104.5	100.6	5.4
	0.807			102.9
	0.741			94.5

 

OD = Optical density

SD = Standard deviation

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

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not classified for skin irritation.

Executive summary:

The potential of the test material to cause skin irritation was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions using a human skin model.

Prior to treatment the test material was examined for its potential to interfere with MTT. The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT. The pale green colour was attributed to the intrinsic colour of the test material itself, it was therefore unnecessary to run colour correction tissues.

During the study, 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 viability of the test material treated tissues was 100.6% 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.

Under the conditions of this study, the test material was found not to require classification as a skin irritant.

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

Study period:

14 February 2017

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:

2013

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)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: PR2O3-4/16
- Expiration date of the lot/batch: 21 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark over SiGel

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test material was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution. The test material was formulated within 2 hours of being applied to the test system.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular: 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:

other: 0.9% w/v sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20% w/v

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

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 replicates

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 (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1°C for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
- At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
- The medium from both chambers of each holder was replaced with fresh complete MEM.
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 negative control. Three corneas were also allocated to the test material and three corneas to the positive control.

NUMBER OF REPLICATES: 3

VEHICLE CONTROL USED: 0.9% w/v sodium chloride solution

POSITIVE CONTROL USED: Imidazole 20% w/v in 0.9% w/v sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes

TREATMENT METHOD
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material preparation or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1°C for 240 minutes.

REMOVAL OF TEST SUBSTANCE
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1°C for 90 minutes. 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: 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:
- 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 (IVIS) = mean opacity value + (15 x mean permeability OD492 value)
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

DECISION CRITERIA
The test material was classified according to the following prediction model:
- IVIS ≤ 3: No category. Not requiring classification to UN GHS or EU CLP
- IVIS > 3; ≤ 55: No prediction of eye irritation can be made
- IVIS > 55: Category 1. UN GHS or EU CLP Causes serious eye damage

ACCEPTABILITY OF THE TEST
- 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 2015 for this testing facility (50.8 to 100.4).
- Sodium chloride 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 2015 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤ 5.4 and for permeability ≤ 0.070.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

6.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
The corneas treated with the test material were clear post treatment. The corneas treated with the negative control were clear post treatment. The corneas treated with the positive control were cloudy post treatment.

ACCEPTABILITY OF THE TEST
The positive control In Vitro Irritancy Score was within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤ 5.4 and permeability ≤ 0.070. The negative control acceptance criteria were therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	2	3	1		0.012
	4	2	3	1		0.035
	7	2	5	3		0.010
				1.7*		0.019**		2.0
Positive Control	2	3	65	62	60.3	1.552	1.533
	3	6	84	78	76.3	1.037	1.018
	5	1	67	66	64.3	1.420	1.401
					67.0***		1.317***	86.8
Test Material	6	1	12	11	9.3	0.080	0.061
	8	2	6	4	2.3	0.024	0.005
	9	5	11	6	4.3	0.148	0.129
					5.3***		0.065***	6.3

 

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

** = Mean permeability

*** = Mean corrected value

Interpretation of results:

other: No prediction of eye irritation can be made.

Conclusions:

Under the conditions of this study, no prediction of eye irritation can be made.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) test.

During the study, 0.75 mL test material was applied to the corneas at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control materials were tested concurrently and the study was performed in triplicate. 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 positive control In Vitro Irritancy Score was within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 5.4 and permeability ≤ 0.070. The negative control acceptance criteria were therefore satisfied.

The In Vitro Irritancy Score for the test material was 6.3 and as a result, under the conditions of this study, no prediction of eye irritation can be made.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion

The potential of the test material to cause skin corrosion was determined in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions. The corrosivity potential of the test material was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, duplicate tissues were treated with discs of the test material 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 material 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).

The quality criteria required for acceptance of results in the test were satisfied. The 3 minute exposure time gave a relative mean viability of 111.6% and the 60 minute exposure time was 100.4%.

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

Skin Irritation

The potential of the test material to cause skin irritation was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions using a human skin model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Prior to treatment the test material was examined for its potential to interfere with MTT. The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT. The pale green colour was attributed to the intrinsic colour of the test material itself, it was therefore unnecessary to run colour correction tissues.

During the study, 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 viability of the test material treated tissues was 100.6% 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.

Under the conditions of this study, the test material was found not to require classification as a skin irritant.

Eye Irritation

The eye irritation potential of the test material was determined in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, 0.75 mL test material was applied to the corneas at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control materials were tested concurrently and the study was performed in triplicate. 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 positive control In Vitro Irritancy Score was within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 5.4 and permeability ≤ 0.070. The negative control acceptance criteria were therefore satisfied.

The In Vitro Irritancy Score for the test material was 6.3 and as a result, under the conditions of this study, no prediction of eye irritation can be made.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin corrosion/irritation or eye irritation.