Neodymium trichloride

Endpoint summary

Description of key information

Skin Corrosion

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation (in vitro)

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Skin Irritation (in vivo)

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

Eye Irritation (in vitro/ex vivo BCOP)

Under the conditions of the study the test material induced an IVIS of 8.1 hence no prediction of eye irritation can be made.

Eye irritation (in vivo)

Under the conditions of the study the test material requires classification for irreversible effects on the eye (Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 1984 to 14 April 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: 83040

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.5 kg ± 200 g
- Housing: individually housed in polystyrene cages (540 x 360 x 315 mm) with a perforated polystyrene floor
- Diet: 150 g per rabbit per day
- Water: ad libitum
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity: 30 to 70%
- Air changes: 12 to 14 times per hour
- Photoperiod: 12 hours lighting per day, 7.30 am to 7.30 pm

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

no

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 hours after removal of the patch

Number of animals:

6

Details on study design:

METHOD
The day before the application of the test material, the backs and flanks of six rabbits were clipped free of fur with an electric clipper, to expose a surface of 14 x 14 cm. Only animals with perfectly healthy intact skin showing no macroscopic sign of irritation, after a rest period of 24 hours were used for the study.
0.5 g of the test material was moistened with water and applied directly onto the animal skin and spread evenly over an area of ca. 6 cm² and then covered with a gauze pad about 2.5 cm².
The test material and gauze pad were held in contact with the skin with a semi-occlusive patch: 10 cm wide adhesive perforated tape applied on a crimped gauze bandage covering the whole clipped surface and wrapped around the animal.
The test material was kept in contact with the skin for 4 hours whilst the animals were kept in plastic restraining boxes.
After the exposure treatment the bandages were removed and if necessary any excess test material wiped from the skin with a gauze pad moistened with deionised water.
The cutaneous irritation was evaluated 1, 24, 48 and 72 hours after removal of the test material.

DESCRIPTION OF REACTIONS
Erythema and Eschar formation:
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well defined erythema
3 = Moderate to severe erythema
4 = Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Oedema formation:
0 = No oedema
1 = Very slight oedema (barely perceptible)
2 = Slight oedema (edges of area well defined by definite raising)
3 = Moderate oedema (edges raised approximately 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond the area of exposure)

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0.17

Max. score:

1

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

Very slight erythema was noted in one of the six treated animals at 1, 24, 48 and 72 hours following removal of the test material.

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

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

Executive summary:

The potential of the test material to cause irritation to the skin was determined in accordance with the standardised guideline OECD 404 using an in-vivo method.

During the study, six male New Zealand White rabbits were treated with the test material under a semi-occlusive dressing for four hours. After the removal of the test material the skin was observed at 1, 24, 48 and 72 hours for cutaneous irritation.

Very slight erythema was observed in 1 out of 6 animals but no oedema was observed.

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2017 to 16 May 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:

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

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-019
- Delivery date: 10 May 2017

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

ASSESSMENT OF POSSIBLE MTT REDUCTION
- 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 according to the following procedure: 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.

MAIN STUDY

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, whether tissues were satisfactory, whether 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.

APPLICATION OF TEST MATERIAL (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 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°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.

NUMBER OF REPLICATE TISSUES: 3

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS (DAY 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

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). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of test material/ mean OD570 of negative control) x100.

Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to:
- Relative mean tissue viability is ≤ 50%: Irritant (H315 Category 2)
- 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:
- 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 OD570 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18%.
- Test Material: 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(s) applied: 10 mg (26.3 mg/cm^2)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

93.1

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 MTT
The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

TEST MATERIAL AND CONTROLS
- The individual and mean OD570 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also shown in Table 1.
- The relative mean viability of the test material treated tissues was 93.1% 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 11.7% relative to the negative control treated tissues and the standard deviation value of the viability was 3.1%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.664 and the standard deviation value of the viability was 7.1%. 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 15.7%. The test material acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₇₀Values and Viabilities for the Negative Control, Positive Control and Test Material

 

Treatment	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.636	0.664	0.048	95.8	100*	7.1
	0.637			95.9
	0.719			108.2
Positive Control	0.061	0.078	0.021	9.2	11.7	3.1
	0.073			11.0
	0.101			15.2
Test Material	0.652	0.618	0.105	98.2	93.1	15.7
	0.501			75.5
	0.702			105.7

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

OD = Optical Density

SD = Standard Deviation

 

 

 

 

 

 

 

 

 

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Executive summary:

The skin irritation potential of the test material was determined in accordance with the standardized guidelines OECD439 and EU Method B.46. under GLP conditions using a human skin model.

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. 

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. The test material was found to have the potential to cause colour interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. 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 570 nm.

The relative mean viability of the test material treated tissues was 93.1% 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 is non-irritant in the in vitro skin irritation test.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2017 to 06 May 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)

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): 25810
- Delivery date: 03 May 2017

TEST FOR MTT REDUCTION
A test material may interfere with the MTT endpoint if it is able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test is 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 MTT
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.

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.
- 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. 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.

REMOVAL OF TEST MATERIAL AND CONTROLS AND MTT LOADING
- 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. The plate was incubated (37°C, 5% CO2) for 3 hours. Once the 60 minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
- After the 3 hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

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

NUMBER OF REPLICATE TISSUES: 2

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 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 570 nm (OD570) of each well was measured using the Lab tech LT4500 microplate reader.

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 OD570 of test material/ mean OD570 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 A
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:
- Negative control
The absolute OD570 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 OD570 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.
- Positive control
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%.
- Coefficient of Variation
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 (with 25 µL sterile water to increase tissue surface contact)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

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

Duration of treatment / exposure:

3 minutes or 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

80.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

59.3

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
The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

TEST MATERIAL AND CONTROLS
Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.

QUALITY CRITERIA
- The mean OD570 for the negative control treated tissues was 1.403 for the 3 minute exposure period and 1.364 for the 60 minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 4.8% 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 OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue	Exposure period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative control	3 minutes	1.415	1.403	0.017	1.2	100*
		1.391
	60 minutes	1.342	1.364	0.030	2.2
		1.385
Positive control	3 minutes	0.063	0.066	0.004	N/A	4.7
		0.068
	60 minutes	0.061	0.066	0.006	N/A	4.8
		0.070
Test material	3 minutes	1.056	1.129	0.103	9.1	80.5
		1.202
	60 minutes	0.878	0.810	0.097	12.0	59.3
		0.741

OD = optical density

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

N/A = not applicable

 

Relative mean % tissue viability = (mean OD570 of test material/mean OD570 of negative control) x 100

Coefficient of variation = (standard deviation/ mean OD570 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 not corrosive in the in vitro skin corrosion test.

Executive summary:

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

During the study, duplicate tissues were treated with 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 570 nm (OD570).

The mean viability of the test material treated tissues after 3 minutes was 80.5% and after 60 minutes was 59.3%. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was not corrosive in the in vitro skin corrosion test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 1984 to 10 May 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: 83040

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.5 kg ± 200 g
- Housing: individually housed in polystyrene cages (540 x 360 x 315 mm) with a perforated polystyrene floor
- Diet: 150 g per rabbit per day
- Water: ad libitum
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity: 30 to 70%
- Air changes: 12 to 14 times per hour
- Photoperiod: 12 hours lighting per day, 7.30 am to 7.30 pm

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

The eyelids were held together for ten seconds to avoid any loss of test material. The animals were then restrained for one hour.

Observation period (in vivo):

1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation

Number of animals or in vitro replicates:

6

Details on study design:

Any animals showing possible ocular lesions were eliminated from the study. 100 mg of the test material was placed into the conjunctival sac of the right eye of the animal, the upper and lower eyelids are held together for ten seconds to avoid the loss of any test material. The left eye served as a control. Each animal was immobilised in a plastic restraining box during treatment and for one hour afterwards.

Animals were immobilised for examination, the examinations were always carried out under the same conditions (especially lighting) and the treated eye was compared to the control at 1, 24, 48 and 72 hours post treatment and again at 7, 14 and 21 days after treatment

Observations of the cornea was made with the aid of a Heine's ophthalmoscope which was also used to observe the iris, pupil and lens, if required. If further examination was necesarry a slit lamp was used.

Observations were made on the following: conjunctivae (oedema and redness), pupil, iris congestion, corneal lesions and opacity degree.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

2.28

Max. score:

3

Reversibility:

not fully reversible within:

Remarks:

21 days

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within:

Remarks:

21 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

3.5

Max. score:

4

Reversibility:

not fully reversible within:

Remarks:

21 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within:

Remarks:

21 days

Interpretation of results:

other: Irreversible effects on the eye (Category 1) in accordance with EU criteria

Conclusions:

Under the conditions of the study the test material requires classification for irreversible effects on the eye (Category 1).

Executive summary:

The potential of the test material to cause irritation to the eye was determined in accordance with the standardised guideline OECD 405 using an in-vivo method.

Six male New Zealand White rabbits were treated with the test material which was placed into the conjunctival sac of the right eye of the animal, the upper and lower eyelids are held together for ten seconds to avoid the loss of any test material. The left eye served as a control. The eye was observed at 1, 24, 48 and 72 hours and at days 7, 14 and 21 for signs of ocular irritation.

Chemosis and conjunctival redness were observed in the treated eyes of all animals from 1 hour post-treatment and the effects were not reversible within the 21-day observation period. Iridial effects (score 1) were noted in all animals at all timepoints. Corneal opacity was observed in the treated eyes of all animals from 24 hours post-treatment and the effects were not reversible within the 21-day observation period.

Therefore, under the conditions of the study the test material requires classification for irreversible effects on the eye (Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 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 Ocular Corrosives and Severe Irritants)

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:

- Storage conditions: Room temperature in the dark over silica gel

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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

other: sodium chloride 0.9 % w/v

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
- Lot/batch no.: 3012488

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with sodium fluorescein solution (5 mg/mL)

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- 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 65 minutes. 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.

NUMBER OF REPLICATES: 3

NEGATIVE (VEHICLE) CONTROL USED: 0.9% sodium chloride w/v

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT
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 MATERIAL:
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. A post-treatment opacity reading was taken and each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A pre and post-treatment opacity reading was taken using a calibrated opacitometer 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. 1.0 mL of media representing each cornea was dispensed into separate 1.5 mL cuvettes, with 1.0 mL complete EMEM being used for blank correction. Optical density at 492 nm (OD492) was measured using the Camspec Model M108 Spectrophotometer.
- Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

DATA EVALUATION

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

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

- In Vitro Irritancy Score
The 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 material induced a response through only one of the two endpoints.

- Visual Observation
The condition of the cornea was visually assessed post treatment.

- Data Interpretation:
The test material was classified according to the following criteria:
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 CRITERIA
For an acceptable test the following positive 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 collated during 2015 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 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:

8.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

In vitro irritancy scores, as well as Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

CORNEAL EPITHELIUM CONDITION
The corneas treated with the test material were slightly cloudy 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.

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 and in vitro irritancy scores

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	3	5	2	-	0.037	-	-
	2	3	5	2	-	0.074	-	-
	4	3	4	1	-	0.018	-	-
	-	-	-	1.7*	-	0.043**	-	2.3
Positive control	3	4	70	66	64.3	1.764	1.721	-
	5	3	58	55	53.3	1.916	1.873	-
	6	7	78	71	69.3	1.028	0.985	-
	-	-	-	-	62.3***	-	1.526***	85.2
Test material	7	4	9	5	3.3	0.071	0.028	-
	8	3	14	11	9.3	0.029	0.000	-
	9	4	17	13	11.3	0.014	0.000	-
	-	-	-	-	8.0***	-	0.009***	8.1

OD = optical density

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

** = Mean permeability

*** = Mean corrected value

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, as the test material induced an IVIS of 8.1, no prediction of eye irritation can be made.

Executive summary:

The potential of the test material to cause eye irritation 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.

The 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 material is assessed by quantitative measurements of changes in corneal opacity and permeability.

During the study the test material was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control materials were tested concurrently. All treatments were tested 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 acceptability criteria of the assay were met and the test was considered to be valid.

Under the conditions of this study, as the test material induced an IVIS of 8.1, no prediction of eye irritation can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions 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 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 570 nm (OD570).

The mean viability of the test material treated tissues after 3 minutes was 80.5% and after 60 minutes was 59.3%. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material was not corrosive in the in vitro skin corrosion test.

Skin Irritation (in vitro)

The skin irritation potential of the test material was determined in accordance with the standardized guidelines OECD439 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).

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. 

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. The test material was found to have the potential to cause colour interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. 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 570 nm.

The relative mean viability of the test material treated tissues was 93.1% 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 is non-irritant in the in vitro skin irritation test.

Skin Irritation (in vivo)

The potential of the test material to cause irritation to the skin was determined in accordance with the standardised guideline OECD 404 using an in-vivo method.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, six male New Zealand White rabbits were treated with the test material under a semi-occlusive dressing for four hours. After the removal of the test material the skin was observed at 1, 24, 48 and 72 hours for cutaneous irritation.

Very slight erythema was observed in 1 out of 6 animals but no oedema was observed.

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

Eye Irritation (in vitro/ex vivo BCOP)

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guidelines OECD437 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 the test material was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control materials were tested concurrently. All treatments were tested 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 acceptability criteria of the assay were met and the test was considered to be valid.

Under the conditions of this study, as the test material induced an IVIS of 8.1, no prediction of eye irritation can be made.

Eye Irritation (in vivo)

The potential of the test material to cause irritation to the eye was determined in accordance with the standardised guideline OECD 405 using an in-vivo method. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Six male New Zealand White rabbits were treated with the test material which was placed into the conjunctival sac of the right eye of the animal, the upper and lower eyelids are held together for ten seconds to avoid the loss of any test material. The left eye served as a control. The eye was observed at 1, 24, 48 and 72 hours and at days 7, 14 and 21 for signs of ocular irritation.

Chemosis and conjunctival redness were observed in the treated eyes of all animals from 1 hour post-treatment and the effects were not reversible within the 21-day observation period. Iridial effects (score 1) were noted in all animals at all timepoints. Corneal opacity was observed in the treated eyes of all animals from 24 hours post-treatment and the effects were not reversible within the 21-day observation period.

Therefore, under the conditions of the study the test material requires classification for irreversible effects on the eye (Category 1).

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 but does require classification for irreversible effects on the eye (Category 1).