Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Description of key information
SKIN CORROSION / IRRITATION
Skin corrosion (Read-across from ytterbium triflouride, Lacey, 2017):
The test material was considered to be non-corrosive to the skin.
Skin irritation (Read-across from ytterbium triflouride, Lacey, 2017):
The test material was determined to be a non-irritant to skin.
Skin irritation (Read-across from ytterbium oxide, Orovecz, 2016):
The test material was determined to be a non-irritant to skin.
EYE IRRITATION
Eye irritation (Read-across from ytterbium triflouride, Lacey, 2017):
No prediction on eye irritation could be made.
Eye irritation (Read-across from ytterbium oxide, Richez, 2017):
The test material is not classified as an eye irritant.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 November to 24 November 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- 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:
- EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
- 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: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature over silica gel, in the dark - 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, MatTek
- Tissue lot number: 23380
- Delivery date: 22 November 2016, upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C
NUMBER OF REPLICATE TISSUES: 2
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 according to the procedure below:
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 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 AND RINSING
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing 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. 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.
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 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.
QUANTITATIVE MTT ASSESSMENT (% 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
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 vehicle
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount applied: 25 mg wetted with 25 µL of sterile water
VEHICLE CONTROL
- Amount applied: 50 µL
POSITIVE CONTROL
- Amount 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:
- The test was performed in duplicate.
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes exposure
- Value:
- 95.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60 minutes exposure
- Value:
- 95.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- 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 did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.
RELATIVE MEAN VIABILITIES - TEST MATERIAL
The relative mean viabilities for each treatment group are shown in Table 2.
QUALITY CRITERIA
-The mean OD562 for the negative control treated tissues was 1.709 for the 3 minute exposure period and 1.692 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 3.1% 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. - Interpretation of results:
- other: Not corrosive in accordance with EU criteria
- Conclusions:
- Under the conditions of the study the test material was 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 B40.bis, under GLP conditions using the EpiDerm Human Skin Model.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test material.
Before the main test was performed the test material was tested for 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. The test material was also assessed for its potential to cause colour interference with the MTT endpoint. 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.
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 562 nm (OD562).
The negative control, positive control and coefficient of variation were all within the acceptability criteria and so the study was valid.
Under the conditions of the study the test material was considered to be non-corrosive to the skin.
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.
- Reason / purpose for cross-reference:
- read-across source
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60 minute exposure
- Value:
- 95.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes exposure
- Value:
- 95.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Interpretation of results:
- other: Not classified as corrosive in accordance with EU criteria.
- Conclusions:
- Under the conditions of the study the test material was 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 B40.bis, under GLP conditions using the EpiDerm Human Skin Model.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test material.
Before the main test was performed the test material was tested for 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. The test material was also assessed for its potential to cause colour interference with the MTT endpoint. 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.
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 562 nm (OD562).
The negative control, positive control and coefficient of variation were all within the acceptability criteria and so the study was valid.
Under the conditions of the study the test material was considered to be non-corrosive to the skin.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15 December 2016 to 20 December 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- 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: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018
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 in international guidelines
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue lot number: 16-EKIN-050
- Delivery date: 14 December 2016
TEST FOR DIRECT MTT REDUCTION
The 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 or purple it 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
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert, if the tissues were satisfactory, if the temperature indicator colour was satisfactory and if the agar medium colour was satisfactory.
On day 0, 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 STUDY
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C
On 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 center). After a 7 minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (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
- Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with 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.
MTT LOADING/ FORMAZAN EXTRACTION
On 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 EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
At the end of the formazan extraction period on day 6, 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
DECISION CRITERIA
The relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x 100
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 the following:
- Relative mean tissue viability is ≤ 50 %: Irritant
- Relative mean tissue viability is > 50 %: Non-irritant
QUALITY CRITERIA
-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) wetted with 5 µL of sterile distilled water
NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration (if solution): used as supplied
POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aqueous solution (w/v) - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- The test was performed in triplicate
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 113.2
- 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 or purple which indicated that the test material did not directly reduce MTT.
ASSESSMENT OF THE COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The solution containing the test material was a white colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.
MAIN STUDY
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 113.2 % 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 11.4% relative to the negative control treated tissues and the standard deviation value of the viability was 4.4%. The positive control acceptance criteria were therefore satisfied.
The mean OD562 for the negative control treated tissues was 0.625 and the standard deviation value of the viability was 14.1%. The negative control acceptance criteria were therefore satisfied. - Interpretation of results:
- other: Not classified in accordance with EU criteria
- Conclusions:
- Under the conditions of this study, the test material is not classified as a skin irritant.
- Executive summary:
The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 using the human epidermis model, under GLP conditions.
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. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls.
Interference checks were performed before the main study. 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 solution prepared containing the test material was a white colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.
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 113.2% 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 all satisfied.
Under the conditions of the study, the test material was found not to be irritating to skin.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.
- Reason / purpose for cross-reference:
- read-across source
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 113.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- other: Not classified in accordance with EU criteria
- Conclusions:
- Under the conditions of this study, the test material is not classified as a skin irritant.
- Executive summary:
The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 using the human epidermis model, under GLP conditions.
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. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls.
Interference checks were performed before the main study. 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 solution prepared containing the test material was a white colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.
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 113.2% 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 all satisfied.
Under the conditions of the study, the test material was found not to be irritating to skin.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 July 2016 to 08 July 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 2009
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- No correction for purity of the test material was applied
- 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™ (SM) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 16-EKIN-027
- Expiry date: 2016-07-11
- Date of initiation of testing: 2016-07-06
TEST FOR DIRECT MTT REDUCTION
The 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: Approximately 10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
- Test materials which do not react with MTT: yellow
- Test material reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test material had not reacted with MTT and therefore the use of additional controls was not necessary.
ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
Prior to treatment, the test material was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour% (NSCliving%) was determined in order to evaluate the ability of test materials to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.
PRE-INCUBATION (DAY -1)
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.
MAIN STUDY
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.4-27.4°C)
- Temperature of post-treatment incubation: 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (80-100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
On Day 0, as the test material was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 20 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface. Similarly, 50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.4-27.4°C).
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After 15 min incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: no
MTT LOADING/ FORMAZAN EXTRACTION
After the 42 hours incubation, all EPISKINÖ (SM) units (except of two colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN™ (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 in a >95% RH% protected from light.
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit using a biopsy punch. The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol. The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate. The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
NUMBER OF REPLICATE TISSUES: 3
PREDICTION MODEL / DECISION CRITERIA
- The test material is considered to be non-irritant to skin if the relative mean viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 50% of the mean variability of the negative controls. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
NEGATIVE CONTROL (PBS)
- Amount(s) applied (volume or weight): 50 μL
POSITIVE CONTROL (SDS)
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v) - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- The test was performed in triplicate
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 90.6
- 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:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was 0.021, consequently, Non Specific Colour % was calculated to be 3.0%. This value was below 5%, therefore additional data calculation was not necessary.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was in the recommended range (0.699). Standard deviation of the viability results for negative control samples was 4.1.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 6.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.9.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 2.2.
- The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid. - Interpretation of results:
- other: not classified according to EU criteria
- Conclusions:
- Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test material was determined to be non-irritant to skin.
- Executive summary:
The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 using the human epidermis model, under GLP conditions.
An in vitro skin irritation test of Diytterbium trioxide test material was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Disks of EPISKIN™ (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% RH%. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO2 in a >95RH% protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test material is considered to be irritant to skin.
Following exposure with Diytterbium trioxide, the mean cell viability was 90.6% compared to the negative control. This is above the threshold of 50%, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test material was determined to be non-irritant to skin.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.
- Reason / purpose for cross-reference:
- read-across source
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean of 3 replicates
- Value:
- 90.6
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- other: not classified according to EU criteria
- Conclusions:
- Under the conditions of this study (in vitro EPISKIN model material according to OECD guideline 439), the test item was determined to be non-irritant to skin.
- Executive summary:
The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 using the human epidermis model, under GLP conditions.
An in vitro skin irritation test of Diytterbium trioxide test material was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Disks of EPISKINTM (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% RH%. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO2 in a >95RH% protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test material is considered to be irritant to skin.
Following exposure with Diytterbium trioxide, the mean cell viability was 90.6% compared to the negative control. This is above the threshold of 50%, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test material was determined to be non-irritant to skin.
Referenceopen allclose all
Table 2: relative mean viabilities for each treatment group
Exposure Period |
Percentage Viability |
||
Negative Control |
Positive Control |
Test Material |
|
3 minute |
100* |
3.1 |
95.7 |
60 minute |
100* |
2.5 |
95.8 |
*The mean viability of the negative control tissues is set at 100%
Table 3: 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 |
1.720 |
1.709 |
0.016 |
1.0 |
100* |
1.697 |
||||||
60 Minutes |
1.649 |
1.692 |
0.060 |
3.6 |
||
1.734 |
||||||
Positive Control |
3 Minutes |
0.058 |
0.053 |
0.007 |
N/A |
3.1 |
0.048 |
||||||
60 Minutes |
0.055 |
0.043 |
0.018 |
N/A |
2.5 |
|
0.030 |
||||||
Test Material |
3 Minutes |
1.658 |
1.635 |
0.033 |
2.0 |
95.7 |
1.611 |
||||||
60 Minutes |
1.621 |
1.620 |
0.001 |
0.1 |
95.8 |
|
1.619 |
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
Table 1: Mean OD562 Values and Viabilities for the Negative Control, Positive Control and Test Material
Item |
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 |
0.632 |
0.625 |
0.088 |
101.1 |
100* |
14.1 |
0.710 |
113.6 |
|||||
0.534 |
85.4 |
|||||
Positive Control |
0.103 |
0.071 |
0.027 |
16.5 |
11.4 |
4.4 |
0.054 |
8.6 |
|||||
0.057 |
9.1 |
|||||
Test Material |
0.719 |
0.707 |
0.010 |
115.0 |
113.2 |
1.6 |
0.700 |
112.0 |
|||||
0.703 |
112.5 |
OD = Optical Density
SD = Standard deviation
* = The mean viability of the negative control tissues is set at 100%
Table 1: Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissue
| Additional control | Optical Density (OD) | NSC% | ||
| Measured | Blank corrected | |||
| Treated with 0.025 | 1 | 0.071 | 0.025 | |
| Diytterbium trioxide | 2 | 0.064 | 0.018 | 3.0 |
| Mean | ---- | 0.021 |
Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each
sample
(rounded to three decimal places).
Table 2: Optical Density (OD) and the calculated relative viability % of the samples
| Substance | Optical Density (OD) | Viability (% RV) |
||
| Measured | Blank corrected | |||
| Negative control | 1 | 0.718 | 0.672 | 96.1 |
| Phosphate buffered saline | 2 | 0.775 | 0.729 | 104.2 |
| 3 | 0.743 | 0.697 | 99.7 | |
| mean | -- | 0.699 | 100 | |
| Positive control | 1 | 0.087 | 0.041 | 5.8 |
| 5% (w/v) SDS solution | 2 | 0.089 | 0.043 | 6.1 |
| 3 | 0.099 | 0.053 | 7.5 | |
| mean | -- | 0.045 | 6.5 | |
| Test Material: | 1 | 0.673 | 0.627 | 89.7 |
| Diytterbiumtrioxide | 2 | 0.697 | 0.651 | 93.1 |
| 3 | 0.669 | 0.623 | 89.1 | |
| mean | -- | 0.633 | 90.6 |
Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each
sample (rounded to three
decimal places).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29 November 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- 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: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature over silica gel, in the dark
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: A local abattoir as a by-product from freshly slaughtered animals
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle typically 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. 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 applied: 0.75 mL
- Concentration: 20 %
VEHICLE
- Concentration (if solution): 0.9 % (w/v)
- Batch no.: 3011424 - 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. 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 material.
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 items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the test 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.
POST-EXPOSURE INCUBATION: 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.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken after the test material had been removed and each cornea was visually observed.
- Corneal permeability: 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)
- 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
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.
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 ASSAY
- 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 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 66.9 to 101.4.
- 0.9 % w/v sodium chloride solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤ 4.1 and for permeability ≤ 0.105. - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- mean
- Value:
- 9.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- A summary of individual and mean corneal opacity and permeability measurements can be seen 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 material were clear post treatment. The corneas treated with the positive control material were cloudy post treatment.
- ACCEPTANCE OF RESULTS
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied. - Interpretation of results:
- other: No prediction on eye irritation can be made
- Conclusions:
- Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.
- Executive summary:
The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 437 and EU method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) test method.
The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability.
The 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, the study was performed in triplicate. Negative and positive control materials were tested concurrently, in triplicate also. 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 in vitro irritancy score for the test material was 9.3 and as a result it falls into the band of IVIS > 3 - ≤55 in the classification system and so no prediction of eye irritation can be made.
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.
Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.
- Reason / purpose for cross-reference:
- read-across source
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- mean
- Value:
- 9.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Interpretation of results:
- other: No prediction on eye irritation can be made
- Conclusions:
- Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.
- Executive summary:
The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 437 and EU method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) test method.
The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability.
The 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, the study was performed in triplicate. Negative and positive control materials were tested concurrently, in triplicate also. 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 in vitro irritancy score for the test material was 9.3 and as a result it falls into the band of IVIS > 3 - ≤55 in the classification system and so no prediction of eye irritation can be made.
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.
Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 June 2017 to 30 June 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- No correction factor was applied for this type of study
- Species:
- human
- Strain:
- other: Reconstructed human cornea-like epithelium (tissues)
- Details on test animals or tissues and environmental conditions:
- - Source: MatTek, Bratislava, Slovak Republic
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.
- Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Storage conditions: At receipt, the living EpiOcular tissues were stored on their day of arrival, at 37°C, 5% CO2, in a humidified incubator.
- Description of the cell system used: The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinised epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotype 3D cornea-like model. The 3D tissue consists of highly organised cell layers similar to those found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue. - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51 mg (± 1 mg)
NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL
POSITIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL - Duration of treatment / exposure:
- 6 hours (± 15 minutes)
- Observation period (in vivo):
- 18 hours (± 15 minutes)
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- TEST FOR DIRECT MTT REDUCTION
To identify any test material interference with the MTT endpoint, the following preliminary test was performed:
- 51 mg (± 1 mg) of the test material were weighed and added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the colour of the solutions obtained was evaluated.
If the MTT solution colour turns blue/purple when compared to the negative control, the test material was presumed to reduce MTT. In this case, additional controls were performed on freeze-dead tissues in parallel to the main test (performed on viable tissues) to evaluate the part of OD due to the non-specific reduction of the MTT. Otherwise, no additional tissue controls were used.
TEST FOR THE DETECTION OF THE COLOURING POTENTIAL OF THE TEST MATERIAL
As a test material may be coloured or become coloured in contact with water and/or isopropanol, it is necessary to test its potential interference with the MTT determination in these two conditions.
The ability of the test material to absorb significantly light at the wavelength used for MTT determination was tested.
The maximum amount of the test material, 51 mg (± 1 mg) was weighed and added first to 1 mL of water and incubated for at least one hour in the dark at +37°C, 5% CO2 and then to 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After that, the presence and intensity of the colouration were evaluated. If the solution changes colour significantly, additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.
MAIN TEST
PRE-INCUBATION
The tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The underside of each tissue was inspected for air bubbles between the agarose gel and the insert. Tissues with air bubbles under the insert covering greater than 50% of the insert area were not used. Any unusual observation was noted separately. The plastic bag containing the 24-well plate shipping container was disinfected by wiping with 70% ethanol-soaked tissue paper. Each 24-well shipping container was then removed from its plastic bag under sterile conditions. A volume of 1 mL assay medium pre-warmed (+37°C) was added to 2 wells per pre-labeled 6-well plate. The tissues were carefully removed from the 24‐well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze.
The insert was then transferred aseptically into the 6-well plate and pre-incubated at +37°C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
TREATMENT
Following the pre-incubation period, the tissues were pre-wetted with 20 μL of D-PBS. If the D-PBS do not spread across the tissue, the plate was tapped to ensure that the entire tissue surface was wetted. The tissues were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes.
As the test material was solid, the insert was removed from the medium and placed onto a sterile surface (e.g. the lid of a microtiter plate) to avoid that test material was spilled into the assay medium under the tissue insert. The test material was then applied by pouring it onto the tissue surface so that the surface was completely covered by the test material. The test material, negative and positive controls were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues. Inserts were then tapped on the wall of the plate to ensure that the items were applied evenly to the surface of each tissue.
The tissues were placed back into the assay medium after treatment with the test material.
All tissues (test material, negative and positive controls) were incubated at +37°C, 5% CO2 in a humidified incubator for 6 hours (± 15 minutes).
The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue receives an equal exposure period.
RINSING
At the end of the treatment period, each tissue was removed from the well of the treatment plate and rinsed to gently remove any residual test material or control items. A set of three clean beakers containing a minimum of 100 mL each of D-PBS was used per test material or control item. The test or control items were firstly removed from the tissue surface by tapping upside down each insert onto a clean absorbent paper. The tissues were then dipped into the first beaker of D-PBS, swirled in a circular motion during approximately 2 seconds, lifted out and decanted back into the beaker. This process was performed three times per beaker. Any remaining liquid was decanted onto an absorbent paper.
Residual amounts of test item were noted on all test item-treated tissues at the end of the rinsing step.
POST-SOAK AND POST-INCUBATION
The rinsed tissues were transferred to new wells of a pre-labelled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature. This incubation in assay medium was intended to remove any test material from the tissue.
Each tissue was incubated for 25 minutes (± 2 minutes) at room temperature to remove any solid test material or negative and positive controls from the tissue. Residual amounts of test material were still noted on all test material-treated tissues.
At the end of the post-soak immersion, each insert was blotted on absorbent material and transferred to appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 18 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator for test material, negative and positive controls. At the end of this incubation, residual amounts of test item were still noted on all test material-treated tissues.
MTT VIABILITY ASSAY
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labeled 24-well plates.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at +37°C, 5% CO2 in a humidified incubator.
At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. At this step, the residual amounts of test item were still observed on all test item-treated tissues.
For the negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well so that isopropanol was flowing into the insert on the tissue surface.
As the test material was a non-colourant solid, the inserts were transferred to a 6-well plate containing 2 mL of isopropanol in each well so that no isopropanol was flowing into the insert. This avoided any potential contamination of the isopropanol solution with any test material that may have remained on the tissue.
Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed during 2 to 3 hours at room temperature by placing the plates on an orbital plate shaker.
OD MEASUREMENTS
At the end of the formazan extraction period, tissues (test item, negative and positive control-treated tissues) were not pierced.
The extract solution was mixed using a pipette and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate), and a separate 96-well plate was used for test item-treated tissues.
For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.
DATA ANALYSIS
As the test material was found in the preliminary test not to have any colouring potential and any direct MTT reducing properties, no additional controls were run during the main test. Therefore, the mean blank OD value (mean ODblank) was calculated from the four replicates.
Then, the mean ODblank was subtracted from each OD value and the corrected mean OD values (mean cOD) of the two aliquots were calculated for each tissue.
The corrected mean OD of the two negative controls-treated tissues (mean cODNC) was set to 100% viability and was used as a reference.
For the tissues treated with the test material or negative or positive controls, the relative viabilities for each tissue were expressed as percentages of the reference viability and were calculated as follows:
TI relative viability (%) = (cODTI or NC or PC / mean cODNC) x 100
where
cODTI = corrected OD of each tissue treated with test material.
cODNC = corrected OD of each tissue treated with negative control.
cODPC = corrected OD of each tissue treated with positive control.
The difference of viability between the two tissue replicate was calculated.
ACCEPTANCE CRITERIA
- Acceptable variability between tissue replicates for positive and negative controls: Negative control acceptance criteria: mean cOD between 0.8 and 2.5. Positive control acceptance criteria: relative mean viability of the positive control is < 50% of the relative mean viability of the negative control.
- Acceptable variability between tissue replicates for the test chemical: Acceptable if the difference of viability between the two tissue replicates is < 20%.
- Data interpretation: A test substance is predicted as ocular irritant, if the mean relative tissue viability (%) of two tissues exposed to the test item is ≤ 60%.
CRITERIA FOR CLASSIFICATION:
- Mean viability is ≤ 60%: Irritant
- Mean viability is > 60%: Non-irritant - Irritation parameter:
- mean percent tissue viability
- Run / experiment:
- mean of duplicate tissues
- Value:
- 99
- 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:
- PRELIMINARY TESTS
- Visible damage on test system: none
- Test for direct MTT reduction with the test material: The MTT solution containing the test material did not turn blue/purple compared to the negative control. The test material was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
- Test for the detection of the colouring potential of the test material: During this test, as both water and isopropanol solutions containing the test material did not change colour, the test material was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.
MAIN TEST
The relative mean viability of the tissues treated with the test material was 99% with a difference of 3% between duplicate tissues.
As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 2.117.
- Acceptance criteria met for positive control: Yes. Mean viability observed in the positive control was 14%, whereas a mean viability of 100% was observed for the two tissue replicates in the negative control. The relative mean viability in the positive control was hence < 50% of that in the negative control.
- Acceptable variability between replicate tissues treated with test material: Yes. A difference of only 3% was obtained between the replicate tissues. - Interpretation of results:
- other: not classified according to EU criteria
- Conclusions:
- Under the conditions of this study, the test material is not classified as an eye irritant.
- Executive summary:
The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 492 under GLP conditions using a Reconstructed Human Cornea-like Epithelium assay.
Preliminary tests were performed to detect the ability of the test material to directly reduce MTT as well as its colouring potential.
Following the preliminary tests, the eye irritation potential of the test material was assessed in the main test.
The test material and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test material from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2 in a humidified incubator.
The cell viability was then assessed by means of the colorimetric MTT reduction assay.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
In the preliminary tests, the test material was neither found to have direct MTT reducing properties nor colouring potential.
In the main test, the relative mean viability of the tissues treated with the test item was 99% with a difference of 3% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.
Under the conditions of this study, the test material is not classified as an eye irritant.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.
- Reason / purpose for cross-reference:
- read-across source
- Irritation parameter:
- mean percent tissue viability
- Run / experiment:
- mean of duplicate tissues
- Value:
- 99
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Interpretation of results:
- other: not classified according to EU criteria
- Conclusions:
- Under the conditions of this study, the test material is not classified as an eye irritant.
- Executive summary:
The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 492 under GLP conditions using a Reconstructed Human Cornea-like Epithelium assay.
Preliminary tests were performed to detect the ability of the test material to directly reduce MTT as well as its colouring potential.
Following the preliminary tests, the eye irritation potential of the test material was assessed in the main test.
The test material and both negative and positive controls were applied topically on duplicate tissues and incubated at +37 °C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test material from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37 °C, 5% CO2 in a humidified incubator.
The cell viability was then assessed by means of the colorimetric MTT reduction assay.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
In the preliminary tests, the test material was neither found to have direct MTT reducing properties nor colouring potential.
In the main test, the relative mean viability of the tissues treated with the test item was 99% with a difference of 3% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.
Under the conditions of this study, the test material is not classified as an eye irritant.
Referenceopen allclose all
Table1: 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 |
3 |
4 |
1 |
- |
0.029 |
- |
- |
2 |
3 |
3 |
0 |
- |
0.021 |
- |
- |
|
3 |
3 |
3 |
0 |
- |
0.020 |
- |
- |
|
Mean |
- |
- |
0.3* |
- |
0.023** |
- |
0.7 |
|
Positive |
4 |
3 |
73 |
70 |
69.7 |
1.860 |
1.837 |
- |
5 |
3 |
74 |
71 |
70.7 |
1.570 |
1.547 |
- |
|
6 |
2 |
73 |
71 |
70.7 |
1.422 |
1.399 |
- |
|
Mean |
- |
- |
- |
70.3*** |
- |
1.594*** |
94.2 |
|
Test Material |
7 |
3 |
13 |
10 |
9.7 |
0.058 |
0.035 |
- |
8 |
2 |
13 |
11 |
10.7 |
0.033 |
0.010 |
- |
|
9 |
4 |
11 |
7 |
6.7 |
0.033 |
0.010 |
- |
|
Mean |
- |
- |
- |
9.0*** |
- |
0.018*** |
9.3 |
|
OD= Optical density
* = Mean of the post-treatment - pre-treatment values
** = Mean permeability
*** = Mean corrected value
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Read-across argument
The read across substances are structurally similar, both are ytterbium compounds that are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed for the read across substances as for the substance object of this registration.
An overview of the available information on the read across substances is provided below.
SKIN CORROSION / IRRITATION
Skin corrosion (Read-across from ytterbium triflouride, Lacey, 2017):
The potential of the test material to cause skin corrosion was determined in accordance with the standardised guidelines OECD 431 and EU method B40.bis, under GLP conditions using the EpiDerm Human Skin Model.
The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test material treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test material.
Before the main test was performed the test material was tested for 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. The test material was also assessed for its potential to cause colour interference with the MTT endpoint. 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.
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 562 nm (OD562).
The negative control, positive control and coefficient of variation were all within the acceptability criteria and so the study was valid.
Under the conditions of the study the test material was considered to be non-corrosive to the skin.
Skin irritation (Read-across from ytterbium triflouride, Lacey, 2017):
The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 using the human epidermis model, under GLP conditions.
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. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls.
Interference checks were performed before the main study. 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 solution prepared containing the test material was a white colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.
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 113.2% 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 all satisfied.
Under the conditions of the study, the test material was found not to be irritating to skin.
Skin irritation (Read-across from ytterbium oxide, Orovecz, 2016):
The skin irritation potential of the test material was determined in accordance with the standardised guidelines OECD 439 and EU Method B.46 using the human epidermis model, under GLP conditions.
The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
An in vitro skin irritation test of Diytterbium trioxide test material was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Disks of EPISKINTM (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% RH%. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO2 in a >95RH% protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test material is considered to be irritant to skin.
Following exposure with Diytterbium trioxide, the mean cell viability was 90.6% compared to the negative control. This is above the threshold of 50%, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test material was determined to be non-irritant to skin.
EYE IRRITATION
Eye irritation (Read-across from ytterbium triflouride, Lacey, 2017):
The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 437 and EU method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) test method.
The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability.
The 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, the study was performed in triplicate. Negative and positive control materials were tested concurrently, in triplicate also. 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 in vitro irritancy score for the test material was 9.3 and as a result it falls into the band of IVIS > 3 - ≤55 in the classification system and so no prediction of eye irritation can be made.
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.
Under the conditions of this study, as the test material induced an IVIS score of 9.3, no prediction on eye irritation can be made.
Eye irritation (Read-across from ytterbium oxide, Richez, 2017):
The potential of the test material to cause eye irritation was investigated under the standardised guidelines OECD 492 under GLP conditions using a Reconstructed Human Cornea-like Epithelium assay.
The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Preliminary tests were performed to detect the ability of the test material to directly reduce MTT as well as its colouring potential.
Following the preliminary tests, the eye irritation potential of the test material was assessed in the main test.
The test material and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 25 minutes at room temperature to remove any remaining test material from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2 in a humidified incubator.
The cell viability was then assessed by means of the colorimetric MTT reduction assay.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
In the preliminary tests, the test material was neither found to have direct MTT reducing properties nor colouring potential.
In the main test, the relative mean viability of the tissues treated with the test item was 99% with a difference of 3% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.
Under the conditions of this study, the test material is not classified as an eye irritant.
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.
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