Aluminium cerium lutetium oxide

Endpoint summary

Description of key information

The test item was not irritating to skin in the In Vitro Skin Irritation Test (Reconstructed Human Epidermis Model) according to OECD Guideline 439.

The test item was not irritating to eye in the Bovine Cornea Opacity and Permeability Assay (BCOP) according to OECD Guideline 437.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 16, 2015 - December 18, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SkinEthic Skin Irritation Test (42bis) Standard Operating Procedure (SOP): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

To reduce animal testing, this alternative in vitro method was used. The human skin RHE-model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number(s): 15-RHE-151
- Date of initiation of testing: December 16, 2015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 25 mL DPBS using a pipene

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.0 (Acceptance criteria: OD > 0.7)
- Barrier function: 5.4 h (Acceptance criteria: 4 h <= ET50 <= 10 h
- Morphology: 6.0 cell layers, absence of significant histological abnormalities, satisfactory (acceptance criteria: number of cell layers >= 4; absence of significant histological abnormalities; well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum)

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One experiment in triplicate

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL

Duration of treatment / exposure:

42 minutes (+/- 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (+/- 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1/experiment 1

Value:

2.111

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2/experiment 1

Value:

2.004

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3/experiment 1

Value:

2.105

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

The tissue viability after treatment with the test item was higher than 50% (mean viability: 88.73%). Therefore, the test item is not considered to possess an irritant potential to skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis (RHE) Test.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 μL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item, 10 μL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 2.074 (study acceptance criteria: > 1.423). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.10% (study acceptance criteria: <3.42%).

Therefore, the study ful fi lled the validity criteria.

The tissue viability after treatment with the test item was 88.73% and, thus, higher than 50%, i.e. according to UN GHS classification the test item is considered to be  not irritanting to skin (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15, 2016 - January 15, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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:

Commission Regulation (EU) No. 1152/2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals: Age: 14 - 36 months; Corneal diameter: 25 - 27 mm
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL of the suspended test item (i.e. 150 mg/750 µL)
- Concentration: 20% (w/v) suspension in a 0.9% sodium chloride solution

VEHICLE
- Amount applied: 750 µL

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without Stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES: 3


NEGATIVE CONTROL USED: No


SOLVENT CONTROL USED: 0.9% sodium chloride solution


POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of the suspended test item (i.e. 150 mg/750 µL) and 240 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments. To remove the solvent and positive control, the corneal surface was washed three times.

METHODS FOR MEASURED ENDPOINTS:

- Corneal opacity: calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: IVIS <= 3 (no UN GHS category), IVIS > 3; <= 55 (No prediction can be made), IVIS > 55 (UN GHS category 1)

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1/experiment 1

Value:

0.104

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2/experiment 1

Value:

2.391

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3/experiment 1

Value:

1.962

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

The IVIS obtained after treatment with the test item was 1.5 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.

Executive summary:

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Cornea Opacity and Permeability Assay).

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 μL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes.

After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro lrritancy Score (IVIS).

All validity criteria were fulfilled.

The IVIS obtained after treatment with the test was 1.5 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis (RHE) Test.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 μL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues.

The tissue viability after treatment with the test item was 88.73% and, thus, higher than 50%, i.e. according to UN GHS classification the test item is considered to be  not irritanting to skin (UN GHS: No Category).

Eye Irritation/Corrosion

This in vitro study was performed to evaluate the eye hazard potential of the test item by means of the BCOP (Bovine Cornea Opacity and Permeability Assay).

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. The incubation time was 240 minutes.

The IVIS obtained after treatment with the test was 1.5 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Based on the data provided, the test item is not classified and labelled for skin and eye irritation according to Regulation (EC) No 1272/2008.