Cerium doped lutetium yttrium orthosilicate

Endpoint summary

Description of key information

In an acute dermal toxicity study conducted according to OECD 402, no indication of skin irritation up to the relevant limit dose level was observed. Thus, the substance can be considered as non-irritating to the skin and in accordance with Colum 2 of the standard information requirement 8.1 of the REACH Regulation 1907/2006 it is not necessary to conduct an in vitro skin irritation/corrosion study. The potential of LYSO to induce eye irritation (OECD 492, OECD 437) was tested in suitable in vitro test methods and the results from both in vitro eye irritation tests were assessed in a weight-of-evidence approach. Based on results, the substance can be considered as non-irritating to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)

Justification for type of information:

Referring to Column 2 of REACH Annex VII (standard information requirements for substances manufactured or imported in quantities of 1 tons per year or more) of Regulation (EC) No 1907/2006, studies on skin irritation/corrosion do not need to be conducted if an acute study by the dermal route does not indicate skin irritation up to the limit dose level of 2,000 mg/kg body weight. According to the results of the acute dermal toxicity limit test performed with lutetium-yttrium oxyorthosilicate, cerium doped performed in accordance with OECD Guidelines for Testing of Chemicals (Section 4, No. 402, "Acute Dermal Toxicity"), single dermal application of lutetium-yttrium oxyorthosilicate, cerium doped to rats at a dose of 2,000 mg/kg body weight was not associated with significant signs of skin irritation or corrosion (see section 7.2.3 of the IUCLID dossier). Based on these findings conducting of a study to determine the skin irritation potential of lutetium-yttrium oxyorthosilicate, cerium doped is considered as scientifically not necessary under Regulation (EC) No 1907/2006 (REACH).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-04-13 to 2016-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted: 26th July 2013

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: C14-193
- Purity: theoretical composition based on starting materials: 81.25% Lu2O3, 13.51% SiO2, 5.2% Y2O3 and 0.039% CeO2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability in water at room temperature: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: A. Moksel AG, Buchloe, Germany
- Number of animals: 3 corneas
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in the Hank’s balanced salt solution containing Pen/Strep on ice to the laboratories. Immediately after arrival of eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 1406788

Duration of treatment / exposure:

4 hours ± 5 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

The optical density was measured upon 90 minutes of incubation with sodium fluorescein solution after exposure to the test item.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The eyes were carefully examined for defects and defectives eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C.

TREATMENT METHOD: closed chamber

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I>I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 μL of the test item preparation or the control substance was introduced into the anterior chamber(closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32±1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4 hours incubation either the test substance or the control substance was removed and the epithelium washed at least three times with minimum essential medium (MEM) (containing phenol red).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1 (see "Any other information on materials and methods").
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490. The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

39.42

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no prediction can be made

Other effects / acceptance of results:

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
For detailed results please refer to Tables 2 to 4 in box “Any other information on results incl. tables”.

Table 2: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	2.77	3.05	0.27
2		3.24	4.51	1.27
3		2.66	3.89	1.23
MV		2.89	3.82	0.93
4	Positive Control	3.77	75.02	71.26	70.33
5		3.44	87.04	83.59	82.67
6		3.77	74.45	70.69	69.76
MV		3.66	78.84	75.18	74.25
7	Test Item	1.60	37.39	35.79	34.86
8		1.38	42.04	40.65	39.73
9		3.32	47.78	44.45	43.53
MV		2.10	42.40	40.30	39.37

MV = mean value

Table 3: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.013
2		0.010
3		0.020
MV		0.014
4	Positive Control	2.715	2.701
5		2.240	2.226
6		1.444	1.430
MV		2.133	2.119
7	Test Item	0.020	0.006
8		0.020	0.006
9		0.013	-0.001
MV		0.018	0.003

Table 4: In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.27	0.013
2		1.27	0.010
3		1.23	0.020
MV		0.93	0.014	1.14
4	Positive Control	70.33	2.701
5		82.67	2.226
6		69.76	1.430
MV		74.25	2.119	106.03
7	Test Item	34.86	0.006
8		39.73	0.006
9		43.53	-0.001
MV		39.37	0.003	39.42

MV = mean value

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, based on the mean in vitro irritation score of 39.42 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437) no prediction can be made regarding the classification of the test substance.

Executive summary:

The eye irritation potential of LYSO was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item was suspended with physiological saline 0.9 % NaCl to gain a 20 % concentration. A mean in vitro irritation score of 39.42 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of the test substance. For the purpose of classification, further testing with another suitable method is required.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-07-18 to 2017-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

adopted: 28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch-No.: C14-193; Source: Saint-Gobain Cristaux et détecteurs, 2, rue des Essarts, 38610 Gières, France
- Purity: theoretical composition based on starting materials: 81.25% Lu2O3, 13.51% SiO2, 5.2% Y2O3 and 0.039% CeO2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability in Water at Room Temperature: stable
- Storage Conditions: at room temperature


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Approximately 50 mg (83.3 mg/cm^2) of the test item was applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue.

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.
- Description of the cell system used: The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium that is morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

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): 50 mg

Duration of treatment / exposure:

6 ± 0.25 h

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation, the tissues were pre-treated with 20 μL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing, the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 10 min at 37 ± 1 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out either after storage overnight in the dark at 2 - 8 °C or immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27001),
1x bottle EpiOcularTM assay medium (Lot No.: 082117ISA),
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 0222172ZSB),

- Doses of test chemical and control substances used:
1. Negative Control 50 µL Aqua dest. (Sigma, Lot No.: RNBF7110)
2. Positive Control 50 µL methyl acetate (CAS No. 79-20-9; Merck, Lot No.: 56943111)
3. Test Item 50 mg

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm ± 30 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): UN GHS “Category 1” or “Category 2”
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”


Test Acceptance Criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positve control is < 50%
- relative tissue viability difference of replicate tissues is < 20%

Irritation parameter:

other: Relative tissue viability (%)

Run / experiment:

Mean of replicates

Value:

68.4

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:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (68.4 %). For detailed information please refer to Table 1 in box "Any other information on results incl. tables".

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
See also Table 2 in box "Any other information on results incl. tables".

Table 1: Result of the test substance, LYSO

Name	Negative Control	Positive Control	Test Item
Total mean OD570 of 2 replicate tissues (blank-corrected)	1.545*	0.389	1.057
SD OD570	0.02	0.10	0.09
Relative tissue viability difference [%]***	1.6	11.6	9.8
CV [% viability]	1.6	45.8	14.4
Mean relative tissue viability [%]	100.0	25.2**	68.4
NSCCV [%]	-	-	67.9

 

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

** mean relative tissue viability of the positive control is < 50%

*** relative tissue viability difference of replicate tissues is < 20%.

 

Table 2: Acceptance Criteria
		Value	Cut-off	pass/fail
Mean absolute OD570 NK		1.661	0.8 < NK < 2.5	pass
Mean relative viability PC [%]		18.6	< 50%	pass
Max difference of % viability [%]		18.6	< 20%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item, LYSO showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation.

Executive summary:

In the present study the eye irritant potential of the test item, LYSO was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 hours exposure and 18 hours post-incubation period and compared to those of the concurrent negative controls. The test item showed non-specific reduction of MTT and showed colouring potential after mixture with isopropanol. Therefore, NSCliving was determined (0.49%) and used for correction of the results. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (68.4 %). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an acute dermal toxicity study conducted according to OECD 402, no indication of skin irritation up to limit dose of 2000 mg/kg bw was observed. Thus, the substance can be considered as non-irritating to the skin. Referring to Column 2 of Annex VII standard information requirement 8.2 of the REACH Regulation 1907/2006, studies on skin irritation/corrosion do not need to be conducted as in an acute dermal toxicity no skin irritation up to the limit dose level of 2000 mg/kg body weight was observed.

The potential of LYSO to induce eye irritation (OECD 492, OECD 437) was tested in suitable in vitro test methods and the results from both in vitro eye irritation tests were assessed in a weight-of-evidence approach.

The eye irritation potential of LYSO (target substance) was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). A mean in vitro irritation score (IVIS) of 39.42 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of the test substance. Thus, a second in vitro eye irritation was conducted, namely the three-dimensional human corneal epithelium model EpiOcular (OECD 492). Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was >60% (68.4 %). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”. Based on these data, the substance can be considered as non-irritating to the eye, especially by consideration of the high false positive rate of 69% of the OECD 437 for substances, which do not require classification.

Justification for classification or non-classification

Based on the available data, no classification for skin and/or eye irritation is warranted.