Gadolinium zirconium oxide (Gd2Zr2O7)

Endpoint summary

Description of key information

The skin irritant potential of Gadolinium zirconium oxide (> 98% purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SMTM). Hereby, the test item was applied topically. In this study under the given conditions the test item showed no irritant effects (92.5 % mean relative tissue viability) after 15 min exposure and 42 h post-incubation. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”. The eye irritancy potential of Gadolinium zirconium oxide was investigated in a bovine corneal opacity and permeability assay according to OECD 437. Following an exposure period of 4 h a mean in vitro irritation score of 0.32 was calculated, clearly classifying Gadolinium zirconium oxide as non-irritant 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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-22 to 2016-09-22

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and 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: EPISKIN-SM^TM (SkinEthic)
- Tissue batch number(s): 16-EKIN-035

SkinEthic Kit:
- EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm²); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 16-EKIN-035):
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 16-MAIN3-056 / 16-MAIN3-060)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 16-ESSC-038)

- Validity controls as provided by the supplier (SkinEthic):
Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 22.5 ± 0.4, CV = 2.0%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n= 14):
Specification ≥ 1.5 mg/mL
Result: 1.8 mg/mL

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C, 5.0 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µL aqua dest.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat No. 14040-91, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium dodecyl sulfat (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277)
- Concentration (if solution): 5% in aqua dest

Duration of treatment / exposure:

15 min ± 0.5 miin

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3 tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

92.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

For detailed results see "Any other information on results" Table 1

Results of the Pre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC_livingequalled 0%.

Results of the main experiment

Table1:  Result of the test Item

 

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.742	0.816	0.676	0.069	0.086	0.086	0.746	0.625	0.724
	0.767	0.855	0.689	0.074	0.086	0.081	0.730	0.651	0.746
OD570(Blank-Corrected)	0.699	0.773	0.634	0.027	0.044	0.044	0.703	0.583	0.682
	0.724	0.813	0.647	0.031	0.044	0.039	0.688	0.609	0.704
Mean OD570Of The Duplicates (Blank-Corrected)	0.712	0.793	0.641	0.029	0.044	0.041	0.696	0.596	0.693
Total Mean OD570Of 3 Replicate Tissues (Blank-Corrected)	0.715*			0.038			0.662
SD OD570	0.076			0.008			0.057
Relative Tissue Viabilities [%]	99.5	110.9	89.6	4.1	6.1	5.8	97.3	83.3	96.9
Mean Relative Tissue Viability [%]	100.0			5.3**			92.5
SD Tissue Viability [%]^***	10.6			1.1			8.0
CV [% Viability]	10.6			20.6			8.6

^* Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is 40%.

^*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570 nmBlank	0.042	< 0.1	pass
Mean Absolute OD570 nmNK	0.757	0.6 ≤ NK ≤1.5	pass
Mean Relative Viability [%] PC	5.3	≤ 40%	pass
Max. SD of % Viability [%]	10.6	≤ 18%	pass

 

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In the present study the skin irritant potential of Gadolinium zirconium oxide (> 98% purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects (92.5 % mean relative tissue viability). As the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%, the test item is therefore classified as “non-irritant” in accordance with 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:

2016-08-09 to 2016-10-19

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)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL:
- 0.75 g of the test item was applicated directly onto the cornea and moistened with one drop of physiological saline 0.9 % NaCl (open-chamber-method). 750 µL of the control substance was introduced into the anterior chamber (closed-chamber-method)

VEHICLE
- Concentration (if solution): physiological saline (0.9 % NaCl)

Duration of treatment / exposure:

4 h ± 5 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 120 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

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 for controls
- Open chamber for the test item

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Washing with MEM containing phenol red until the medium was free of test substance (at least 3 times), then completly rinsed with RPMI without phenol red.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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. After the 4 hours incubation period and subsequent washing the anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490):
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 120 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 (Jenway 6405 UV/VIS).

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.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (triplicates)

Value:

0.32

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

Table 2: In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.33	0.05	1.02
2		0.33	0.035
3		0.41	0.048
MV		0.36	0.044
4	Positive Control	99.37	3.311	142.3
5		94.74	3.451
6		83.13	3.216
MV		92.41	3.326
7	Test Item	-0.21	0.034	0.32
8		0.71	-0.019
9		0.75	-0.033
MV		0.42	-0.0006

MV = mean value

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the mean in vitro irritation score of 0.32 obtained in the bovine corneal opacity and permeability assay (OECD 437), the test item is considered to be not irritating under the test conditions reported.

Executive summary:

The eye irritation potential of the test item (> 98% purity) was investigated in the bovine corneal opacity and permeability assay in accordance with OECD 437. The test item was applied directly to the cornea and moistened with one drop of 0.9% physiological saline. The corneal opacity was measured before and after treatment (4 hours). The mean in vitro irritation score was determined with 0.32. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, Gadolinium zirconium oxide can be considered as not irritating to the eye (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

The irritancy potential of Gadolinium zirconium oxide to the skin and eye was addressed by using two in vitro assays, the EPISKIN-SM, a reconstituted three-dimensional human epidermis model (OECD 439) and the bovine corneal opacity and permeability (BCOP) test (OECD 437).

In the in vitro skin irritation test, the target substance (> 98% purity) showed no irritant effects (92.5 % mean relative tissue viability) after 15 min exposure and 42 h post-incubation, classifying Gadolinium zirconium oxide as non-irritant to the skin.

The eye irritancy potential of the target substance (> 98% purity) was investigated in a bovine corneal opacity and permeability assay according to OECD 437. Following an exposure period of 4 h a mean in vitro irritation score of 0.32 was calculated, clearly classifying Gadolinium zirconium oxide as non-irritant to the eye.

Justification for classification or non-classification

The in vitro methods used are suitable methods for testing the irritancy potential of Gadolinium zirconium oxide to the skin and eye (OECD 439 and OECD 437). For both target organs, addressed by the methods used, the target substance can be considered as non-irritating. Therefore, based on the available data, Gadolinium zirconium oxide does not warrant classification for skin and/or eye irritation.