Gadolinium zirconium oxide (Gd2Zr2O7)

Endpoint summary

Description of key information

The dermal sensitization potential of Gadolinium zirconium oxide (>98% purity) was tested in a murine local lymph node assay according to OECD 429. Neither mortality nor any other adverse effects were observed during the study. Based on the results, the target substance is not a dermal sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-02 to 2017-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

OlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 18 - 22 g
- Housing: in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): ad libitum; Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum; tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days
- Indication of any skin lesions: Prior to the application all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

ENVIRONMENTAL CONDITIONS
- Full barrier in an air-conditioned room
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12; artifical light

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50, 100 % (w/v)

No. of animals per dose:

5 mice / group
5 mice / pre-screen test

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:
1. Solubilitiy test with the unground test item in AOO (4:1 acetone/ olive oil)
The obtained suspension could not be applied to the animal.
2. Solubility test with AOO (4:1 acetone/ olive oil)
Concentrations: 200, 150, 100, 50, 25 % (w/v)

- Irritation:
4 animals were treated by topical application with the test item (25 µL of the suspension, 2 mice per dose (100% (w/v), 50% (w/v))) on three consecutive days to the entire dorsal surface of each ear. One further animal was treated with the vehicle AOO and served as negative control.
- Systemic toxicity:
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
- Ear thickness measurements: day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6
- Erythema scores: see Table 1 in box "Any other information in material and methods"
Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%

For the results of the pre-screen tests please refer to the box "Any other information on results"

MAIN STUDY

- Controls
AOO was used as vehicle and served as negative control. Due to animal welfare the negative control was shared. A shared positive control was performed concomitantly (1% phenylenediamine in AOO was used as positive-control substance.
-Other Materials
3H-methyl thymidine (TRK 300, 20 Ci/mmol; PerkinElmer, lot no. 201604E), diluted to a working concentration of 80 µCi/mL.
Phosphate buffered saline (PBS), Eurofins Munich, lot no. 30082016, expiry date: 26/03/2017.
Trichloroacetic acid (TCA), Sigma-Aldrich, lot no. BCBN7603V, expiry date: 14/01/2017.
- Preparation of the Test Item
The preparations were made immediately prior to each dosing. All applied formulations were suspensions
- Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.). Identification was ensured by cage number and individual marking (tail).
- Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.
- Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).
- Test Regime
-> Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.
-> Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80µCi/mL.
-> Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
-> Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
- Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3= c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, sixth revised edition, 2015:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):

other: phenylenediamine

Positive control results:

The positive-control substance exceeded the stimulation index of 3 (see Table 2 in box "Any other information on results")

Parameter:

SI

Remarks:

mean of five animals

Value:

0.9

Variability:

SD = 0.2

Test group / Remarks:

25 % (w/v)

Parameter:

SI

Remarks:

mean of five animals

Value:

1.4

Variability:

SD = 0.7

Test group / Remarks:

50 % (w/v)

Parameter:

SI

Remarks:

mean of five animals

Value:

1.5

Variability:

SD = 0.4

Test group / Remarks:

100 % (w/v)

Cellular proliferation data / Observations:

The EC3 value could not be calculated as the stimulation idices of all concentrations were below 3. There was no mortality and there were no significant clinical observations or effects on body weights.

Results of the pre-screen tests

Solubility tests

At the concentrations of 200 % (w/v) and 150 % (w/v) the obtained suspension was too viscous, i.e. it was similar to a paste, and therefore could not be applied to the animal. At the concentration of 100 % (w/v) and lower in AOO a suspensions was obtained which was considered to be applicable to the animals.

The maximum technically applicable concentrations of the test item was found to be 100 % (w/v) in AOO.

Irritation and toxicity test

Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. All animals showed the expected weight development.

Results of the main study

Table 2: Stimulation Indices obtained for the Positive-Control Group

Test Item	Conc. [%]	Animal number	Stimulation Index
Negative Control (AOO)	100	101
		102
		103
		104
		105
		MV	1.0
		SD
Positive Control Phenylene-diamine in AOO	1	101	17.4
		102	10.2
		103	6.3
		104	5.1
		105	9.3
		MV	9.7
		SD	4.3

SD = standard deviation; MV = mean value

Table 3: Stimulation Indices obtained in the main experiment

Test Item	Conc. [%]	Animal number	Stimulation Index
Negative Control (AOO)	100	101
		102
		103
		104
		105
		MV	1.0
		SD
Gadolinium zirconium oxide (w/v) in AOO	25	1	0.9
		2	0.5
		3	0.7
		4	1.0
		5	1.2
		MV	0.9
		SD	0.2
Gadolinium zirconium oxide (w/v) in AOO	50	6	1.8
		7	2.5
		8	0.8
		9	1.1
		10	1.0
		MV	1.4
		SD	0.7
Gadolinium zirconium oxide (w/v) in AOO	100	11	2.2
		12	1.0
		13	1.2
		14	1.7
		15	1.2
		MV	1.5
		SD	0.4

SD = standard deviation; MV = mean value

Interpretation of results:

GHS criteria not met

Conclusions:

Gadolinium zirconium oxide is not sensitizing to the mouse skin under the conditions of this LLNA study.

Executive summary:

In a dermal sensitization study according to OECD 429 with Gadolinium zirconium oxide (> 98 % purity) suspended in AOO (4:1, acetone/olive oil), young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 25 % (w/v), 50 % (w/v) and 100 % (w/v) in a local lymph node assay (LLNA). Due to animal welfare the negative control (AOO) was shared and a periodically performed positive control (1% phenylenediamine in AOO) was used. There was no mortality and no significant clinical observations or effects on body weights were observed. None of the tested concentrations of the test substance reached the stimulation index threshold of 3. Therefore the EC3 value could not be calculated. In this study, Gadolinium zirconium oxide is not a dermal sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a dermal sensitization study according to OECD 429 Gadolinium zirconium oxide (target substance) was tested at concentrations of 25 % (w/v), 50 % (w/v) and 100 % (w/v). There was no mortality and no significant adverse clinical signs of toxicity or adverse effects on body weights were observed. None of the tested concentrations of the test substance reached the stimulation index threshold of 3. Based on the results Gadolinium zirconium oxide can be considered as not dermal sensitizing.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a dermal sensitization study according to OECD 429, female mice were tested negative for Gadolinium zirconium oxide. Based on the results, no classification of Gadolinium zirconium oxide is warranted.