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Description of key information

Based on the results of the available in vivo skin sensitisation studies for praseodymium(III,IV) oxide (Henzell, 2012), yttrium zirconium oxide (Japanese Chemical Inspection and Testing Institute, 1999), and the reaction mass of cerium dioxide and zirconium dioxide (De Jouffrey, 1996d), it could be concluded that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is not a skin sensitiser and does not need to be classified for this endpoint under the CLP Regulation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 16-OCT-1995 to 6-MAR-1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Modified Buehler test
Justification for non-LLNA method:
Study was performed before the LLNA method became the preferred method for skin sensitisation testing.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Lebeau, 78950 Gambais, France
- Age at study initiation: 1 - 3 months
- Weight at study initiation: 320 +/- 25 g for the males; 328 +/- 17 g for the females
- Housing: Housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Sifted and dusted sawdust was provided as litter (SICSA, 92142 Alfortville, France). An analysis of potential residues and major contaminants is performed periodically (Laboratoire Wolff, 92110 Clichy, France).
- Diet: Free access to "guinea-pigs sustenance reference 106 diet" (U.A.R., 91360 Villemoison-sur-Orge, France). Food was periodically analysed (composition and contaminants) by the supplier.
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.
- Acclimation period: At least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 2 °C
- Humidity: 30 to 70%
- Air changes: The air was non-recycled and filtered
- Photoperiod: light/dark cycle: 12 h/12 h

IN-LIFE DATES: From: 13-NOV-1995 To: 14-DEC-1995
Route:
epicutaneous, occlusive
Vehicle:
other: aqueous solution of methylcellulose at 0.5%
Concentration / amount:
20% (w/w) in 0.5% methylcellulose aqueous solution
Amount applied: 0.5 mL
Day(s)/duration:
Day 1, 3, 5, 8, 10, 12, 15, 17 and 19 (6 h per exposure)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: aqueous solution of methylcellulose at 0.5%
Concentration / amount:
20% (w/w) in 0.5% methylcellulose aqueous solution
Day(s)/duration:
Day 28, 6 h of exposure
Adequacy of challenge:
not specified
No. of animals per dose:
No. of animals per dose: 10 animals for control group (5M/5F) and 20 animals for treated group (10M/10F).
Details on study design:
RANGE FINDING TESTS:
A preliminary test was performed to define the maximum concentration to be tested. 0.5 mL of the test substance at a concentration of 20% (w/w) in 0.5% methylcellulose aqueous solution was applied to a clipped area of the skin of approximately 4 cm2. The test substance was prepared on a dry gauze pad and then held in place by means of an occlusive dressing for 6 hours. 24 and 48 hours after application of the test substance, scoring of cutaneous reactions was performed. No residual test substance was observed upon removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 9
- Type of epicutaneous induction: occlusive
- SLS application: No
- Exposure period: On days 1, 3, 5, 8, 10, 12, 15, 17 and 19 (6 h per exposure)
- Test groups: 0.5 mL of the test substance at a concentration of 20% (w/w) in the vehicle. The maximum concentration of the test substance which could be obtained in the vehicle was 20% (w/w).
- Control group: vehicle alone
- Site: anterior left flank
- Frequency of applications: 3 times a week for 3 consecutive weeks
- Duration: 19 days. Following the induction period, the animals received no treatment for 10 days, from day 19 to day 28 inclusive.
- Concentrations: 20% (w/w) in 0.5% methylcellulose aqueous solution

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 29
- Exposure period: 6 h, right flank
- Test groups: Application of 0.5 mL of the test substance at a concentration of 20% (w/w) in the vehicle on an area of 4 cm2 (2 cm x 2 cm) of the posterior right flank (not treated before) under occlusive dressing.
- Control group: Application of 0.5 mL of the test substance at concentration of 20% (w/w) in the vehicle on an area of 4 cm2 (2 cm x 2 cm) of the posterior right flank (not treated before) under occlusive dressing.
- Site: right flank (test substance), left flank (vehicle)
- Concentrations: 20% (w/w) in the vehicle
- Evaluation: 24 and 48 hours after patch removal
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene
Positive control results:
The sensitivity of guinea pigs was checked with a positive sensitiser: 2,4-dinitrochlorobenzene. Induction application: from 0.5 to 0.1% (9 applications). Challenge application: 0.5% (right flank), paraffin oil (left flank).
Under the experimental conditions and according to the modified Buehler method (9 exposures), 2,4-dinitrochlorobenzene at a concentration of 0.5% (w/w) induced positive skin sensitisation reactions in 80% of the guinea pigs.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20% (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20% (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.5%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
not reported
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.5%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
not reported
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The test item (reaction mass of cerium dioxide and zirconium dioxide) is not to be classified as a skin sensitiser according to the results of the Modified Buehler test.
Executive summary:

The sensitisation potential of the reaction mass of cerium dioxide and zirconium dioxide was evaluated on Dunkin-Hartley guinea pigs by cutaneous application according to the modified Buehler test and in compliance with Good Laboratory Practice.

 

During the induction period, 10 male and 10 female animals received 0.5 mL of 20% w/w reaction mass of cerium dioxide and zirconium dioxide in 0.5 % methylcellulose aqueous solution, applied to the back on the left side of the spinal column. The substance was held in place for 6 hours with an occlusive dressing. This procedure was repeated 3 times a week for 3 weeks (total: 9 inductions). Control animals (5 males and 5 females) received the vehicle on the left flank using the same experimental conditions. After a 10-day rest period, a cutaneous challenge application of 0.5 mL of the test substance at a concentration of 20% (w/w) in 0.5% methylcellulose aqueous solution (right flank) and the application of the vehicle (left flank) were performed on a non-treated area of the posterior region of all the animals. The cutaneous reactions were evaluated 24 and 48 hours after the challenge application.

 

No clinical signs and no deaths were observed during the study. 24 and 48 hours after removal of the challenge pads, no cutaneous reactions were observed in both control and treated groups. The susceptibility of the guinea pigs lot was confirmed by the positive result obtained with dinitrochlorobenzene in a recent study.

 

The reaction mass of cerium dioxide and zirconium dioxide is not classified as a sensitiser according to the criteria laid down in the CLP Regulation.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint:
skin sensitisation, other
Remarks:
in vivo skin sensitisation (both LLNA and non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The in vivo skin sensitisation studies on praseodymium(III,IV) oxide, yttrium zirconium oxide (i.e. zirconium dioxide with a small amount of yttrium oxide added to its crystal lattice), and the reaction mass of cerium dioxide and zirconium dioxide are considered relevant for drawing a conclusion on the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reading:
other: read across conclusion
Remarks on result:
other: The reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is considered not to be a skin sensitiser.
Remarks:
This conclusion is based on the results of the in vivo studies performed with praseodymium(III,IV) oxide, yttrium zirconium oxide (i.e. zirconium dioxide with a small amount of yttrium oxide added to its crystal lattice), and the reaction mass of cerium dioxide and zirconium dioxide. The tests with the two latter substances were performed according to non-LLNA methods.
Parameter:
other: read across conclusion
Remarks on result:
other: The reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is considered not to be a skin sensitiser.
Remarks:
This conclusion is based on the results of the in vivo studies performed with praseodymium(III,IV) oxide, yttrium zirconium oxide (i.e. zirconium dioxide with a small amount of yttrium oxide added to its crystal lattice), and the reaction mass of cerium dioxide and zirconium dioxide. The test with praseodymium(III,IV) oxide was performed according to the LLNA method.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1999-03-08 to 1999-05-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study.
Qualifier:
according to guideline
Guideline:
other: Maximization Test of the Guideline for Toxicity Studies of Drugs (Notification No. 1-24 of Pharmaceuticals and Cosmetics Division dated September 11, 1989)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was performed before the LLNA method became the preferred method for skin sensitisation testing.
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 4 weeks old
- Weight at study initiation: 272 g - 302 g
- Housing: Five guinea pigs were kept together in each aluminum bracket cage (360 W x 520 D x 330 H mm, Bottom: 320 W x 480 D mm) until the elicitation treatment, then were kept individually in aluminum bracket cages (220 W x 380 D x 250 H mm) after the elicitation treatment. The cages were changed once a week.
- Diet: Solid feed (RC4, Oriental Yeast Col, Ltd)
- Water: Hita municipal water supply was used for water, which was provided freely by automatic water-supply equipments.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 2 degrees C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): 10 ~ 15 ventilation per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and dark period (light on at 7 am - off at 7 pm)

IN-LIFE DATES: From: 1999-02-23 To: 1999-05-24
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
2.5% w/v in physiological saline
Amount applied: 0.1 mL per injection
Day(s)/duration:
Day 1
Adequacy of induction:
not specified
Route:
epicutaneous, occlusive
Vehicle:
other: distilled injection water
Concentration / amount:
25% w/v in distilled injection water
Amount applied: 0.2 mL
Day(s)/duration:
Day 8, 48 h of exposure
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: distilled injection water
Concentration / amount:
25% and 2.5% w/v in distilled injection water
Amount applied: 0.1 mL
Day(s)/duration:
Day 22, 24 h of exposure
Adequacy of challenge:
not specified
No. of animals per dose:
Control group: 5 animals
Test agent group: 10 animals
Positive conrol group: 5 animals
Details on study design:
Intradermal sensitisation:
Suprascapular fur was shaved by an electric clipper in order to establish 2x4 cm sensitisation regions, and with the midline as the axis of symmetry, 0.1 mL of below preparations were injected per region of each left-right pair.
1) Test agent group
E-FCA
2.5% test agent
2.5% test agent/FCA emulsion
2) Control group
E-FCA (2 pairs in total)
3) Positive control group
E-FCA
0.1% DNCB/olive oil
0.1% DNCB/FCA emulsion

Patch sensitisation:
Six days after the intradermal sensitisation, the fur in the sensitisation regions of the animals in the control groups and the test agent group were shaved by an electric clipper and an electric shaver, then sodium lauryl sulfate (contains 10% petrolatum) was applied. Seven days after the intradermal sensitisation, the control group was applied with distilled injection water, the test agent group with 25% test agent, and the positive control group with 0.5% DNCB, by placing 2x4 cm lints (Nankai Sangyou Co.) moistened with 0.2 mL each of the preparation on the shaved sensitisation regions and by covering them with rubber dam sheets (Nihon Rikagaku Industry Co., Ltd.), then the trunks were wrapped with Dermicel (Johnson & Johnson K.K.) for 48 hours for occlusive dressing.

Elicitation treatment (challenge):
Fourteen days after the start of the patch sensitisation, the flank fur of the animals was shaved by an electric clipper and an electric shaver, and for the control group and the test agent group, the areas were applied with 25% test agent and 2.5% test agent to each group, respectively, by placing 2x2 cm lints moistened with 0.1 mL of the preparation, and by covering them with oil paper and rubber dam sheets, then the trunks were wrapped with Dermicel (Johnson & Johnson K.K.) for 24 hours for occlusive dressing. For the positive control group, 0.1% DNCB was applied by placing 2x2 cm lints moistened with 0.1 mL of the preparation and by covering them with rubber dam sheets, then the trunks were wrapped with Dermicel (Johnson & Johnson K.K.) for 24 hours for occlusive dressing.
Challenge controls:
25% test agent
- 2.5 g of the test agent was suspended in distilled injection water to make 10 mL.
2.5% test agent
- 0.25 g of the test agent was suspended in distilled injection water to make 10 mL.
0.1% DNCB (2,4-dinitrochlorobenzene, positive control substance)
- 0.01 g of DNCB was dissolved in ethanol to make 10 mL.
Positive control substance(s):
yes
Remarks:
DNCB (2,4-dinitrochlorobenzene)
Positive control results:
0.1% DNCB elicited region: Diffuse moderate erythema or severe erythema and oedema were acknowledged in every sample 24 and 48 hours after the elicitation patch removal. Furthermore, scab formations were acknowledged in 4/5 cases after both 24 and 48 hours, and desquamation was acknowledged in all cases after 48 hours. The average scores 24 and 48 hours after the elicitation patch removal were 3.0 and 2.8, respectively.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2.5% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2.5% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2.5% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2.5% w/v in distilled injection water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1% w/v DNCB in ethanol
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Diffuse moderate erythema or severe erythema and oedema were acknowledged in every animal. Furthermore, scab formations were acknowledged in 4 out of 5 animals.
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1% w/v DNCB in ethanol
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Diffuse moderate erythema or severe erythema and oedema were acknowledged in every animal. Furthermore, scab formations were acknowledged in 4 out of 5 animals, and desquamation was acknowledged in all cases.
Remarks on result:
positive indication of skin sensitisation

Skin Reaction

2.1 Test Agent Group

1) 25% test agent elicited region

No skin reaction was acknowledged 24 and 48 hours after the elicitation patch removal.

The average scores 24 and 48 hours after the elicitation patch removal were both 0.

2) 2.5% test agent elicited region

No skin reaction was acknowledged 24 and 48 hours after the elicitation patch removal.

The average scores 24 and 48 hours after the elicitation patch removal were both 0.

2.2 Control Group

1) 25% test agent elicited region

No skin reaction was acknowledged 24 and 48 hours after the elicitation patch removal.

The average scores 24 and 48 hours after the elicitation patch removal were both 0.

2) 2.5% test agent elicited region

No skin reaction was acknowledged 24 and 48 hours after the elicitation patch removal.

The average scores 24 and 48 hours after the elicitation patch removal were both 0.

Interpretation of results:
GHS criteria not met
Conclusions:
Since no skin reaction was acknowledged in the elicited region of either the test agent group or the control group, it was concluded that the test substance does not have skin sensitising potential under the conditions of this test. On the other hand, it was confirmed that 2,4-dinitrochlorobenzene, the positive control agent, has an extreme skin sensitising potential.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 July 2012 - 3 December 2012
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 - 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (e.g. ad libitum): ad libitum access to mains tap water
- Acclimation period: at least 5 days
- Source: Harlan Laboratories UK Ltd., Oxon, UK


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (0600 to 1800) and twelve hours darkness.

IN-LIFE DATES: From: 17 July 2012 To: 26 September 2012
Vehicle:
propylene glycol
Concentration:
25, 10 and 5% w/w
No. of animals per dose:
4 animals per dose
Details on study design:
PRELIMINARY SCREENING TEST
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL at a concentration of 25% w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer, pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN TEST
- Test Material Administration
The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

- 3H-Methyl Thymidine Administration
Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

- Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

- Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 x gravity) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 x gravity) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

INTERPRETATION OF RESULTS
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".
Positive control substance(s):
other: phenylacetaldehyde (CAS: 122-78-1) at 2.5% v/v in propylene glycol.
Positive control results:
The positive control was administered at 2.5% v/v in propylene glycol and produced a stimulation index of 11.23. This corresponds to a positive result.
Therefore, the positive control was found to be a sensitiser under the conditions of the test.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Disintegrations per minute and disintegration per minute per node are displayed in Table 1. The highest dpm was found in the 25% w/w group, which had a dpm of 14723.36 (dpm/node: 1840.42)
Parameter:
SI
Value:
1.37
Test group / Remarks:
5% of test item in propylene glycol
Remarks on result:
other: At the given concentration, the result was negative.
Parameter:
SI
Value:
1.31
Test group / Remarks:
10% of test item in propylene glycol
Remarks on result:
other: At the given concentration, the result was negative.
Parameter:
SI
Value:
1.43
Test group / Remarks:
25% of test item in propylene glycol
Remarks on result:
other: At the given concentration, the result was negative.

Table 1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in propylene glycol

dpm

dpm/node

Stimulation Index

Result

Vehicle

10311.57

1288.95

-

-

5

14177.08

1772.14

1.37

Negative

10

13556.23

1694.53

1.31

Negative

25

14723.36

1840.42

1.43

Negative

Positive Control

115794.60

14474.33

11.23

Positive

Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (the total number of lymph nodes).

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study.

 

Body weight

Body weight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of this study in accordance with the criteria of the CLP regulation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

1. Information on praseodymium(III,IV) oxide

In a study (Henzell, 2012; Klimisch 1) conducted to test the skin sensitisation properties of praseodymium(III,IV) oxide following OECD guideline 429 (LLNA), the results for all concentrations of the test material were negative. The positive control material yielded the expected result. Therefore, praseodymium(III,IV) oxide was considered to be a non-sensitiser under the conditions of this study in accordance with the CLP criteria.

2. Information on zirconium dioxide

Since no skin reaction was acknowledged in the elicited region of either the test agent group or the control group in the OECD 406 study (GPMT) performed by the Japanese Chemical Inspection and Testing Institute (1999; Klimisch 2 (non-GLP)), it was concluded that the test substance yttrium zirconium oxide (i.e. zirconium dioxide with a small amount of yttrium oxide incorporated into its crystal lattice) does not have any skin sensitising potential under the conditions of this test. On the other hand, it was confirmed that 2,4-dinitrochlorobenzene, the positive control agent, has an extreme skin sensitising potential. Since the result for the yttrium zirconium oxide test item was negative, it can be concluded that zirconium dioxide is also not a sensitiser.

3. Information on the reaction mass of cerium dioxide and zirconium dioxide

The skin sensitisation potential of the reaction mass of cerium dioxide and zirconium dioxide was evaluated on Dunkin-Hartley guinea pigs by cutaneous application according to the Modified Buehler test and in compliance with GLP (De Jouffrey, 1996d; Klimisch 1). In this study, no clinical signs and no deaths were observed, and 24 and 48 hours after removal of the challenge pads, no cutaneous reactions were observed in both control and treated groups. The susceptibility of the guinea pigs lot was confirmed by the positive result obtained with the 2,4-dinitrochlorobenzene in a recent study.

4. Conclusion on the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide

Based on the negative results obtained for praseodymium(III,IV) oxide, yttrium zirconium oxide (i.e. zirconium dioxide with a small amount of yttrium oxide incorporated into its crystal lattice), and the reaction mass of cerium dioxide and zirconium dioxide in the available in vivo skin sensitisation studies, it is safe to conclude that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide does not need to be classified for skin sensitisation either.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide does not need to be classified as skin sensitiser. This conclusion is based on the negative results of experiments performed with praseodymium(III,IV) oxide, zirconium dioxide (with a small amount of yttrium oxide incorporated into its crystal lattice), and the reaction mass of cerium dioxide and zirconium dioxide.

No information is available on respiratory sensitisation.