lambda4-cerium(4+) zirconium(4+) tetraoxidandiide

Endpoint summary

Description of key information

Not sensitising (Modified Buehler test with a representative nanoform of the reaction mass). This conclusion is considered relevant for the bulk forms as well, should such forms be placed on the market.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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.

Key result

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

Key result

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% (w/w)

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% (w/w)

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.

Reference 2

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

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1982 - February 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Guinea Pig Maximisation Test (OECD TG 406) was selected based on the fact that insoluble inorganic forms such cerium dioxide are often not able to penetrate the skin. Furthermore, OECD Guideline 429 (LLNA) test was not validated in 1983.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Iffa-Crédo, France or Shamrock Farms, UK or Gwen Meur, France
- Age at study initiation: 5 to 7 weeks old
- Weight at study initiation: 300 to 500 g
- Housing: by group of 5 of the same sex, in polystyrene cages (54 x 36 x 31.5 cm)
- Diet: 50 g of pelleted diet/animal/day
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity: 50 ± 20%
- Air changes: 12 per hr
- Photoperiod: 12 hrs dark / 12 hrs light (7.30 am - 7.30 pm)

IN-LIFE DATES: From November 1982 To December 1982

Route:

intradermal

Vehicle:

water

Concentration / amount:

- Preliminary assay: 10%, 25% and 50% (w/w)
- Main assay: 50% (w/w) in treated group and vehicle only for control group

Day(s)/duration:

1 day

Adequacy of induction:

other: maximal non-irritating concentrations

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

- Preliminary assay: 0.5 g as such (100%) and 50% (w/w)
- Main assay: 0.5 g as such (100%) in treated group and vehicle only for control group

Day(s)/duration:

48 h

Adequacy of induction:

other: maximal non-irritating concentrations; skin pre-treated with 10% SLS

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

- Preliminary assay: 0.5 g as such (100%) and 50% (w/w) in water
- Main assay: 0.5 g as such (100%) in treated (left flank) group and vehicle only for control group (right flank)

Day(s)/duration:

1 day

Adequacy of challenge:

other: maximal non-irritating concentrations

No. of animals per dose:

- Preliminary assay: 6 per sex
- Main assay: 10 treated + 5 controls per sex

Details on study design:

RANGE FINDING TESTS:
6 animals per sex were used to determine maximal non irritating concentrations by intradermal route or topical occlusive patch application for 24 or 48 hours.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 series of 2 intradermal injections (adjuvant, test substance and test substance in adjuvant) + one 48-hour topical application (test substance)
- Exposure period: 1 days for intradermal injections, 2 days for topical applications
- Test groups: Freund's adjuvant + Test substance in vehicle
- Control group: Freund's adjuvant + Vehicle alone
- Site: Interscapular area
- Frequency of applications: single topical application
- Duration: 10 days total
- Concentrations: 50% (w/w) in water for intradermal injections, 0.5 g as such (100%) for dermal applications
- Before dermal application, 0.5 mL of a Sodium Lauryl Sulfate solution (10% in vaseline) were applied to the application site to create local irritation

B. CHALLENGE EXPOSURE
- No. of exposures: one 24-hour topical application (test substance)
- Day(s) of challenge: day 21
- Exposure period: 1 day
- Test groups: Vehicle alone + Test substance in vehicle
- Control group: Vehicle alone + Test substance in vehicle
- Site: Left flank (test substance) + right flank (vehicle)
- Concentrations: 0.5 g as such (100%)
- Evaluation (hr after challenge): 1, 6, 24 and 48

OTHER:
Macroscopic dermal reactions (erythema and edema) were graded using the following scale:
No reaction -> 0
Slight erythema -> 1
Moderate erythema -> 2
Sever erythema -> 3

Challenge controls:

Vehicle controls on the same animals (left flanks) + Control group (no contact with test substance during induction phase)

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

1

Group:

negative control

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 1.0. Group: negative control. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

1

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Slight skin erythema in 4 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 1.0. Group: negative control. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Slight skin erythema in 4 animals.

Reading:

1st reading

Hours after challenge:

6

Group:

negative control

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 6.0. Group: negative control. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

6

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Slight skin erythema in 5 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 6.0. Group: negative control. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Slight skin erythema in 5 animals.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 24.0. Group: negative control. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Slight skin erythema in 5 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 24.0. Group: negative control. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Slight skin erythema in 5 animals.

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 48.0. Group: negative control. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 48.0. Group: negative control. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

1

Group:

test chemical

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 1.0. Group: test group. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

1

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Slight to moderate skin erythema in 10 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 1.0. Group: test group. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Slight to moderate skin erythema in 10 animals.

Reading:

1st reading

Hours after challenge:

6

Group:

test chemical

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 6.0. Group: test group. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

6

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Slight to moderate skin erythema in 13 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 6.0. Group: test group. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Slight to moderate skin erythema in 13 animals.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 24.0. Group: test group. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Slight skin erythema in 8 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 24.0. Group: test group. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Slight skin erythema in 8 animals.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0 (vehicle alone, right flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 48.0. Group: test group. Dose level: 0 (vehicle alone, right flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Slight skin erythema in 3 animals

Remarks on result:

other: Reading: 1st reading. Hours after challenge: 48.0. Group: test group. Dose level: 0.5 g (left flank). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Slight skin erythema in 3 animals.

Group:

positive control

Remarks on result:

not measured/tested

Slight erythema (grade 1) on the treated flank (0.5 g as such) was noted among control animals between 1 hour and 24 hours following challenge, with incidences varying between 4/10 and 5/10 control animals. No sign of irritation was observed on the control flank (vehicle only) of the control animals.

Slight to moderate erythema (grade 1 or 2) on the treated flank (0.5 g as such) was noted among treated animals, with incidences of 10/20, 13/20, 8/20 and 3/20 animals at 1, 6, 24 and 48 hours following challenge, respectively.

No sign of irritation was observed on the control flank (vehicle only) of the treated animals.

None of these animals were considered to show positive skin sensitization reactions, using confirmation by histopathological examination.

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of delayed hypersensitivity in this Guinea Pig Maximisation Test using concentrations of 50% (w/w) in water (intradermal) or 0.5 g as such (epicutaneous).

Executive summary:

In a dermal sensitization study using the Guinea Pig Maximisation Test method (Magnusson & Kligman), Cerium Oxide was administered to 5 to 7-week old Dunkin-Hartley Guinea pigs (6/sex used in preliminary assay, 5 controls and 10 treated/sex). Induction phase consisted of intradermal injections of 0.1 mL of a 50% (w/w) suspension of Cerium Oxide in water, a suspension of Cerium Oxide in a 50% (v/v) mixture of complete Freund's adjuvant and saline, or Freund's adjuvant alone (vehicle alone and adjuvant in controls), followed by dermal application of 0.5 g (100%) of Cerium Oxide on a skin surface of ~8cm² previously irritated by Sodium Lauryl Sulfate, and kept under occlusive dressing for 48 hours. Following an 11-day resting period, the challenge phase consisted of a dermal application of 0.5 g of Cerium Oxide on a skin surface of ~4cm² kept under occlusive dressing for 24 hours, and application of the vehicle alone (same for controls). Skin reactions (erythema and edema) indicative of potential sensitization were monitored 1, 6, 24 and 48 h after occlusive dressing removal.

 

In the preliminary assay, slight irritation was observed following intradermal injection of 50% (w/w) suspension in water (3/4 animals) or 25% (w/w) suspension in water (1/4 animals). The concentration of 50% (w/w) in water was therefore selected for the main assay. The maximal non-irritating concentration for induction and challenge phases was selected at 100% (0.5 g of the test substance as such). In the main assay, minimal to slight erythema was noted only on the treated flank among test animals, mostly within 24 h after challenge application. None of the test animals were considered to be positive for sensitization using histopathological examination for confirmation.

 

The test substance was therefore considered as non-sensitizing. No classification for skin sensitization is warranted based on the absence of positive reactions in the Guinea Pig Maximisation Test, according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.

 

This study is classified as acceptable. It satisfies the OECD 406 guideline requirements for skin sensitization.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Popliteal Lymph Node Assay

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 1996 - August 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Similarly as LLNA, PLNA is based on 3H-thymidine incorporation measurements in draining lymph nodes following subcutaneous injection of the test substance in footpad.
Ig E determinations are an indication of immediate hypersensitivity (allergy) reactions where this immune mediator is released.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Popliteal Lymph Node Assay combined with IgE determinations

Species:

other: rat

Strain:

other: Brown Norway

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld Germany
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: ca. 150 g
- Housing: 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: > 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22.0 - 24.5°C
- Humidity: 48 - 83%
- Air changes: ca. 10 per hr
- Photoperiod (hrs dark / hrs light): Not specified

IN-LIFE DATES: From 18 June 1996 To 13 August 1996

RATIONALE FOR STRAIN SELECTION:
Brown Norway rats are known to be high Ig E responders.

No. of animals per dose:

- PLNA: 5 groups of 5 rats each
- IgE assay: 3 groups of 5 rats each

Details on study design:

PLNA:
- 5 groups of 5 rats each: 3 treated (Cerium Oxide suspended in DMSO), 1 positive control (TMA), 1 negative control (vehicle: DMSO)
- Tested concentrations: 1, 10 and 100 mg/mL, equivalent to 0.35, 3.5 and 35 mg/kg bw
- Technical procedure:
Day 0: subcutaneous injection (50 µL) into the right hind footpad
Day 7: intravenous injection (100 µCi/animal) of 3H-thymidine
Five hours later: euthanasia and sampling of ipsi- and controlateral popliteal lymph nodes (PLN)
PLN weight measurements
Calculation of PLN weight index = weight ratio of injected (ipsilateral) over non-injected (controlateral) PLN
Determination of 3H-thymidine incorporation by Liquid Scintillation
Calculation of PLN incorporation index = 3H-thymidine incorporation ratio of injected (ipsilateral) over non-injected (controlateral) PLN

Ig E assay:
- 3 groups of 5 rats each: 1 treated (Cerium Oxide suspended in DMSO), 1 positive control (TMA), 1 negative control (vehicle: DMSO)
- Tested concentration: 300 mg/mL, equivalent to 300 mg/kg bw
- Technical procedure:
Day 0: intradermal injections (2 x 50 µL) in the abdomen skin
Day 7: subcutaneous injection (50 µL) into the right hind footpad
Days 14, 21, 28 and 35: collection by orbital puncture of approximately 1 mL blood for determination of serum Ig E concentrations using a sandwich ELISA technique and determination of albumin/globulin ratios
Day 35: euthanasia and sampling of ipsi- and controlateral popliteal lymph nodes (PLN) for histopathological examination

Concentrations tested:
- PLNA: 1, 10 and 100 mg/mL, equivalent to 0.35, 3.5 and 35 mg/kg bw
- IgE assay: 300 mg/mL, equivalent to 300 mg/kg bw

Challenge controls:

Not relevant

Positive control substance(s):

yes

Remarks:

trimellitic anhydride (TMA) 100 mg/mL

Positive control results:

- PLNA:
TMA treatment resulted in consistently, although not statistically significant, increased PLN weights (mean PLN weight index = 3.00) and in a non-significantly increased 3H-thymidine incorporation in ipsilateral PLN.

- Ig E :
The TMA treated animals showed markedly and consistently increased serum Ig E levels from day 14 to day 35.
At microscopic examination, the ipsilateral PLN of TMA treated animals were distinctly activated as was apparent from a markedly increased germinal center development.

Remarks on result:

other: see section "Any other information on results"

- PLNA:

Comparison of the ipsilateral PLN weights, PLN weight index and relative changes showed no significant differences between treated and negative control groups (cf. table below).

Similarly for 3H-thymidine incorporation, no statistically significant differences between groups were observed. There was a non-significantly increased 3H-thymidine incorporation in the PLN from rats given Cerium Oxide but with no clear dose-response relationship (cf. table below).

 

	Lymph node weight (Mean ± SD)				3H-Thymidine incorporation (Mean ± SD)
Dose (mg/mL)	Left PLN (untreated side, mg)	Right PLN (treated side, mg)	PLN weight index	Relative difference (% vs. controls)	Left PLN (untreated side, dpm)	Right PLN (treated side, dpm)	PLN incorporation index	Relative difference (% vs. controls)
0 (vehicle)	7.0 ± 0.9	11.6 ± 3.4	1.71 ± 0.64	-	215 ± 146	669 ± 719	5.92 ± 8.54	-
1	10.9 ± 4.6	11.9 ± 9.9	1.58 ± 2.11	-7.6 ± 123.1	1065 ± 1173	2964 ± 5946	11.54 ± 24.46	94.8 ± 412.9
10	8.8 ± 2.2	13.7 ± 7.2	1.80 ± 1.39	4.7 ± 81.0	293 ± 70	2745 ± 2921	9.22 ± 9.94	55.6 ± 167.7
100	6.7 ± 2.0	11.1 ± 3.9	1.68 ± 0.55	-2.1 ± 32.1	154 ± 116	1620 ± 1569	15.15 ± 21.82	155.8 ± 368.3
Positive control (TMA 100 mg/mL)	6.0 ± 1.6	16.3 ± 4.2	3.00 ± 1.58	74.8 ± 91.9	321 ± 428	6137 ± 8168	24.81 ± 24.83	318.8 ± 419.2

 

- Ig E determinations:

Serum Ig E levels from animals given Cerium Oxide were similar to those of the negative control group.

At necropsy, all ipsilateral PLN of animals given Cerium Oxide showed a white discoloration. At microscopic examination, those PLN exhibited fine-granulated material which had accumulated predominantly at the base of the lymphoid follicles (in 3 rats) or in the paracortex and subcapsular sinus (in 2 rats), without associated germinal center development or plasma cell accumulation.

Mean globulin/albumin ratios in animals given Cerium Oxide were not statistically different from control rats on days 14, 21, 28 and 35.

	Serum IgE concentration (Mean ± SD)
Dose (mg/mL)	Day 14	Day 21	Day 28	Day 35
0 (vehicle)	620 ± 320	562 ± 113	604 ± 88	844 ± 521
300	494 ± 62	1744 ± 1706	1064 ± 589	498 ± 118
Positive control (TMA 100 mg/mL)	2342 ± 580**	2464 ± 971*	1984 ± 597**	1421 ± 621

 

* p < 0.05 ** p < 0.01

Interpretation of results:

GHS criteria not met

Conclusions:

Cerium Oxide administration did not result in significant increases in PLN weights or in statistically significant increases in 3H-thymidine incorporation. The serum Ig E concentrations were not significantly increased by Cerium Oxide administration. At microscopic examination, the Cerium Oxide particles were clearly present in the draining PLN of animals administered Cerium Oxide, but with no associated cell proliferation or increased antibody production.
Therefore, there was no indication that Cerium Oxide is able to generate an antigen specific immune response.

Executive summary:

In a Popliteal Lymph Node Assay (PLNA), the sensitization potential of Cerium Oxide was tested by administering the test substance at 0 (vehicle: DMSO), 1, 10 or 100 mg/mL (equivalent to 0, 0.35, 3.5 or 35 mg/kg bw, respectively) to 5 female Brown Norway rats per dose as a single subcutaneous injection into the right hind footpad. One week later, tritiated thymidine was intravenously injected, animals euthanasied and ipsi- and controlateral popliteal lymph nodes (PLN) sampled and weighed. 3H-Thymidine incorporation was determined by liquid scintillation. In addition, following a single intradermal injection and one week later a single subcutaneous injection into the right hind footpad of Cerium Oxide at 300 mg/mL (equivalent to 300 mg/kg bw), blood was sampled on days 14, 21, 28 and 35 for determination of Immunoglobulins (Ig)E using sandwich ELISA technique. On day 35, animals were euthanasied and ipsi- and controlateral PLN were sampled for histopathological examination.

In the PLNA phase, the comparison of the ipsilateral PLN weights, PLN weight index and relative changes versus controls showed no significant differences between treated and negative control groups. Similarly, for 3H-thymidine incorporation, no statistically significant differences between groups were observed. There was a non-significantly increased 3H-thymidine incorporation in the PLN from rats given Cerium Oxide but with no clear dose-response relationship.

In the IgE determination phase, serum Ig E levels from animals given Cerium Oxide were similar to those of the negative control group. At necropsy, all ipsilateral PLN of animals given Cerium Oxide showed a white discoloration. At microscopic examination, those PLN exhibited fine-granulated material which had accumulated predominantly at the base of the lymphoid follicles or in the paracortex and subcapsular sinus, without associated germinal center development or plasma cell accumulation.

Therefore, under the conditions of this study, there was no indication that Cerium Oxide is able to generate an antigen specific immune response.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999-03-08 to 1999-05-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

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. Moreover, the GPMT test is preferred specifically for zirconium compounds.

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 and epicutaneous

Vehicle:

physiological saline

Concentration / amount:

Intradermal sensitisation
1) Test agent group

- E-FCA (equal volume (v/v) of "distilled injection water" and Freund's complete adjuvant (FCA) were mixed and emulsified in water-in-oil style
- 2.5% test agent
- 2.5% test agent/FCA emulsion

2) Control group

- E-FCA

3) Positive control group

- E-FCA
- 0.1% DNCB/olive oil
- 0.1% DNCB/FCA emulsion

Patch sensitization
1) Test agent group
- 25% test agent
2) Control group
- distilled injection water
3) Positive control group
- 0.5% DNCB
Elicitation Treatment
1) Test group and control group
- 25% test agent
- 2.5% test agent
2) Positive control group
- 0.1% DNCB

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Intradermal sensitisation
1) Test agent group

- E-FCA (equal volume (v/v) of "distilled injection water" and Freund's complete adjuvant (FCA) were mixed and emulsified in water-in-oil style
- 2.5% test agent
- 2.5% test agent/FCA emulsion

2) Control group

- E-FCA

3) Positive control group

- E-FCA
- 0.1% DNCB/olive oil
- 0.1% DNCB/FCA emulsion

Patch sensitization
1) Test agent group
- 25% test agent
2) Control group
- distilled injection water
3) Positive control group
- 0.5% DNCB
Elicitation Treatment
1) Test group and control group
- 25% test agent
- 2.5% test agent
2) Positive control group
- 0.1% DNCB

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:
Supra scapular fur was shaved by an electric clipper in order to establish 24 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 Sensitization:
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 animals were applied with distilled injection water, the test agent group with 25 % test agent, and the positive control group with 0.5 % DNCB, by placing 24 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 were 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 22 cm lints moistened with 0.1 mL of the preparation, and by covering them with oilpaper 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 22 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
- 0.01 g of DNCB was dissolved in ethanol to make 10 ml.

Positive control substance(s):

yes

Remarks:

DNCB

Positive control results:

0.1% DNCB elicited region: Diffused moderate erythema or severe erythema and edema 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 were 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:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

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 surmised that TZ-3y does not have skin sensitising potential under the conditions of this test. On the other hand, it was confirmed that DNCB, the positive control agent, has an extreme skin sensitising potential.

Reference 6

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:

supporting study

Justification for type of information:

The in vivo skin sensitisation studies on cerium dioxide and zirconium dioxide (or rather zirconium dioxide with a small w/w % of yttrium oxide in it) are considered relevant for supporting the conclusion drawn from the study conducted on the reaction mass of cerium dioxide and zirconium dioxide itself.
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 and zirconium dioxide is considered not to be a skin sensitiser.

Remarks:

This conclusion is based on data obtained with the constituents of the reaction mass, i.e. cerium dioxide and zirconium dioxide (or, for this endpoint, zirconium dioxide with a small w/w % of yttrium oxide in it), which supported the results of the study performed with the reaction mass itself.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A skin sensitisation study (Klimisch 1) is available on a representative nanoform of the reaction mass of cerium dioxide and zirconium dioxide. Further information on skin sensitisation potential is also available on the bulk constituents of the reaction mass: cerium dioxide as well as zirconium dioxide (or, for this endpoint, rather zirconium dioxide with a small w/w % of yttrium oxide in it). As these constituents showed similar physicochemical, toxicological, ecotoxicological and environmental properties, results of studies performed on both constituents are used as supporting evidence.

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 GLP. This study was selected as the key study.

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 the dinitrochlorobenzene in a recent study.

 

Based on the results of this study, the reaction mass of cerium dioxide and zirconium dioxide is not classified as sensitising according to the CLP criteria. This conclusion is considered to hold for both the nanoforms and the bulk forms, as a representative nanoform was tested and no different response would be expected with larger particles. This is further supported by data on the individual constituents of the reaction mass (cerium dioxide and zirconium dioxide - for zirconium dioxide a study performed with zirconium dioxide containing a small % w/w of yttrium oxide in it was included).

The skin sensitisation potential of cerium dioxide was assessed in vivo in two distinct tests (Klimisch 1): one Guinea-Pig Maximisation Test (GPMT) and one Popliteal Lymph Node Assay (PLNA) in rats.

Results of both studies were consistent in the absence of skin sensitisation potential for cerium dioxide. In the GPMT, no test animals were positive for sensitisation and this was confirmed by performing histopathology. In the PLNA conducted on Brown-Norway rats, a rat strain well known for its high sensitivity to immunotoxicants, there were no significant differences in popliteal lymph node weights and tritiated thymidine incorporation, as well as blood Ig E concentrations, for animals treated up to 35 mg/kg bw, indicating that cerium dioxide is unlikely to be able to generate an antigen-specific immune response. Therefore cerium dioxide is considered not to be a skin sensitiser.

For zirconium dioxide, the study of the Japanese Chemicals Inspection and Testing Institute, 1999; Klimisch 2), performed on zirconium dioxide with a small w/w % of yttrium oxide in it, was included as supporting information. The traditional skin sensitisation test was performed according to OECD Guideline 406 and the Maximization Test of the Guideline for Toxicity Studies of drugs (Notification No. 1-24 of Pharmaceuticals and Cosmetics Division dated September 11, 1989). Female guinea pigs of the Hartley strain were treated by intradermal sensitisation and elicitation. The test material was found to be not sensitising to the skin at levels up to 25% whilst the positive control substance, DNCB, was found to have an extreme skin sensitising potential.

Skin sensitisation	Reaction mass (nano)	Cerium dioxide (bulk)	Yttrium zirconium oxide
Conclusion	Not sensitising	Not sensitising	Not sensitising
Method	Modified Buehler test	Guinea Pig Maximisation test and PLNA	Guinea Pig Maximisation test

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Not formally assessed but no indication of specific immunotoxicity in a 90-day inhalation study in rats (Viau, 1994) performed with cerium dioxide (which could be found at a level up to 80% in the reaction mass of cerium dioxide and zirconium dioxide).

Justification for classification or non-classification

Based on the CLP classification criteria, and given the absence of positive reactions in a modified Buehler test, the reaction mass of cerium dioxide and zirconium dioxide is not to be classified as a skin sensitiser. This conclusion is supported by results obtained for the individual constituents of the reaction mass.

No reliable data are available for respiratory sensitisation, therefore no conclusion can be drawn on the classification for this endpoint.