Cerium dioxide

Endpoint summary

Description of key information

No skin senitisation oberved in a GMPT study and in a PLNA study by analogy to bulk CeO2.

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:

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

Remarks:

The study was performed before the implementation of GLP.

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:

non-irritant substance, but skin pre-treated with 10% SDS

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:

24 hours

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 treated animals (left flanks) + Control group (no contact with test substance during induction phase)

Positive control substance(s):

not specified

Remarks:

No positive control was reported in this study.

Positive control results:

No positive control has been reported in this study performed in 1983.

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

Reading:

1st reading

Hours after challenge:

1

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

Slight skin erythema (Grade 1) observed 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

Reading:

1st reading

Hours after challenge:

6

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

Slight skin erythema (Grade 1) was observed 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

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 g (left flank)

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

Slight skin erythema (Grade 1) was observed 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

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

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

Reading:

1st reading

Hours after challenge:

1

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

10

Total no. in group:

20

Clinical observations:

Slight to moderate (Grade 1 or 2) skin erythema in 10 animals.

Remarks on result:

no indication of skin sensitisation

Remarks:

Based on histological analysis.

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

Reading:

1st reading

Hours after challenge:

6

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

13

Total no. in group:

20

Clinical observations:

Slight to moderate (Score 1 to 2) skin erythema in 13 animals

Remarks on result:

no indication of skin sensitisation

Remarks:

Bases on histological analysis.

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

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

8

Total no. in group:

20

Clinical observations:

Slight skin erythema (Grade 1) in 8 animals

Remarks on result:

no indication of skin sensitisation

Remarks:

Based on histological analysis.

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

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5 g (left flank)

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

Slight skin erythema (Grade 1) in 3 animals

Remarks on result:

no indication of skin sensitisation

Remarks:

Based on histological analysis.

Group:

positive control

Remarks on result:

not measured/tested

Remarks:

No positive control were reported in this study.

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 after skin contact in guinea pig. No classification for skin sensitization is warranted based on the absence of positive reactions in the Guinea Pig Maximisation Test, according to the UN/EU GHS criteria.

 

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

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

PLNA

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

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

Justification for non-LLNA method:

At the time being, the LLNA guideline was not available.

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.

Vehicle:

dimethyl sulphoxide

Concentration:

- 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

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 determinati on 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

Positive control substance(s):

other: 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.

Parameter:

other: PLN incorporation index

Value:

ca. 5.92

Variability:

+/- 8.54

Test group / Remarks:

control (vehicle)

Parameter:

other: PLN incorporation index

Value:

ca. 11.54

Variability:

+/- 24.46

Test group / Remarks:

1 mg/ml

Parameter:

other: PLN incorporation index

Value:

ca. 9.22

Variability:

+/- 9.94

Test group / Remarks:

10 mg/ml

Parameter:

other: PLN incorporation index

Value:

ca. 15.15

Variability:

+/- 21.82

Test group / Remarks:

100 mg/ml

Parameter:

other: PLN incorporation index

Value:

ca. 24.81

Variability:

+/- 24.83

Test group / Remarks:

Positive control (TMA 100 mg/ml)

- 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 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Data from bulk cerium dioxide, that presents qualitatively similar properties with the nanoceria cerium dioxide, was used to cover this endpoint. See the Read-across justification document (Justification for category approach) attached in IUCLID Section 13.2 for the justification of the read-across.

Reason / purpose for cross-reference:

read-across source

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

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

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

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

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

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

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

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

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

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

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

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

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

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:

no indication of skin sensitisation

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

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:

no indication of skin sensitisation

Group:

positive control

Remarks on result:

not measured/tested

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of delayed hypersensitivity in this Guinea Pig Maximisation Test using concentration of 0.5 g as such of bulk CeO2. Based on the qualitatively similar properties between target and source substances, the same conclusions are assumed for nano cerium oxide.

Executive summary:

In a dermal sensitization study using the Guinea Pig Maximisation Test method (Magnusson & Kligman), Bulk 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.

Based on the qualitatively similar properties between target and source substances, the same conclusions are assumed for nano cerium oxide.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

PLNA

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Justification for type of information:

Data from bulk cerium dioxide, that presents qualitatively similar properties with the nanoceria cerium dioxide, was used to cover this endpoint. See the Read-across justification document (Justification for category approach) attached in IUCLID Section 13.2 for the justification of the read-across.

Reason / purpose for cross-reference:

read-across source

Parameter:

other: PLN incorporation index

Value:

ca. 5.92

Variability:

± 8.54

Test group / Remarks:

control (vehicle)

Parameter:

other: PLN incorporation index

Value:

ca. 11.54

Variability:

± 24.46

Test group / Remarks:

1 mg/ml

Parameter:

other: PLN incorporation index

Value:

ca. 9.22

Variability:

± 9.94

Test group / Remarks:

10 mg/ml

Parameter:

other: PLN incorporation index

Value:

ca. 15.15

Variability:

± 21.82

Test group / Remarks:

100 mg/ml

Parameter:

other: PLN incorporation index

Value:

ca. 24.81

Variability:

± 24.83

Test group / Remarks:

Positive control (TMA 100 mg/ml)

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.
Based on the qualitatively similar properties between target and source substances, the same conclusions are assumed for nano cerium oxide.

Executive summary:

In a Popliteal Lymph Node Assay (PLNA), the sensitization potential of bulk 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 andipsi- 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 bulk Cerium Oxide is able to generate an antigen specific immune response.

Based on the qualitatively similar properties between target and source substances, the same conclusions are assumed for nano cerium oxide.

Reference 5

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

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Data from bulk cerium dioxide, that presents qualitatively similar properties with the nanoceria cerium dioxide, as well as consideration of the poor dermal penetration of these inorganic insolubles forms (see section 7.1.2 Dermal absorption for nano-CeO2), were used to cover this endpoint. See the Read-across justification document (Justification for category approach) attached in IUCLID Section 13.2 for the justification of the read-across.

First, in a dermal sensitization study using the Guinea Pig Maximisation Test method (Magnusson & Kligman), Bulk 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.

Based on the qualitatively similar properties between target and source substances, the same conclusions are assumed for nano cerium oxide.

In a second study, the sensitization potential of bulk Cerium Oxide was tested using a Popliteal Lymph Node Assay (PLNA) test 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 andipsi- 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 bulk Cerium Oxide is able to generate an antigen specific immune response.

Based on the qualitatively similar properties between target and source substances, the same conclusions are assumed for nano cerium oxide.

Furthermore, inorganic and insoluble substances such as bulk and nano CeO2 are described as having a poor dermal absorption as observed by Mauro M. et al. (2019) in an OECD TG n°428 study using nano-Ce02. Thus, no or few systemic translocation of CeO2 from the skin to the blood is expected, resulting in the absence of CeO2 presentation to the immune system. Therefore, it is expected no induction of immune response and thus a skin sensitisation is not possible. The particle size seems to have no influence here as it is established that intact skin greatly restricts and, in most cases, prevents nanoparticle uptake. Nanomaterials dermally applied do not penetrate deeply the intact skin, at least not as for as the stratum (Yokel and MacPhail, 2011; Mauro M et al., 2019), from which nanomaterials could enter lymphatic or blood circulation. Thus, nano-CeO2 is expected to have no skin sensitising potential.

Moreover, some in vivo and in vitro data show that nanometric CeO2 seems to have no or rare immunological impact. Hirst SM et al. (2011) demonstrated that nano-CeO2 administered by intravenous (i.v.) or intraperitoneal (i.p.) route caused no induction of immune response in mice exposed weekly for 2 or 5 weeks to 0.5 mg nano-CeO2/kg. In addition, Schanen BC et al. (2013) showed that nano-CeO2 had no deleterious impact on immune cells (e.g. dendritic cells, lymphocytes) in vitro. Therefore, it can be hypothesised that nanometric CeO2 has no immunogenic potential, a determining characteristic for sensitisation.

Parameters	Hirst SM et al. (2011)	Schanen BC et al. (2013)
Supplier	None (in-house synthesis)	None (in-house synthesis)
Synthesis method	Wet-chemistry method	Wet-chemistry method
Primary particle size	3 to 5 nm	3 to 5 nm
Particle size distribution	10 to 50 nm	10 nm in culture medium
Stability	Agglomeration (~500 nm)	Agglomeration (~10 nm)
Specific surface area	No data	90 m²/g
Surface charge (zeta potential)	No data	-10.01 mV in culture medium
Isoelectric point	No data	No data
Shape	No data	Round
Crystallinity	Crystalline (fluorite)	Crystalline (fluorite)
Purity and impurities	No data	No data
Solubility	No data	No data
Oxidation degree	Mixing of Ce3+ and Ce4+, with higher abundance of Ce3+	No data
Surface properties	No data	(1) No endotoxin contamination (2) Reductive surface reactivity
pH of nano-CeO2 suspension	< 3.5	-

Based on the qualitative similar properties bewteen bulk and nano cerium dioxide and considering the above mentioned argumentation using the physico-chemical and toxicokinetic properties of nano-CeO2, no classification of nano-CeO2 for skin sensitisation is warranted according to the Regulation (EC) No. 1272/2008 (CLP / EU GHS) and UN GHS.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There is no data available on the respiratory sensitising potential of nanometric cerium dioxide (nano-CeO2). Thus, a classification of nano-CeO2 for respiratory sensitisation is not possible. However, several repeated dose toxicity studies performed on various nano-CeO2 are available (see Repeated dose toxicity section for details). Even though, the respiratory sensitisation potential of nano-CeO2 was not formally assessed in these published studies, there was no description of specific immunotoxicity and/or sensitising effect in rodents exposed by inhalation for up to 28 days.

Therefore, as for micrometric CeO2 (bulk CeO2), nano-CeO2 is expected to cause no respiratory sensitisation. Nevertheless, as the quality of the whole database is insufficient at the date, no definitive classification for respiratory sensitisation can be deduced.

Justification for classification or non-classification

The available data on nanometric cerium dioxide (nano-CeO2) suggest the substance has no potential for skin sensitisation. Furthermore, no skin sensitisation was observed in vivo after treatment with micrometric CeO2 (bulk CeO2). Thus, nano-CeO2 is not classified as a skin sensitizer according to classification criteria of Annex VI of Directive 67/548/EEC, Regulation (EC) No. 1272/2008 and UN GHS.

No reliable data is available on the respiratory sensitising potential of nano-CeO2; therefore no conclusion can be drawn on the classification of nano-CeO2 for this endpoint.