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REACH

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

EC number: 909-709-8 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Oral route

Since no studies are available on the reaction mass of cerium dioxide and zirconium dioxide, a weight-of-evidence approach was followed to cover the endpoint, using a study performed with (bulk) cerium dioxide (Davies, 2010a) and a study performed with (bulk) zirconium acetate (Rossiello, 2013), which can be considered as a representative zirconium compound.

Both studies yielded parental NO(A)EL values of >= 1000 mg/kg bw/day after repeated oral administration to rats. Based on these results it can be concluded that similar results could be expected with the reaction mass of cerium dioxide and zirconium dioxide in a similar OECD 422 study. No studies are available for nanoforms, however, such studies are available for the inhalation route of exposure, which is considered the most important route to be fully covered for repeated dose toxicity in order to be able to draw conclusions on the nanoforms of the reaction mass.

Inhalation route

The endpoint (including both sub-acute and sub-chronic toxicity) was covered using a weight-of-evidence approach, including studies performed with nano zirconium dioxide, nano cerium dioxide, bulk zirconium dioxide or bulk cerium dioxide. One of the studies also performed experiments with nano cerium dioxide containing 27 or 78% w/w of zirconium. The following results were obtained, in summary:

- Viau (1994; Klimisch 1) performed a sub-chronic (OECD 413) study in rats with bulk cerium dioxide and based on the results of this study, a LOAEC of 50.5 mg/m3 was derived, based on the incidence and severity of alveolar epithelial hyperplasia in the lungs.

- Landsiedel et al. (2014; Kimisch 2) performed a standard short-term inhalation study (STIS) in rats with nano cerium dioxide as well as nano zirconium dioxide in which rats were exposed during five days followed by three weeks recovery. Based on the results of this study, the NOAEC for nano zirconium dioxide was >= 10 mg/m3, based on the absence of any loco-regional effects in the lungs, whereas for nano cerium dioxide the NOAEC for loco-regional effects was set at <= 0.5 mg/m3, based on the observed increase in PMN neutrophil and lymphocyte count, and macrophage colony stimulating factor levels.

- Dekkers et al. (2017; Klimisch 2) performed sub-acute studies (cfr. OECD 412) in mouse with nano cerium dioxide and two forms of zirconium-doped cerium dioxide (containing 27% and 78% zirconium, respectively). In this study, the NOAEL was observed to be > 4 mg/m3 based on the general low toxicological activity of the different test materials in mouse.

- Spiegl et al. (1956; Klimisch 2 ) performed 30-day studies in dog, rabbit and rat and 60-day studies in cat, dog, guinea pig, rabbit and rat with bulk zirconium dioxide and did not observe any adverse effects in these studies, resulting in a 30-d NOAEC of >= 100.8 mg/m3 and a 60-d NOAEC of >= 15.4 mg/m3.

Based on these studies, it is clear that these materials (i.e. the individual constituents zirconium dioxide and cerium dioxide, as well as the reaction mass itself), regardless of their nano-status, are not capable of causing systemic toxicity after repeated inhalation exposure. Zirconium dioxide, whether bulk or nano, is consistently found to be toxicologically inert, not resulting in loco-regional adverse effects in the lungs either. For cerium dioxide, whether bulk or nano, loco-regional adverse effects have been consistently observed in rats, but not in mouse. The observed loco-regional effects of cerium dioxide in rats are considered to represent rather a non-specific adaptive response to particle overload of the lung and indicate the sensitivity of rats to this type of condition.

Further, it was demonstrated that the increased presence of zirconium in nano cerium dioxide did not result in the increased observation of obvious adverse effects in mouse.

Taking all evidence together, it is not considered necessary to perform further repeated dose inhalation toxicity studies with the reaction mass of cerium dioxide and zirconium dioxide, as this is not expected to bring further insights, and it is therefore not considered justifiable taking into account the obligation to avoid animal testing where necessary.

Dermal route

No data are available for repeated dose toxicity via the dermal route (for the reaction mass of cerium dioxide and zirconium dioxide and the two constituents separately).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 2008 - August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 10 weeks old
- Weight at study initiation: 402 - 454 g (males) / 244 - 285 g (females)
- Fasting period before study: no
- Housing: individual (except during pairing), in wire-mesh cages (43 x 21.5 x 18 cm) or polycarbonate cages (43 x 21.5 x 20 cm) for females during lactation
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 12 per hr
- Photoperiod: 12 hrs dark / 12 hrs light (7.00 am - 7.00 pm)

IN-LIFE DATES: From 14 May 2008 To 14 July 2008

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

0.5% solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item ground to fine powder using mortar and pestle, suspended in vehicle and homogenized by magnetic stirrer
- Dosing solutions prepared for use for up to 12 days and stored in brown flasks at +4°C, protected from light, prior to use

VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate for oral suspensions
- Concentration in vehicle: 30, 90 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Sigma batches 017K0052 and 066K0129 (methylcellulose)
- Concentration: 0.5% in water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the pre-study period, the homogeneity, stability and concentration of two dosage forms prepared at low and high concentrations (33.6 and 230 mg/mL) were checked using ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry) after validation of the analytical method. The results showed acceptable homogeneity and stability of both concentrations over 12 days of storage at +4°C protected from light.

During the study, the concentration of the test item (0, 33.6, 101.7 and 230 mg/mL in week 1 and 0, 30, 90 and 200 mg/mL in week 6) and homogeneity of the dosage forms were determined in samples of each control and test item dosage form prepared for use in weeks 1 and 6. The results showed acceptable homogeneity and concentration of all dosage forms analyzed. Precision (RSD ≤ 10%) and accuracy (100 ± 10%) of the method were found to be satisfactory. The homogeneity of the dosage form prepared for week 6 at 200 mg/mL was slightly outside the acceptance criteria with a RSD of 12.8% but this was considered to have no impact on the validity of the study.

Duration of treatment / exposure:

- Males: 15 days before mating, during mating (up to 3 weeks), until euthanasia (4 weeks total)
- Females: 15 days before mating, during mating (up to 3 weeks), during pregnancy, during lactation, until day 5 post partum inclusive

Frequency of treatment:

Once daily, 7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

168 mg/kg bw/day (nominal)

Remarks:

nominal up until day 3, due to an error of density measurement

Dose / conc.:

509 mg/kg bw/day (nominal)

Remarks:

nominal up until day 3, due to an error of density measurement

Dose / conc.:

1 150 mg/kg bw/day (nominal)

Remarks:

nominal up until day 3, due to an error of density measurement

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

nominal from day 4 up to the end of the study

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

nominal from day 4 up to the end of the study

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

nominal from day 4 up to the end of the study

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of a previous 10-day dose-range finding study (CIT No. 33178 TSR) at the same dose levels which elicited no treatment-related effects
- Rationale for animal assignment (if not random): computerized stratification procedure so that the average body weight of each group was similar

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (morbidity and mortality) or once daily (clinical signs)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before beginning of treatment period and once weekly during treatment period
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: on first day of treatment and then once weekly
Females: on first day of treatment and then once weekly until mated, and on days 0, 7, 14 and 20 post coitum and days 1 and 5 post partum

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
Males: once weekly over 7-day periods from first day of dosing
Females: once weekly over 7-day periods from first day of dosing through gestation and lactation
Not recorded during pairing period

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight for at least 14 hours)
- How many animals: first 5 males and 5 females to deliver in each group
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology, reticulocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight for at least 14 hours)
- How many animals: first 5 males and 5 females to deliver in each group
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, bile acid

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of treatment period (day 5 post partum for females)
- Dose groups that were examined: all groups (first 5 males and 5 females to deliver)
- Battery of functions tested: sensory activity / grip strength / motor activity / other: standard reflexes and responses, rectal temperature

Details:
The FOB included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity. The animals were randomized in order to ensure "blind" evaluation. All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
- "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Then, the following parameter measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.

OTHER: No other general toxicity parameter

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights (F0 generation, see table below)
- Macroscopic post-mortem examination (F0 generation)

HISTOPATHOLOGY: Yes
On all tissues specified (see table below), macroscopic lesions and females sacrificed because of absence of delivery to investigate possible causes

Statistics:

- Comparison of mean values by one-way variance analysis and Dunnett's test
- Percentage values compared by Fisher's exact probability test
- Specific sequences of tests used for clinical chemistry and organ weight data

Clinical signs:

no effects observed

Description (incidence and severity):

Isolated and non-dose-related incidences of soft feces, loud breathing, chromorhinorrhea and reflux at dosing were observed in all groups treated with Cerium oxide. Generally only one animal was affected and the signs were short-lived. It was considered that none of these represented signs of toxicity of the test item.
In addition, hairloss and cutaneous lesions were observed in the control group and the groups treated at 150 or 450 mg/kg/day. These signs are commonly observed in laboratory rats of this strain and are considered not to be related to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on mean male or female body weight or body weight gains during the premating, gestation or lactation phases.
The female group treated at 450 mg/kg/day had a slightly lower mean body weight gain over the gestation period when compared with the controls but the difference was mainly due to one female with a low body weight gain which skewed the group mean.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on mean male or female food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The males treated at 450 mg/kg/day showed increases when compared with the controls for red blood cell count, hemoglobin level and hematocrit. These differences were not statistically significant and were not observed at 150 or 1000 mg/kg/day. It was considered that the differences did not represent an effect of treatment with the test item.
The female group treated at 1000 mg/kg/day showed a statistically significant increase in hemoglobin concentration and monocyte count. Neither was observed in the males treated at the same dose-level and no related parameters were affected. It was considered that these differences were not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The male group treated at 1000 mg/kg/day had increased concentrations of creatinine and albumin (three or four of the males treated at 1000 mg/kg/day had values outside the control range). Related parameters (for example urea and total proteins) were not affected and the female group treated at the same dose-level did not show any differences to controls. It was considered that neither of these parameters had been affected by treatment with the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The first five males and the first five delivered females of each group treated with Cerium oxide were assessed for Functional Observation Battery. All groups achieved normal scores compared to the controls for touch escape, reactivity to handling, touch response, fur appearance, pupil size, grooming, palpebral closure, gait, posture, breathing, defecation, urination, visual stimulus response, pupillary reflex, auditory startle reflex, tail pinch response, forelimb grip strength, righting reflex, landing foot splay and rectal temperature.
All animals had absence of salivation, lacrimation, piloerection, exophthalmos, tremors, twitches, clonic or tonic convulsions, hyperactivity, hypoactivity, ataxia, hypotonia, stereotypy and abnormal behavior.
There were no marked differences in the mean number of movements made by the assessed males or females of all test item-treated groups when compared with the controls. No dose-relationship was observed and the small increases and decreases observed in all test item-treated groups were considered not to represent an effect of treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weight changes (principally of adrenals, spleen and thymus) were considered not to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

In a few rats given the test item, intestinal distension with gas was described in the cecum (1/10 high-dose males), colon (2/10 high-dose and 1/10 low-dose males), and ileum (2/10 high-dose and 1/10 low-dose males). In the absence of microscopic correlates, this finding was considered not to be of to xicological significance.
In one high-dose female there was a dilatation of the cervix associated with serous contents in the uterine horns. These findings were explained at microscopic examination by the presence of a congenital anomaly involving the cervix and distal vagina.
The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental. One mid-dose female showed a thymic mass which correlated with a chronic abscess at microscopic examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

A detailed, stage-aware qualitative evaluation of the testes was conducted in control and high dose males.
There were no microscopic findings related to the test item administration.
In the few low-, mid-, and high-dose females which were sacrificed because of no delivery, no estrous cycle abnormalities were found at microscopic examination of the genital organs. However, in one high-dose female, a congenital anomaly involving the cervix and distal vagina explained the absence of mating/pregnancy. Both the cervix and the distal vagina were dilated with presence of a large mucosal protrusion in the cavity.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOEL

Remarks:

parental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No relevant effects up to highest dose tested

Critical effects observed:

not specified

Conclusions:

No significant systemic toxicity was observed up to the highest tested dose, i.e. 1000 mg/kg bw/day. The NOEL for parental toxicity is >= 1000 mg/kg bw/day.

Executive summary:

In an OECD TG 422-compliant study, the potential general and reproductive or developmental toxicity of Cerium Oxide were tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0 (0.5% aqueous methylcellulose solution), 150, 450 or 1000 mg/kg (except for the first 3 days of dosing when an error in density measurements resulted in dose levels of slight overdosing at 168, 509 or 1150 mg/kg, respectively).

No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects on body weight, body weight gain or food consumption at any dose level. The Functional Observational Battery assessment, hematology and blood chemistry parameters revealed no treatment-related effects. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters. Pups showed no effects of treatment on survival or body weight performance. Macroscopic and microscopic examinations at necropsy did not reveal any treatment-related findings and there were no treatment-related changes in organ weights.

The NOELs for parental toxicity, for reproductive performance (mating and fertility) and for toxic effects on progeny were therefore all considered to be >= 1000 mg/kg.

No classification for repeat-dose toxicity or reproductive or developmental toxicity is warranted based on the absence of relevant effects in this study, 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 422 guideline requirements on repeated dose toxicity testing and reproduction/developmental toxicity screening.

Reference 2

Endpoint:: short-term repeated dose toxicity: oral
Remarks:: combined repeated dose and reproduction / developmental screening
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 2012-12-06 to 2013-02-07
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: Study performed under Good Laboratory Practices (GLP) and according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) without significant deviation.
Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 204.5 to 212.8 g (males); 164.8 to 180.2 g (females)
- Fasting period before study: none
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5 x 38 x 20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polycarbonate cages measuring approximately 43 x 27 x 18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating while females were transferred to individual solid bottomed cages for the gestation period, birth and lactation.
- Diet: ad libitum, except prior to drawing of blood for clinical chemistry examinations
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15%
- Air changes (per hour): 15 to 20
- Photoperiod (hours dark / hours light): 12/12
Route of administration:: oral: gavage
Vehicle:: other: purified water
Details on oral exposure:: PREPARATION OF DOSING SOLUTIONS
The required amount of zirconium acetate solution (containing 40.7% of zirconium acetate anhydrous) was dissolved in the vehicle (purified water) to obtain final concentrations of 10, 30 and 100 mg/mL. The formulations were prepared daily or up to 7 days before dosing according to stability data. The concentrations were calculated and expressed in terms of zirconium acetate content (40.7%).

VEHICLE
- Concentration in vehicle: 10, 30, 100 mg/L (expressed as active compound content)
- Amount of vehicle (if gavage): 10 mL/kg body weight (for males, dose volumes were adjusted once per week for each animal according to the last recorded body weight; for females, dose volumes were calculated according to individual body weight on days 0, 7, 14 and 20 post coitum and on day 1 post partum, thereafter individual dose volumes remained constant)
- Purity: not required
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (check of concentration). Samples of dosing formulations prepared on Weeks 1 and 5 [when 10 females per group were present] were also analysed to verify the concentrations. Samples of the formulations were collected and sent at ambient temperature to the analytical laboratory. Chemical analyses were carried out according to an Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) method.
Duration of treatment / exposure:: - Males were treated two weeks prior to pairing, throughout pairing and thereafter through the day before scheduled sacrifice (32 days of dosing).
- Females were treated two weeks prior to pairing, throughout pairing until day 3 post partum or the day before scheduled sacrifice (up to 50 days of dosing).
Frequency of treatment:: Once daily
Remarks:: Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
other: actual ingested (zirconium acetate anhydrous)
Remarks:: Doses / Concentrations:
53, 159, 530 mg/kg bw/day
Basis:
other: actual ingested (zirconium)
No. of animals per sex per dose:: 10
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a non-GLP 2 week preliminary toxicity study (RTC Study no. 94150EXT).
- Rationale for animal assignment: Rats were allocated to groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Rationale for selecting satellite groups: not applicable (satellite group not included)
- Post-exposure recovery period in satellite groups: not applicable (satellite group not included)
Positive control:: None
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked each morning and afternoon for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical sign was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
- Parameters checked: the weight of food consumed by each cage of males and females

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of the sacrificial procedure
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 per sex (females with viable litters if possible)
- Parameters checked: haematocrit; haemoglobin; red blood cell count; reticulocyte count; mean red blood cell volume; mean corpuscular haemoglobin; mean corpuscular haemoglobin concentration; white blood cell count; differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells); platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of the sacrificial procedure
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 per sex (females with viable litters i possible)
- Parameters checked: alkaline phosphatase; alanine aminotransferase; aspartate aminotransferase; gamma-glutamyltransferase; urea; creatinine; glucose; triglycerides; bile acids; phosphorus; total bilirubin; total cholesterol; total protein; albumin; globulin; A/G Ratio; sodium; potassium; calcium; chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: for males 5 days before necropsy and for females on Day 3 post partum.
- Dose groups that were examined: from each group, 5 males and 5 females were randomly selected.
- Battery of functions tested: grip strength; sensory reactivity to stimuli; motor activity assessment

FUNCTIONAL OBSERVATION BATTERY TESTS
- Time schedule for examinations: once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.
- Battery of functions tested: removal (from cage); handling reactivity; lachrymation; palpebral closure; salivation; piloerection; rearing; spasms; myoclonia; mobility impairment; arousal (animal activity); vocalisation; stereotypies; unusual respiratory pattern; bizarre behaviour; urination; defecation; tremors; gait.
Sacrifice and pathology:: SACRIFICE: Yes
- Males were killed after the mating of all females (after 32 days of treatment period).
- Females with live pups were killed on Day 4 post partum while females which did not give birth 25 days after positive identification of mating were killed shortly (Day 27 post coitum).
- All parental animals were killed by exsanguination under isofluorane anaesthesia.
- Pups were euthanised by intraperitoneal injection of thiopenthal.

TISSUE PRESERVATION: Yes
- Procedure: Samples of tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
- Organs / tissues preserved: all abnormalities; adrenal glands; bone marrow (from sternum); brain; caecum; colon; duodenum; heart; ileum; jejunum (including Peyer’s patches); kidneys; liver; lungs (including mainstem bronchi); lymph nodes - cervical ; lymph nodes - mesenteric; nasal cavity; oesophagus; pituitary gland; prostate gland; rectum; sciatic nerve; spinal column; spinal cord (cervical, thoracic, lumbar); spleen; stomach; thymus (where present); thyroid ; trachea; urinary bladder
- Reproductive organs / tissues preserved: epididymides; ovaries with oviducts; seminal vesicles with coagulating glands; testes; uterus - cervix; vagina.

GROSS PATHOLOGY: Yes
- Time schedule for examinations: Terminal sacrifice. All animals.
- Organs / tissues examined: All parent animals and pups wee examined macroscopically for any structural changes.
- Reproductive organs / tissues examined: Sexual organs were specifically examined. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY: Yes
- Time schedule for examinations: Tissues were collected from 5 males and 5 females (randomly selected) in the control and high dose group killed at terminal sacrifice and from all animals with abnormalities in all dose groups.
- Procedure: Tissues were dehydrated and embedded in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. Testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS) and morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
- Organs / tissues examined: all abnormalities; adrenal glands; bone marrow (from sternum); brain; caecum; colon; duodenum; heart; ileum; jejunum (including Peyer’s patches); kidneys; liver; lungs (including mainstem bronchi); lymph nodes - cervical ; lymph nodes - mesenteric; pituitary gland; prostate gland; rectum; sciatic nerve; spinal cord (cervical, thoracic, lumbar); spleen; stomach; thymus (where present); thyroid ; trachea; urinary bladder
- Reproductive organs / tissues examined: epididymides; ovaries with oviducts; seminal vesicles with coagulating glands; testes; uterus - cervix; vagina

ORGAN WEIGHT: Yes
- Time schedule for examinations: Organs were collected from all animals surviving the scheduled test period.
- Procedure: Organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
- Organs / tissues examined: adrenal glands; brain; heart; kidneys; liver; prostate gland; spleen; thymus
- Reproductive organs / tissues examined: epididymides; ovaries with oviducts; testes
Other examinations:: VAGINAL SMEARS: Yes
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine anomalies of the oestrous cycle and pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

REPRODUCTIVE INDICES: Yes
- The following reproductive indices were calculated: copulatory index; fertility index; pre-coital interval (mean number of days between pairing and mating); pre-implantation loss; pre-birth loss; pup loss at birth; cumulative pup loss on Day 4 post partum; sex ratios on Day 4 psot partum.

SPERMATOGENIC CYCLE: Yes
- A detailed qualitative examination of the testes was performed in control and high dose groups. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.
Statistics:: - Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
- Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
- The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
- The criterion for statistical significance was p<0.05
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: no effects observed
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: (effects cannot conclusively be attributed to treatment)
Urinalysis findings:: not examined
Behaviour (functional findings):: no effects observed
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: no effects observed
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: (toxicologically irrelevant)
Histopathological findings: neoplastic:: not examined
Details on results:: CLINICAL SIGNS AND MORTALITY
- No mortality occurred in the study.
- No clinical findings of toxicological significance were observed. Hair loss was occasionally recorded throughout the study including control animals. One female of the mid-dose group had salivation on Day 20 post coitum. One high dose female, that did not give birth, showed prolapse of the uterus on Day 27 post coitum. Another high dose female had rales during pairing.

BODY WEIGHT AND WEIGHT GAIN
- Body weight and body weight gain did not show relevant differences between groups. In particular, body weight gain was in some occasions higher in treated groups compared to the control group.

HAEMATOLOGY
- No changes of toxicological relevance were recorded.
- A statistically significant decrease of lymphocytes recorded in some females dosed with 300 mg/kg bw/day (up to 42% below controls) was not dose-related and, therefore, considered incidental.
- No changes were observed in the coagulation test.

CLINICAL CHEMISTRY
- A number of males dosed with 1000 mg/kg bw/day showed a slight decrease of protein and globulin (approximately 10%). Due to the low severity, these changes were considered of no toxicological importance.
- In addition, one animal showed high triglycerides (6.2 fold compared with controls). Due to the low incidence, this finding cannot be conclusively attributed to treatment; however, it also cannot be ruled out that it was related to treatment.

NEUROBEHAVIOUR
- Motor activity recorded at the end of treatment did not show significant differences between control and treated groups.

ORGAN WEIGHTS
- A slight significant reduction of epididymides weight occurred in the high-dose group; however, this reduction was found to be related to the higher terminal body weight in the high-dose group compared to controls. In addition, this change was minimal and no histological associated-findings were found. Therefore, it was considered of no toxicological relevance.

TERMINAL BODY WEIGHT AND ORGAN WEIGHTS
Body weight at term and organ weights did not show differences of toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Minimal, focal vacuolation of squamous epithelium (limiting ridge) of the non-glandular region of the stomach was observed in the high and mid-dose males with an increased incidence in the high dose males and similar severity levels across treatment groups. However, such gastric change was noted only in males, in a specific zone of the forestomach (limiting ridge) with focal and minimal severity and since humans do not have forestomach (squamous epithelium), such change could be considered toxicologically as a minor change.
- The remaining lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.

OTHER FINDINGS:
Spermatogenic cycle:
- A detailed qualitative examination of the testes was performed in control and high dose groups. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Dose descriptor:: NOAEL
Remarks:: (systemic effects)
Effect level:: >= 1 000 mg/kg bw/day (actual dose received)
Based on:: act. ingr.
Remarks:: anhydrous zirconium acetate
Sex:: male/female
Basis for effect level:: other: Based on a lack of toxicologically relevant systemic effects in male or female parental animals in any dose group.
Critical effects observed:: not specified
Conclusions:: On the basis of the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be >=1000 mg/kg bw/day (expressed as zirconium acetate anhydrous) for males and females.

Reference 3

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: supporting study
Justification for type of information:: Read across based on two OECD 422 studies performed with bulk cerium dioxide and zirconium acetate.
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
: Key result
Dose descriptor:: NOAEL
Effect level:: >= 1 000 mg/kg bw/day (nominal)
Based on:: test mat.
Remarks on result:: other: The reaction mass of cerium dioxide and zirconium dioxide is not expected to cause toxicologically relevant adverse effects in a short-term repeated dose toxicity test at a test dose of 1000 mg/kg bw/day.
Remarks:: Conclusion based on the results of the read across OECD 422 studies of Davies (2010a) performed with (bulk) cerium dioxide and of Rossiello (2013) performed with (bulk) zirconium acetate (as representative zirconium compound).
: Key result
Critical effects observed:: no

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 1993 - December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., St Constant, Quebec, Canada
- Age at study initiation: 7 weeks old
- Weight at study initiation: 206 - 270 g (males) / 135 - 179 g (females)
- Fasting period before study: no
- Housing: individually in stainless-steel wire mesh-bottomed cages
- Diet: ad libitum (except during exposure, urinalysis, prior to bleeding and prior to necropsy)
- Water: ad libitum
- Acclimation period: 2 (males) or 3 (females) weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 50 ± 20%
- Air changes: 12 - 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From August 31, 1993 To December 20, 1993

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Mass Median Aerodynamic Diameter (MMAD) ± Gravimetric Standard Deviation (GSD):
- Males at 0.0050 mg/L = 1.9 ± 1.9
- Females at 0.0050 mg/L = 1.8 ± 1.9
- Males at 0.0505 mg/L = 2.0 ± 1.9
- Females at 0.0505 mg/L = 2.0 ± 1.9
- Males at 0.5082 mg/L = 2.2 ± 1.8
- Females at 0.5068 mg/ L = 2.2 ± 1.8

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Four standard stainless-steel cylindrical "flow-through" nose-only inhalation chambers (approx. 80.6 L per chamber)
- Method of holding animals in test chamber: polycarbonate restraint cones
- Source and rate of air: laboratory compressed air supply
- Method of conditioning air: pre-dried compressed air
- System of generating particulates/aerosols: Venturi T-section (connected to powder feed nozzle and compressed air system)
- Temperature, humidity, pressure in air chamber: 20-24°C, 30-70% relative humidity, at least 19% O2
- Air flow rate: set at a level determined in preliminary work to be adequate to maintain chamber environment conditions
- Air change rate: at least 10 per hour
- Method of particle size determination: Andersen 1 ACFM cascade impactor operated at a flow rate of 28.3 L/min
- Treatment of exhaust air: purifying system

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric aerosol concentrations measured hourly using vertically oriented open-faced glass fiber filters. Test atmosphere continually monitored throughout exposure period by a precalibrated real-time aerosol monitor.
- Samples taken from breathing zone: yes

VEHICLE
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Filters from 2nd day of exposure and monthly thereafter used for chemical analysis by Sponsor.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Dose / conc.:

0.005 mg/L air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

0.051 mg/L air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

0.507 mg/L air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

5 mg/m³ air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

50.5 mg/m³ air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

507.5 mg/m³ air

Remarks:

Basis: mean achieved chamber concentrations

No. of animals per sex per dose:

Control animals:

yes

Details on study design:

- Dose selection rationale: Existing toxicity data, limitations imposed by the exposure apparatus and procedure as well as the stability of the experimental atmosphere and based on a TLV of 5 mg/m3 for respirable nuisance dust.
- Rationale for animal assignment (if not random): computer-based randomization procedure based on bodyweight (males and females separately)
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (within the cage) and immediately before, during (hourly) and after exposure (outside the cage)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, commencing on the day of randomization and extending through the treatment period (+ on days of behavioral testing and immediately before sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimation and at study termination
- Dose groups that were examined: low and high dose-groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 6 and at study termination
- Anaesthetic used for blood collection: No
- Animals fasted: Yes (overnight)
- How many animals: all surviving animals
- Parameters examined: red blood cell count, hemoglobin, hematocrit, erythrocyte indices, platelet count, mean platelet volume, white blood cell count (total and differential), prothrombin time, blood cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 6 and at study termination
- Animals fasted: Yes (overnight)
- How many animals: all surviving animals
- Parameters examined: Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), total bilirubin, cholesterol, triglycerides, glucose, blood urea nitrogen, creatinine, total proteins, albumin, globulin, albumin/globulin ratio, sodium, chloride, potassium, calcium, inorganic phosphorus

URINALYSIS: Yes
- Time schedule for collection of urine: in week 6 and at study termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: color and appearance, pH, glucose, ketones, hemoglobin, volume, specific gravity, bilirubin, urobilinogen, proteins, nitrite, microscopy of centrifuged deposit

NEUROBEHAVIOURAL EXAMINATION: Yes (Functional Observational Battery)
- Time schedule for examinations: during acclimation, on day 1 (post dosing) and once during each of weeks 4, 8 and 13
- Dose groups that were examined: all animals
- Battery of functions tested:
* Sensory activity: observations in home cage (body position, tremors, twitches, convulsions, bizarre/stereotypic behavior), removal from home cage (ease of removal, vocalization), observations in arena (rearing, ataxic, hypotonic and impaired gait, overall gait incapacity, bizarre/stereotypic behavior, palpebral closure, tremors, twitches, convulsions, piloerection, respiratory rate/pattern, locomotor activity level, arousal, grooming, defecation, urination, olfactory response), handling observations (lacrimation, pupil size, salivation, urinary staining, diarrhea, body and abdominal tones, extensor thrust, corneal reflex, pinna reflex, toe and tail pinch, visual placing), on surface (auricular startle, air righting reflex), on top of box (positional passivity)
* Grip strength: forelimb, hindlimb, hindlimb splay
* Motor activity: activity counts recorded by microcomputer in 6 successive 10-minute sessions
* Other: body temperature

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (external examination, including identification of all clinically recorded lesions, and detailed internal examination)
ORGAN WEIGHTS: Yes (adrenals, brain, heart, kidneys, liver, lungs, ovaries or testes, pituitary, prostate, spleen, thymus, thyroid, uterus)
HISTOPATHOLOGY: Yes (following standard list of tissues + abnormalities): adrenal, aorta, bone marrow, sternum, brain, bronchus, nasal cavity, cecum, colon, duodenum, epididymis, esophagus, eye, heart, ileum, jejunum, kidney, larynx, liver, lung, mandibular lymph node, mesenteric lymph node, mammary gland (M&F), skeletal muscle , optic nerve, sciatic nerve, pancreas, parathyroid, pharynx, pituitary gland, prostate, salivary gland, seminal vesicle, skin, cervical spinal cord, spleen, stomach, testis, thymus, thyroid, tongue, trachea, urinary bladder, bronchial lymph node, mediastinal lymph node, pancreatic lymph node, ovary, uterus.

Other examinations:

Not applicable

Statistics:

- Group mean values (with standard deviations) analyzed for homogeneity of variance using Bartlett's test
- Homogeneous data analyzed using Analysis of Variance and differences from controls assessed using Dunnett's 't' test
- Heterogeneous data analyzed using Kruskal-Wallis test and differences from controls assessed using Dunn's test
- Frequency data, gross pathology and histopathology findings analyzed using Fisher's exact probability test

Clinical signs:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Mortality:

no mortality observed

Description (incidence):

No relevant (treatment-related) effects were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- No relevant changes in females
- Statistically significantly lower mean body weight gains in high-dose males when compared to controls in weeks 2 and 8
- Overall body weight gain of high-dose males marginally inferior to that of controls, although not statistically significant

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- No relevant changes in females
- Food consumption of high-dose males marginally lower than that of controls although without statistical significance

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- No relevant changes in red blood cell count, hemoglobin, hematocrit, erythrocyte indices, platelet count, mean platelet volume, total or differential white blood cell counts except neutrophil counts, prothrombin time, blood cell morphology
- Statistically significantly elevated segmented neutrophil counts (when expressed as percentages of white blood cells) in low-dose and mid-dose females and high-dose males and females at weeks 6 and/or 13 compared to controls
- When expressed in absolute terms, segmented neutrophil counts in low-dose females and mid-dose and high-dose males and females elevated at weeks 6 and 13 compared to controls

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- No relevant changes in adrenals, brain, heart, kidneys, liver, ovaries or testes, pituitary, prostate, spleen (females), thymus, thyroid or uterus weights
- Statistically significant higher lung (absolute and relative) weights in mid-dose and high-dose groups compared to controls
- Change in lung weight seen in all treated groups, although not always statistically significant
- Statistically significantly higher spleen (relative) weights and higher spleen (absolute) weights in high-dose males compared to controls

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- No relevant findings at external examination, including identification of all clinically recorded lesions, and at detailed internal examination, except for lungs and lymph nodes
- Lungs: discoloration or pale areas (30 rats each in mid-dose and high-dose groups), pale foci (4 rats in low-dose group), uncollapsed parenchyma (2 rats in mid-dose group and 30 in high-dose group)
- Lymph nodes: enlargement and/or pale discoloration of the bronchial lymph nodes (28 rats in low-dose group, 30 each in mid-dose and high-dose groups), mediastinal lymph nodes (1 rat in control group, 12 in low-dose group, 18 in mid-dose group and 20 in high-dose group), pancreatic lymph nodes (3 rats in control group, 1 each in mid-dose and high-dose groups)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- No relevant findings in adrenals, aorta, bone marrow, sternum, brain, cecum, colon, duodenum, epididymis, esophagus, eye, heart, ileum, jejunum, kidney, mesenteric lymph node, mammary gland, skeletal muscle , optic nerve, sciatic nerve, pancreas, parathyroid, pharynx, pituitary gland, prostate, salivary gland, seminal vesicle, skin, cervical spinal cord, stomach, testis, thymus, thyroid, tongue, urinary bladder, ovary, uterus
- Pigment accumulation and/or alveolar epithelial/lymphoid hyperplasia in the lungs (in low-dose, mid-dose and high-dose groups)
- Lymphoid hyperplasia of the bronchial or mediastinal (in low-dose, mid-dose and high-dose groups) and pancreatic (in mid-dose group) lymph nodes
- Metaplasia and/or pigment accumulation in larynx (in low-dose, mid-dose and high-dose groups)
- Pigment accumulation in bronchial or mediastinal lymph nodes, nasal cavity and bronchi (in low-dose, mid-dose and high-dose groups), in trachea and pancreatic lymph nodes (in mid-dose and high-dose groups), and in liver, mandibular lymph nodes and spleen (high-dose group only)

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOEC

Effect level:

0.507 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical biochemistry

clinical signs

mortality

ophthalmological examination

urinalysis

Dose descriptor:

NOEC

Effect level:

0.507 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOEC

Effect level:

0.051 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male

Basis for effect level:

haematology

Dose descriptor:

LOAEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

female

Basis for effect level:

haematology

Dose descriptor:

NOAEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

LOAEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

LOAEC

Effect level:

0.051 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Tissue/Lesion	Incidence of microscopic findings (/No. of animals examined)
	Males - 0 mg/L	Females - 0 mg/L	Males - 0.005 mg/L	Females - 0.005 mg/L	Males - 0.0505 mg/L	Females - 0.0505 mg/L	Males - 0.5075 mg/L	Females - 0.5075 mg/L
Bronchi: pigment accumulation	0/15	0/15	1/15	0/15	5/15*	4/15*	15/15*	15/15*
Nasal cavity:
- Goblet cell hypertrophy and/or hyperplasia	0/15	0/15	0/15	1/15	0/15	0/15	1/15	2/15
- Pigment accumulation	0/15	0/15	12/15*	3/15	15/15*	11/15*	15/15*	15/15*
Larynx:
- Metaplasia	0/15	0/15	3/15	3/15	9/15*	6/15*	13/15*	9/15*
- Pigment accumulation	0/15	0/15	6/15*	0/15	9/15*	7/15*	12/15*	9/15*
Liver: pigment accumulation	0/15	0/15	0/9	0/1	0/7	0/5	6/15*	5/15*
Lungs:
- Pigment accumulation	0/15	0/15	15/15*	15/15*	15/15*	15/15*	15/15*	15/15*
- Alveolar epithelial hyperplasia (with severity)	0/15	0/15	1/15(+)	0/15	11/15* (+ to ++)	5/15* (+ to ++)	14/15* (+ to +++)	15/15* (+ to ++)
- Lymphoid hyperplasia	0/15	0/15	0/15	0/15	0/15	1/15	12/15*	7/15*
Mandibular lymph nodes: pigment accumulation	0/15	0/15	0/3	0/5	0/5	0/3	6/15*	6/15*
Spleen: pigment accumulation	0/15	0/15	0/1	0/0	0/0	0/0	6/15*	3/15
Trachea: pigment accumulation	0/15	0/15	0/15	0/15	1/15	1/15	14/15*	14/15*
Bronchial lymph nodes:
- Pigment accumulation	0/15	0/15	13/13*	14/15*	15/15*	15/15*	15/15*	15/15*
- Lymphoid hyperplasia	0/15	0/15	11/13*	13/15*	15/15*	15/15*	15/15*	15/15*
Mediastinal lymph nodes:
- Pigment accumulation	0/0	0/1	2/2	10/10	8/10	9/9	9/9	9/10
- Lymphoid hyperplasia	0/0	0/1	2/2	10/10	9/10	9/9	9/9	9/10
Pancreatic lymph nodes:
- Pigment accumulation	-	0/2	-	0/0	-	1/1	-	1/1
- Lymphoid hyperplasia	-	0/2	-	0/0	-	1/1	-	0/1

* p < 0.05

+: slight

++: mild

+++: moderate

Conclusions:

An overall NOEC was not established in the study report based on changes in hematology (females only), macroscopic observations at necropsy and histopathology at the lowest concentration tested, i.e. 0.005 mg/L. However, considering that lymphoid hyperplasia in the bronchial lymph nodes may not represent a specific toxic effect, but rather a non-specific adaptive response to the overloading of pulmonary alveolar macrophages by inorganic poorly soluble particles, and considering that alveolar epithelial hyperplasia in the lungs represents a more sensitive indication of adverse effects in the rat following inhalatory exposure to very high particulate concentrations, the LOAEC can be set at 0.0505 mg/l (50.5 mg/m3) based on the incidence and severity of alveolar epithelial hyperplasia in the lungs.

Executive summary:

In an OECD TG 413 compliant study, the potential general toxicity of Cerium Oxide was tested following repeated nose-only inhalation of a dry powder aerosol of cerium dioxide (mass median aerodynamic diameter = 1.8-2.2 µm, geometric standard deviation = 1.8-1.9) to 7-week old Sprague-Dawley rats (15/ sex) for 6 hours a day, 5 days a week, for 13 weeks, at the gravimetric concentrations of 0 (air), 0.005, 0.0505 or 0.5075 mg/L (0, 5, 50.5 or 507.5 mg/m3, respectively). Observations and measurements included mortality, clinical signs, body weight, food consumption, Functional Observational Battery, motor activity, hematology, clinical biochemistry, urinalysis, ophthalmological examination, gross pathological examination, organ weights and histopathological examination of selected tissues.

No treatment-related deaths or clinical signs occurred during the study. There were no effects on ophthalmology, clinical chemistry, urinalysis at any dose level. No behavioural changes following either acute or subchronic exposure and no significant differences in motor activity were observed in treated groups in the Functional Observational Battery assessment.

Treatment-related changes included changes in hematology, lung and spleen weights, macroscopic observations at necropsy and histopathology of the respiratory tract and lymphoreticular system.

Statistically significant increases in absolute and differential segmented neutrophil counts were observed at weeks 6 and 13 in females at 0.005 mg/L and above and in males at 0.0505 mg/L and above. Statistically significant increases in the absolute and relative weight of the lungs were noted for both males and females exposed to 0.0505 mg/L and above. Male rats exposed to 0.5075 mg/L also had a statistically significantly increase in the relative spleen weight. At necropsy, discoloration or pale areas and uncollapsed parenchyma in the lungs were observed in males and females at 0.0505 mg/L and above, and pale foci in females at 0.005 mg/L. Enlargement or pale discoloration of the bronchial, mediastinal and pancreatic lymph nodes were also observed in all treated groups.

Microscopically, pigmented material accumulation in the lungs, bronchial, mandibular and mediastinal or pancreatic lymph nodes, trachea, bronchi, larynx, nasal cavity, liver and spleen, as well as alveolar epithelial hyperplasia in the lungs, metaplasia in larynx, and lymphoid hyperplasia in lymph nodes and lungs, correlating with the presence of pigment in these tissues, were seen in all treated groups with a clear dose-response relationship.

Based on the findings at 0.005 mg/L, no NOEC was established in this study. The concentration of 0.005 mg/L (5 mg/m3) was a LOAEC in the study, based on the increased incidence of lymphoid hyperplasia in the bronchial lymph nodes of rats of both genders.

However, considering that lymphoid hyperplasia in the bronchial lymph nodes may not represent a specific toxic effect, but rather a non-specific adaptive response to the overloading of pulmonary alveolar macrophages by inorganic poorly soluble particles, and considering that alveolar epithelial hyperplasia in the lungs represents a more sensitive indication of adverse effects in the rat following inhalatory exposure to very high particulate concentrations, the concentration of 0.0505 mg/l (50.5 mg/m3) can be considered as a LOAEC based on the incidence and severity of alveolar epithelial hyperplasia in the lungs.

Based on the classification criteria of Annex VI Directive 67/548/EEC or UN/EU GHS and considering the interspecies differences between rats and non-rodent mammals regarding location of the inhaled particles during chronic exposure, no significant effect of relevance to human health were observed in this study. No systemic toxic effects specific to cerium dioxide as such were observed. The observed effects were rather consistent with a species-specific phenomenon of "lung overload" inflammatory response in the rat following inhalation of poorly soluble particles of low toxicity and resulting "portal-of- entry" effects, with a limited relevance to the human occupational situation given the levels of exposure. Therefore no classification is warranted for this endpoint.

This study is classified as acceptable. It satisfies the OECD 413 guideline requirements on subchronic inhalation toxicity.

Reference 2

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

standard short-term inhalation study (STIS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

The study was performed neither according to an OECD guideline, nor in compliance with GLP but it was well described and met generally accepted scientific principles. However, this study should be viewed as preliminary work since the study was designed to evaluate the hazard of 13 metal oxide nanomaterials and one micro-scale zinc oxide using a short-term (5 days) inhalation study (STIS).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Repeated exposure of rats to an aerosol of 13 metal oxide nanomaterials and one micro-scale zinc oxide for 5 consecutive days through nose-only inhalation route followed by a recovery period of 3 weeks.
- Pulmonary inflammation: Analysis of the bronchoalveolar lavage fluid, lung tissue homogenates and blood samples (i.e., cytokine profiles)
- Pulmonary tissue injury: Histopathological examination of all respiratory tract tissues using transmission electron microscopy (TEM)
- Cerium (Ce) content in the lung, lung-associated mediastinal lymph nodes and extrapulmonary organs
- Epithelial hypertrophy: Cell proliferation rates
- Apoptosis in the terminal bronchi and alveoli
- Only results on nanometric cerium dioxide (nano-CeO2) will be presented in this summary.

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physicochemical characterisation of CeO2 nanoparticles are presented below in "any other information on materials and methods incl. tables".

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI (Han) (i.e., Wistar), specific pathogen free
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 7 weeks of age
- Weight at study initiation, fasting period before study: No data available
- Housing: Social housing in polysulfonate cages, with a floor area about 2065 cm² (610x435x215 mm)
- Diet: Ad libitum (laboratory diet [Provimi Kliba SA, Basel, Switzerland]), except during the exposure periods
- Water: Ad libitum, except during the exposure periods
- Acclimation period: About 2 weeks before the onset of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 30 to 70%
- Air changes: No data available
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: 0.6 - 0.9 µm / 2.3 - 2.9

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus, method of holding animals in test chamber, source and rate of air, method of conditioning air: No data available
- System of generating particulates/aerosols: Dust aerosols were produced by dry dispersion of powder pellets with a brush dust generator (developed by the Technical University of Karlsruhe, Germany, in cooperation with BASF, Germany). The different target concentrations were achieved by adjustment of the feeding speed of the substance pellet and the rotation speed of the brush. Aerosols were generated with compressed air and diluted with conditioned air. The aerosols were passed via a cyclone (to separate particles > 3 µm) into the head-nose inhalation system.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate: No data available
- Method of particle size determination: The aerodynamic particle diameter of the aerosol (cascade impactor analysis) was determined by gravimetric measurement using a sampling probe with a diameter of 7 mm. The effective aerodynamic cut-off diameters were 21, 15, 10, 6.5, 3.5, 1, 0.7, or 0.4 µm. A backup filter was used to collect particles smaller than 0.4 µm. In addition, particle size distribution in the range from 0.015-0.805 µm was measured with a scanning mobility particle sizer (SMPS).
- Treatment of exhaust air: No data available

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of the aerosol (cascade impactor analysis) was determined by gravimetric measurement using a sampling probe with a diameter of 7 mm. Two samples were drawn from each exposure chamber on each day (total 10 samples per concentration over the 5-day exposure period).
- Samples taken from breathing zone: Yes; sampling was performed adjacent to the location where the animals' noses were positioned in the exposure system.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: No data available
- Composition of vehicle, type and concentration of dispersant aid: Not applicable
- Concentration of test material in vehicle: See below in "Concentrations"
- Lot/batch no. of vehicle, purity of vehicle: Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The atmospheric concentration of the aerosol (cascade impactor analysis) was determined by gravimetric measurement using a sampling probe with a diameter of 7 mm.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

2.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

No. of animals per sex per dose:

3 to 6 per group

Control animals:

yes, concurrent vehicle

other: negative control exposed to nano-BaSO4 at 50 mg/m3

Details on study design:

- Dose selection rationale, rationale for animal assignment, rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Yes, 21-day post-exposure observation
- Section schedule rationale: No data available

Positive control:

Coated nano-TiO2 (T-Lite SF™) at 0.5, 2 and 10 mg/m3

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (according to OECD 412)
- Time schedule for collection of blood: Shortly after the final exposure or after the 3-week post-exposure period (for recovery groups), the blood was obtained by retro-orbital plexus puncture from the same animals used for BALF collection.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight food withdrawal)
- How many animals: 5 animals/test group
- Parameters checked (hematology analyser): red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell and differential blood cell counts, haptoglobin (photometric assay), C-reactive protein (ELISA), α2-macroglobulin (ELISA) and other cell mediators (see in the BALF paragraph).

CLINICAL CHEMISTRY: Yes (according to OECD 412)
- Time schedule for collection of blood: Shortly after the final exposure or after the 3-week post-exposure period (for recovery groups), the blood was obtained by retro-orbital plexus puncture from the same animals used for BALF collection.
- Animals fasted: Yes (overnight food withdrawal)
- How many animals: 5 animals/test group
- Parameters checked: see in the BALF paragraph

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* LUNG INFLAMMATION AND INJURY (see in Table 3)
Bronchoalveloar lavage fluid (BALF) of 5 rats/test groups was analysed, determining BALF cytology, total protein, enzyme activities and (apart from test groups treated with coated nano-TiO2) BALF cytokines and chemokines. Cytokines and chemokines were additionally assessed in tissue homogenates of the lavaged lungs (n = 5). Animals were anesthetised and exsanguinated, and the exposed lungs were washed by 2 instillations of physiologic saline solution. Aliquots of combined BALF were used to determine:
- total cell counts (hematology analyser),
- differential cell counts = macrophages, polymorphonuclear (PMN) neutrophils, lymphocytes, eosinophils, monocytes and atypical cells (manual evaluation by counting at least 400 cells per sample from cytocentrifuge slide preparations),
- total protein concentration (turbidimetric method),
- lactate dehydrogenase (LDH) (kinetic UV test, 340 nm),
- alkaline phosphatase (ALP) (kinetic color test, 450 nm),
- N-acetyl-β-D-glucosaminidase (NAG) (color test, 580 nm),
- γ-glutamyltransferase (GGT) (Szasz method, kinetic color test, 415 nm).
Moreover, the following markers of pulmonary inflammation were assessed in the BALF and tissue homogenates of the lavaged lungs from test groups exposed to nano-CeO2:
- MCP-1,
- CINC-1,
- M-CSF,
- OPN,
- IL-1α,
- TNF-α.

* CELL PROLIFERATION AND APOPTOSIS RATES
From the animals used for necropsy and histopathological evaluation (n = 6), cell proliferation and apoptosis rates were determined in the large and medium bronchi, terminal bronchioli and alveoli. Three days prior to necropsy, rats were subcutaneously implanted with osmotic pumps containing 5-bromo-2-deoxyuridine (BrdU). Making use of histopathological sections that were dewaxed to remove paraffin, cell proliferation was determined after immunostaining with mouse anti-BrdU antibody. Labelling indices (i.e., percentage of nuclei counted undergoing replicative DNA synthesis indicating cells present in the S-phase) were determined for the three pulmonary compartments abovementioned. In each compartment, a minimum of 1,000 cells was evaluated. Apoptosis in lung sections was determined with a TUNEL kit. Counts of apoptotic cells were performed in the three pulmonary compartments abovementioned.

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes

Six animals from all test or control groups from the exposure or recovery groups were submitted to necropsy and histopathological evaluation of the respiratory tract. At necropsy, animals were exsanguinated by section of the aorta abdominalis and vena cava under pentobarbital anaesthesia. In accordance with the provisions of OECD TG 412, the absolute and relative organ weights of the adrenal glands, brain with olfactory bulb, epididymis, heart, kidneys, liver, lungs, spleen, testes, thymus and thyroid glands were assessed. Lungs, nasal cavities (4 levels) and larynxes (3 levels) were then fixed, trimmed and sectioned. Then the paraffin sections stained with hematoxylin and eosin and examined by light microscopy. Full histopathological examinations were performed in the animals of the control and high concentration groups. If changes were observed in the high concentration groups, the respective organs and tissues of the animals exposed to the low and intermediate concentrations were also examined.

Other examinations:

ORGAN BURDEN
In those animals submitted to necropsy, organ burden was determined in 3 animals/test groups, i.e., the content of CeO2 in the lung, mediastinal lymph nodes, and extra-pulmonary organs (i.e., liver). At necropsy, the lungs, the mediastinal lymph nodes and livers were excised. For organ burden analysis, the organs and tissues were digested, and their CeO2 content was analysed either by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) or by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).
In addition to the analytically determined lung burden, the fraction of potential pulmonary deposition was calculated using the MPPD software.

Statistics:

Nano-CeO2 induced effects on body weight were assessed using Dunnett’s test by comparing each dose group with the corresponding control group. All BALF data and the serum mediator data were subjected to non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p value was 0.05 or below, a pairwise comparison of each dose group with the corresponding control group was performed using the two-sided Wilcoxon test or the two-sided Mann–Whitney U-test. Cell proliferation and apoptosis data were analysed by pairwise comparison of each dose group with the corresponding control group using the one-sided Wilcoxon test.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see in "Details on results" below

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see in "Details on results" below

Histopathological findings: neoplastic:

no effects observed

Details on results:

HAEMATOLOGY AND CLINICAL CHEMISTRY
Haematology parameters and acute phase protein levels in the blood were not affected in rats treated with nano-CeO2 (data not shown).

HISTOPATHOLOGY
Histological examination of the nano-CeO2 test groups revealed particles in alveolar macrophages in the exposure (6/6 rats) and recovery groups (6/6 rats), regardless of the concentration tested. Additionally, mild diffuse or multifocal alveolar histiocytosis was observed in 2/6 rats in the exposure group at 10 mg/m3 and 2/6 rats in the recovery groups at all concentrations. Nevertheless, the histological examinations revealed no signs of beginning granulomatous changes or fibrosis.

LUNG INFLAMMATION AND INJURY (see in Table 4)
At aerosol concentrations of 0.5 mg/m3 nano-CeO2, PMN neutrophil and lymphocyte counts, macrophage colony stimulating factor (M-CSF) levels were significantly increased in the BALF, while total protein concentration decreased significantly. Inhalation exposure to 2.5 and 10 mg/m3 nano-CeO2 significantly increased PMN neutrophil and lymphocyte counts in the BALF immediately after the final exposure, whereas macrophage counts were significantly reduced in comparison to the corresponding control values. A significant increase in total protein concentration was observed at the nano-CeO2 concentration of 10 mg/m3.
Several enzyme activities (LDH, GGT, ALP) were increased in the BALF from the 2.5 and 10 mg/m3 exposure groups, whereas NAG activities and total protein levels were only increased at 10 mg/m3. Among the 68 antigens assessed in the same BALF, the level of MCP-1 was prominently increased (approx. 20- to 25-fold over the corresponding control values after exposure to 10 mg/m3), while M-CSF and CINC-1 levels were only moderately elevated (below 10-fold). The macrophage markers MDC and MPO were increased 360-fold and 115-fold, respectively after inhalation to 10 mg/m3. After the 3-week post-exposure period, a partial regression of these effects was observed. Although some of these parameters were still significantly elevated during the recovery period, the increases were in lesser extent than during the exposure period. Also in the lung tissue homogenates, of the 68 antigens assessed, 9 cytokines and chemokines (i.e., CINC-1/IL-8; KC/GROα; MCP-1; MCP-3; M-CSF; MDC; MIP-1α; MIP-2; NGAL) were significantly increased in the 2.5 and 10 mg/m3 nano-CeO2 exposure groups; and, as observed in BALF, increases partially regressed within the post-exposure period.

CELL PROLIFERATION AND APOPTOSIS RATES
Cell proliferation rates and apoptosis in rats exposed to nano-CeO2 were comparable to the rates recorded in the corresponding control groups (data not shown).

ORGAN BURDEN (see in Table 7)
For nano-CeO2, the recorded pulmonary deposition was consistent with the expected deposition calculated making use of the Mulltiple Path Particle Dosimetry Model (MPPD software, version 2.11). At the end of the 3-week recovery period, a slight decrease in CeO2 lung burden was observed. No data were available for mediastinal lymph nodes. In the lung-draining lymph nodes (data not shown), cerium levels were below the detection limit in the exposure groups. In the recovery group, they were 1.4, 2.5, and 4.1 μg nano-CeO2, respectively, for the 3 examined rats exposed to 10 mg/m3 (effective concentration 11.6 mg/m3).

Dose descriptor:

other: NOEC/NOAEC (systemic)

Based on:

other: nano-CeO2 (test mat.)

Sex:

male

Basis for effect level:

other: No NOEC/NOAEC was determined in the publication and no NOEC/NOAEC could be set by the registrant since the study evaluated only lung toxicity.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Dose descriptor:

other: NOAEC (local)

Effect level:

<= 0.5 mg/m³ air (nominal)

Based on:

other: nano-CeO2 (test mat.)

Sex:

male

Basis for effect level:

other: - This NOAEC value was set by the registrant from data available in the publication. - Basis: loco-regional effects, i.e., increase in PMN neutrophil and lymphocyte counts, and macrophage colony stimulating factor (M-CSF) levels

Critical effects observed:

not specified

Table 4: Effects induced by nano-CeO2 in the BALF

	Exposure groups			Recovery groups
Concentration (mg/m3)	0.5	2.5	10	0.5	2.5	10
Protein/Enzymes
Total protein	0.6*	1.4	2.8**	1.1	1.2	1.4
GGT	2.1	6.4**	6.6**	4	4.1	5.4
LDH	1.1	2.2**	4.3**	1.2	1.8*	2.3**
ALP	1.0	2.9*	4.1**	1.7	2.0**	2.4**
NAG	0.4	1.4	3.3**	1.2	1.6*	1.7**
Cell counts
Total cells	1.3	1.3	2.6	1.4	1.2	2.6
Macrophages	1.3	0.4*	0.4**	1.4	1.1	0.9
Lymphocytes	2.7*	10.3**	26.2**	0.8	3.3*	5.5*
PMN neutrophils	4.6*	164.1**	386.6**	0.9	8.0**	46.7**
Mediators
β2-Microglobulin	1.2	2.5**	3.5**	1.0	1.4	1.8*
CINC-1/IL-8	1.1	3.9*	5.3**	1.2	1.7*	2.1**
Clusterin	0.6	1.8	3.6**	1.0	1.4	1.1
Cystatin C	0.9	1.8*	3.8**	2.7**	3.3**	2.6**
Eotaxin	1.2	1.5*	2.3**	1.0	1.1*	1.1
Fibrinogen	1.0	1.4	2.7**	1.1	1.4	0.9
GCP-2	0.7	10.8**	15.4**	1.4*	2.8**	5.8**
IP-10	1.1	1.6	2.3*	0.9	0.9	0.7
MCP-1	1.3	5.2**	21.9**	0.7	1.4**	3.3**
MCP-3	1.3	6.6**	24.8**	1.0	1.3	1.9**
M-CSF	1.3*	3.4**	9.5**	1.0	1.2*	1.4**
MDC	1.7	79.0**	362.5**	1.1	1.2	3.3
MIP-1β	0.8	1.4	2.8**	0.9	1.0	1.0
MIP-2	0.8	2.8*	2.9*	1.0	1.0	0.9
MPO	1.1	55.5**	115.0**	2.9	6.2*	21.3**
VEGF	1.2	3.1**	15.7**	1.7*	1.6*	1.9*

Results are only presented for mediators, for which significant changes were recorded in at least one dose group (mean values expressed as x-fold of concurrent mean control value). Statistical evaluation (Mann-Whitney-U-test): *: p ≤ 0.05; **: p ≤ 0.01.

Table 5: Effects induced by nano-CeO2 in the lung tissue homogenates

	Exposure groups			Recovery groups
Concentration (mg/m3)	0.5	2.5	10	0.5	2.5	10
Mediators
CINC-1/IL-8	1.2	3.2**	4.0**	1.1	1.2*	1.7**
KC/GROα	1.0	4.2**	4.5**	1.0	1.3*	2.0**
MCP-1	0.8	1.8**	2.5**	1.1	1.2	1.3*
MCP-3	1.0	1.7**	2.0**	1.1	1.3*	1.3
M-CSF	1.1	1.4**	2.1**	0.9	1.0	1.1
MDC	0.9	3.0**	7.6**	0.8	1.5	1.8*
MIP-1α	1.5*	1.7*	2.3**	1.2	1.0	1.4
MIP-2	0.9	3.3**	3.0**	1.1	1.2	1.6*
NGAL	1.0	2.0*	2.6**	1.0	1.0	1.4**

Table 6: Summary of incidences and severities of main histopathological pulmonary observations in male Wistar rats exposed to nano-CeO2

	Exposure groups				Recovery groups
Concentration (mg/m3)	0	0.5	2.5	10	0	0.5	2.5	10
No. of animals examined	6	6	6	6		6	6	6
Histiocytosis, diffuse Grade 1								2 2
Histiocytosis, (multi)focal Grade 1 Grade 2						2 2 2	2 1 1	2 2 2
Particles in macrophage Present		6 6	6 6	6 6		6 6	6 6	6 6

Table 7: Measured test substance deposition in the lung, expected deposition calculated by the Multiple Path Particle Dosimetry (MPPD) model, measured (absolute and relative) decrease of lung burden (clearance) after the recovery period

Assumed 100% nano-CeO2 deposition (mg)	0.277	1.069	4.176
(Lung burden) Measured nano-CeO2 on study day 5 (µg/lung)	52.0 ± 5.0	165.8 ± 18.4	417.6 ± 44.3
Measured deposition (%)	18.8%	15.5%	10.0%
Calculated deposition (%)	16.6%	14.3%	15.4%
Measured lung burden after recovery period (µg/lung)	44.2 ± 4.4	157.6 ± 18.7	470.9 ± 51.2
% Decrease of lung burden after recovery	-15%	-5%	+13%

Conclusions:

Overall, inhaled nano-CeO2 caused a transient, concentration-dependent inflammation of the lung at all concentrations: this inflammation was only partially reversible during the 3-week post-exposure period.

Executive summary:

Landsiedel et al. (2014) assessed the hazard of nanometric cerium dioxide (nano-CeO2) by performing a standard short-term inhalation study (STIS).

Nano-CeO2 of 0 to 200 nm was used in this study. In dry state, the globular nanoparticles agglomerated (>10,000 nm). Moreover, the crystalline nano-CeO2 displayed a specific surface area of 33.0 m²/g, a zeta potential of +6 mV and an extremely low solubility (Ce < 0.1 ppm). The nano-CeO2 MMAD ranged between 0.6 and 0.9 µm depending on the concentration tested. At last, although the tested nano-CeO2 was described as pure and uncoated, the surface chemistry showed that Al and Zr were present at 9 and 5%, respectively, on nano-CeO2 surface.

Male Wistar rats (3 to 6/group) were exposed to a nano-CeO2 aerosol at 0 (control), 0.5, 2.5 and 10 mg/m3 for 6 h/day on 5 consecutive days with a 21-day post-exposure period of observation. Control animals were exposed to conditioned air. Blood was collected at the end of exposure and recovery periods; haematology and clinical chemistry were then performed. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined to determine the lung inflammation and injury. Pulmonary deposition and clearance of nano-CeO2 were assessed.

Inhaled nano-CeO2 was found in the lung, in alveolar macrophages, and more rarely in the draining lymph nodes, but none was found in extrapulmonary organs according to the authors. Nano-CeO2 induced a transient pulmonary inflammation which was concentration-dependent. Most effects were at least partially reversible during the post-exposure period. The histopathological analysis of lungs revealed no signs of beginning granulomatous changes or fibrosis. Based on cell counts, protein/enzyme concentrations and mediator levels in BALF, the authors derived a NOAEC < 0.5 mg/m3 based on changes of all cytological and biochemical parameters in BALF, particles in macrophages, partial regression of BALF effects and mild diffuse or multifocal alveolar histiocytosis remaining. Therefore, Landsiedel et al. considered nano-CeO2 in a group of materials having a higher toxic potency to lung although a recovery was visible during the post-exposure period. However, only few of these changes (PMN, lymphocyte counts and M-CSL level slightly increased in BALF) were seen at the concentration 0.5 mg/m3 at the end of exposure and recovery occurred for all these parameters, but some mild multifocal alveolar histiocytosis and particles in macrophages were observed also in animals in the recovery group at this concentration. Nevertheless, according to Keller et al. (2014), this reflected an expected physiological response. And thus it could be considered that the NOAEC is lower than or equal to 0.5 mg/m3 in this study.

In conclusion, inhaled nano-CeO2 induced only loco-regional effects manifested by a transient pulmonary inflammation. However, no systemic toxicity occurred.

As stated by the authors, one reason for effects being only partially regressive might be that the 3-week post-exposure period was not sufficiently long to allow full recovery to take place. However, Landsiedel et al. alternatively suggested that partial regression of effects might be an indication that the development of chronic effects cannot be excluded, especially if the lung clearance mechanism is overloaded.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Some parameters for characterisation of the nano CeO2 are missing and limited information on systemic toxicity are reported in this article.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Systemic toxicity was not deeply evaluated in this study which focussed on the lung effect of nano-CeO2 using 3 different mouse models.

Principles of method if other than guideline:

This study was performed in order to assess the influence of redox activity by modifications of cerium dioxide nanoparticles (nano-CeO2) via zirconium doping on the distribution, pulmonary and cardiovascular effects in mice following sub-acute inhalation. This study further explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. Only results observed with nano CeO2 are reported in this summary. Mice from each strain were exposed to the nanoparticles during 4 weeks and the effects were evaluated 4 weeks after the last exposure day (i.e day 56).
Experiments were conducted at Intravacc (Bilthoven, The Netherlands) under a protocol approved by the Ethics Committee for Animal Experiments of the RIVM and performed according to applicable local and EU regulations.

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physicochemical characterisation of nano CeO2 are presented below in "any other information on materials and methods incl. tables".

Species:

mouse

Strain:

other:

Remarks:

See below: in Details on species / strain selection

Details on species / strain selection:

3 mice models were used in this study to explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice model, a well established model for the study of the vascular disease atherosclerosis (Coleman et al., 2006). ApoE-/- mouse were used to study hematology, pulmonary and cardiovascular effects of the nanoparticles.
- Alzheimer’s disease mouse model (5xFAD), were included to study neurological effects (not included in the article and should be published in another article) and the hematology and pulmonary effects of the nanoparticles.
- C57BL/6J mouse - (non-genetically modified) strain, was used as the background strain of the disease mouse models and was used to study biodistribution, hematology and pulmonary effects of the nanoparticles.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain and source:
* Specific Pathogen free (SPF) ApoE−/− mice come from Taconic, Denmark
* 5xFAD and wild type (WT) cross bread C57BL/6J littermates mice come from Jackson Laboratories
- Age at study initiation:
* ApoE−/− mice: 10-12 weeks
* 5xFAD and C57BL/6J mice: 8-11 weeks
- Weight at study initiation, fasting period before study: No data available
- Housing: macrolon cages
- Diet: Ad libitum, The ApoE-/- mice were fed a commercially available rodent Western (high fat) diet (Purified Diet Western 4021.06, ABdiets, Woerden, The Netherlands), starting at the first day of the exposure period until the end of the experiment. The other mice were fed a standard commercially available rodent diet (SMR-A, ABdiets, Woerden, The Netherlands).
- Water: Ad libitum, except during the exposure periods.
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-2 °C
- Humidity: 40 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 280 nm

Geometric standard deviation (GSD):

1.55

Remarks on MMAD:

Details on inhalation exposure:

Approximately one week before the 4-week exposure period, 20 samples of the nano-CeO2 (one for each day) with a concentration of 1 mg/mL were prepared from the stock dispersions (20 mg/mL), by diluting with ultrapure water to the desired concentration. Stock and sample dispersions were sonicated for 5 minutes in an ultrasonic bath (Branson CPX2800, 40 kHz, 110W) before use to re-disperse any possible agglomerates.
Freshly generated aerosols of NPs were generated using a spray nozzle technique, diluted with pressurised clean particle-free air, and heated to 24-25°C.
Exposure was controlled based on stable particle number counts, mass concentrations, temperature and relative humidity, measured continuously using a condensation particle counter (CPC 3022A from TSI Inc., St. Paul, MN, USA), a tempered element oscillating microbalance (TEOM series1400A from Rupprecht & Patashnick, NY, USA) and M-170 Measurement Indicator (Vaisala M170, Vaisala Oyj, Helsinki, Sweden), respectively, during each exposure period.
During the 3 hour exposure periods to the nanoparticles, the control groups were exposed to filtered air under the same conditions (nose-only tubes) for the same amount of time.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmosphere was characterised at least twice during each exposure session using an optical particle sizer (OPS 3330, TSI Inc., St. Paul, MN, USA) and a scanning mobility particle sizer (SMPS 3936 from TSI Inc., St. Paul, MN, USA). In addition, aerosols were collected on polycarbonate filters for scanning electron microscopy (SEM) analysis. The SEM samples were prepared by placing a small piece of the filter on the SEM stub and coating it with platinum, and visualising with an XL30 Environmental SEM-FEG microscope (Philips XL30 ESEMFEG).
The total mass concentration generated over the 3-hour exposure period was determined by gravimetric analysis of pre-weighed and post-weighed polytetrafluoroethylene (PTFE) filters (Teflo R2PJO47, Pall corporation, Port Washington, New York, USA) using a micro-balance (Mettler MC or ME-5 microbalance, Mettler-Toledo LLC, Columbus, OH, USA).

Duration of treatment / exposure:

4 weeks of exposure. The effects were assessed 4 weeks post the final exposure day (56 days after the initial exposure).

Frequency of treatment:

5 days/week for 3 hours/day

Dose / conc.:

4 mg/m³ air (nominal)

No. of animals per sex per dose:

- 8 ApoE-/- mice per group
- 16 5xFAD mice per group
- 10 C57BL/6J mice per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

To explore the (patho)physiological effects of the nanoparticles on multiple organ systems, three different mouse models were exposed:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice are a well established model for the study of the vascular disease atherosclerosis (Coleman et al., 2006), a disease characterised by the build-up of lipid- and inflammatory cell-rich plaques within arteries, which underlies the majority of cardiovascular diseases. The 4-week exposure protocol was integrated into an 8-week high-fat feeding regime that has been shown to generate complex atherosclerotic plaques with many of the hallmarks of the human disease in specific arterial locations (Cassee et al., 2012, Miller et al., 2013).
ApoE−/− mice were used to study hematology, pulmonary and cardiovascular effects.
- The 5xFAD mice are an Alzheimer’s disease mouse model. Although these mice were included to study neurological effects that will be published in a separate paper, the hematology and pulmonary effects were also studied within the same animals and reported in this paper.
- C57BL/6J mice were used as the background (non-genetically modified) strain of the disease mouse models and used to study biodistribution, hematology and pulmonary effects.
Hematology, pulmonary and cardiovascular effects were assessed 4 weeks post exposure. This period was included, firstly, to provide the extra four weeks necessary for mice to develop sufficient plaque formation in arteries and secondly, to investigate the persistency of the pulmonary and cardiovascular effects.
One group of each mouse model was exposed nose-only for four weeks to 4 mg/m3 nano-CeO2 for 5 days/week for 3 hours/day and one control group to clean air under the same experimental conditions (nose-only tube, 3h). Effects were assessed 4 weeks post the final exposure (56 days after the initial exposure).
The number of animals per group was different for each of the mouse models, to provide sufficient statistical power to detect differences between exposed and control animals in the most important effect parameter of each mouse model. Based on previous experiments, 8 ApoE-/- mice per group were expected to be sufficient to detect statistically significant differences in atherosclerotic plaque size and 5 C57BL/6J mice per group were expected to be sufficient to detect statistically significant differences in the number of neutrophils in the bronchoalveolar lavage fluid (BALF). However, 10 C57BL/6J mice per group were included, since 10 C57BL/6J mice per group and 16 5xFAD mice per group were needed to provide sufficient statistical power in the neurological study.

Positive control:

Observations and examinations performed and frequency:

Animals were monitored by cage-side observations and, if necessary, handled to detect signs of compromised health. The body weight of each animal was recorded one day before the start of exposure (day -1), prior to exposure on the first day and weekly thereafter.

Hematology, neurological (results published in a separate article which was not avaible at the time of the dossier preparation), pulmonary and cardiovascular effects were assessed 4 weeks after the last exposure of the mice (i.e. 56 days after the initial exposure).

- QUANTIFICATION OF CERIUM IN TISSUES
During necropsy, organs from half of the C57BL/6J mice per group were obtained to evaluate the distribution of the nano CeO2 throughout the body. Liver, spleen, kidneys, heart and right (exposed mice) or left (control mice) lung, were weighed and immediately frozen in liquid nitrogen for determination of the Ce concentrations. To allow measurement of multiple parameters within the same animal, different parts of the lungs were selected for the exposed compared to the control animals. From the exposed groups the right lung was used for quantification of Cerium, because the left lung was needed for histopathological examination. From the control group the left lung was used for quantification of Cerium, because the right lung was needed for bronchoalveolar lavage. The organs were digested by acidification of each sample with 2 mL nitric acid for 12h. Hydrogen fluoride (0.2 mL) was added, followed by microwave heating for 45 min up to 185°C, and maintained for a further 20 min. Boric acid (2 mL) was added to neutralize the hydrogen fluoride, and the samples were re-heated for 20 min to 160 °C, and maintained for 10 min. Once cooled, samples were filtered with a 450 nm syringe filter, diluted with 10 mL deionized water and stored at room temperature (RT). The presence of Cerium in the lungs, liver, spleen, kidneys and heart was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer NexION 300X instrument operated in standard mode for Cerium. The isotope measured was 140Ce using 115In and 159Tb as internal standards. Calibration standards (0 – 100 μg/L) were prepared from VWR 1000 mg/L stock solutions. Quantities are expressed as µg/g organ tissue.

- HEMATOLOGY
Animals were anaesthetized with a mixture of ketamine and xylazine. Two blood samples were taken by eye extraction. The first sample was collected in a K3-EDTA tube (Minicollect K3EDTA, 1 mL, 450474 Greiner Bio-One) for hematological parameters as determined in a blood auto analyzer (ADVIA 2120 Hematology System, Siemens Healthineers) within 3 hours after collection. The second sample was collected in a serum tube and stored at -20°C for further analysis. Total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cells counts were meaured.

- BRONCHOALVEOLAR LAVAGE
Lung lavage was performed at necropsy. A cannula was placed in the trachea and the diaphragm opened to decrease the amount of air inside the lungs. For the control animals and all ApoE-/- animals, the right lung half was rinsed twice with approximately 0.5 mL (26.7 mL per kg body weight) of physiological saline solution, after ligation of the left lung. The injected volume was inserted and recovered 3 times, after which the lavage liquid was collected and stored on ice for less than 2 hours. To allow necropsy of the planned number of animals within one day, both lungs were lavaged for 10 of the 16 exposed 5xFAD mice and 5 of the 10 exposed C57BL/6J mice, using the same procedure, but approximately 0.8 mL (40 mL per kg body weight) of physiological saline solution.
BALF was centrifuged at 400g for 10 minutes at 4°C. The supernatant was divided into two separate aliquots of 125 µL for total protein (TP; an indicator for acute lung injury), lactate dehydrogenase (LDH; an indicator of cytotoxicity), gammaglutamyl transferase (GGT; an indicator of lung cell damage) and alkaline phosphatase (ALP; an indicator of type II cell damage) measurements using an autoanalyser (LX20- Pro, Beckman-Coulter, Woerden, the Netherlands). The cell pellet was scored for the presence of erythrocytes, re-suspended in 500 μL phosphate-buffered saline (PBS) and kept on ice. Cell counts were determined in the re-suspended pellet using a Coulter counter (Beckman-Coulter, Live Sciences). Cell concentrations were determined using a single sample using at least 150 µL of the re-suspended cells. Cytospins (Cytospin 3, Thermo-Shandon) were prepared and stained using May-Grunwald and Giemsa stain, and cell differentiation was performed by counting 400 cells per slide.

Sacrifice and pathology:

HISTOPATHOLOGY
- Lung tissue:
For the majority of the animals, the right lung was removed after the collection of the BALF and immediately frozen in liquid nitrogen and stored at - 80°C for further analysis. The left lung was removed and, after weighing, cannulated and infused with formaldehyde for 1 hour at a pressure of 20 cm H2O. Lungs were processed for histopathology; embedded in paraffin wax, sectioned at 2-4 µm intervals and stained with hematoxylin and eosin for histopathological examination.
Histopathological changes were described according to distribution, severity and morphological characteristics. The morphological characteristics of chronic inflammation include for example the presence of lymphocytes and macrophages in the lung tissue, while acute inflammation is characterized by the presence of polymorphoneclear neutrophils (PMNs).
Severity scores were assigned as follows: Grade 1 Minimal/very few/very small; Grade 2 Slight/few/small; Grade 3 Moderate/moderate number/moderate size; Grade 4 Marked/many/large; Grade 5 Massive/extensive number/extensive size.

- Assessment of atherosclerosis:
Arteries (brachiocephalic, aortic arch, thoracic aorta) were isolated from ApoE-/- mice. Atherosclerosis was quantified, as previously published (Miller et al., 2013). Briefly, brachiocephalic arteries were fixed in formalin and histological sections were taken in triplicate at 100 µm intervals, beginning at the first section of the artery with a fully intact media. Sections were stained with Masson’s Trichrome. The cross-sectional area of the plaque was measured and standardized to the medial area. A single mean value for atherosclerotic burden for each animal was calculated from the plaque size from each complete serial section throughout the brachiocephalic artery. A single section from each artery (the section exhibiting the largest plaque in cross-section) was chosen for mac-2 immunohistochemistry for macrophage-derived cells. A rat anti-mouse primary antibody was used (1/12000; CL8942AP, VH Bio, Gateshead, UK) with rat IgG (1/12000; I-400, Vector Labs, Peterborough, UK) as a negative control, followed by a goat anti-rat IgG biotinylated secondary antibody (BA-9400, Vector Labs). The area of positive staining was expressed as a proportion of the total plaque area.

- Other organs:
The spleen, liver, heart and kidneys were removed, weighed and stored in 4% formaldehyde for pathological analysis if required based on macroscopic findings.

Statistics:

Statistical analyses were performed using GraphPad Prism v7.00 (GraphPad Software, San Diego, California, USA). Ordinary one-way analysis of variance (ANOVA) analyses including all experimental groups were performed followed by a Tukey’s posthoc multiple comparisons test. A p-value ≤ 0.05 was considered statistically significant.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related effects on organ weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings related to treatment (data not shown) were observed and thus no histopathological analysis was performed on the organs other than lungs.

Neuropathological findings:

not examined

Description (incidence and severity):

Although the authors used the 5xFAD mice, an Alzheimer’s disease mouse model, to study neurological effects of exposure to nanoparticles, they said that the results will be published in a separate paper that was not published at the time of the preparation of this dossier.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- LUNG: While modest, an increased incidence in minimal chronic bronchoalveolar or alveolar inflammation was observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in seven of the eight ApoE-/- mice exposed to nano CeO2 NP but not in the other strains (see table 1 in "Any information on results incl. tables").
- BALF: In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the NP exposed animals but not in the control groups

Other effects:

no effects observed

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE (BALF)
- Total and differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytesin) from BALF measured 4 weeks after the last exposure day (mean of 5 to 16 animals) in the 3 mice strains: No statistically significant differences were observed in the total cell counts (data not shown) or differential cell counts in BALF of the exposed groups compared to the controls for any strain of mouse.

- Total protein, Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP) and Gamma-Glutamyl Transpeptidase (GGT) levels in bronchoalveolar lavage fluid (BALF) 4 weeks after exposure (mean of 5 to 16 mice from the each tested strains):
Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels between the exposed and control groups.

No constitutive differences in differential cell counts or protein levels were observed between the different mice strains.

EFFECTS OF NANO CeO2 ON ATHEROSCLEROSIS IN THE BRACHIOCEPHALIC ARTERY OFApoE-/- MICE AFTER INHALATION
ApoE-/- mice exhibited regions of dense plaques in the aortic arch and branch points of large arteries. Plaques were composed of fibroblastic matrix, smooth muscle cells, lipid cavities and cholesterol crystals. Atherosclerotic burden was quantified in the brachiocephalic artery, with control (air-exposed) mice having a mean plaque size of 94 ± 9% (standardized to the area of the vascular media). Exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries (p=0.62; One-way ANOVA) and in the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

Dose descriptor:

NOAEL

Effect level:

> 4 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: NanoCeO2 had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

Critical effects observed:

Estimated deposition of inhaled nano-CeO2 in different regions of the lungs:

The total inhaled dose, estimated as tidal volume (0.203 mL) x breathing frequency (353 min-1) x exposure concentration (≈ 4 x 10-3 μg/mL) x exposure duration (3600 min), was calculated to be 1020 µg. The total deposited dose (estimated using the MPPD model) was found to be 618 µg. The deposited fraction per region, estimated using the MPPD model, was found to be 48.8 % for the head, 3.5 % for the tracheobronchial region and 8.0 % for the alveolar region of the inhaled mass dose. The retained dose in lung, corresponding to the retained dose in the tracheobronchial and alveolar region 4 weeks post-exposure, estimated using the MPPD model, was found to be 16.2 µg.

Quantification of cerium in tissues (en µg/g of tissues, mean of 4 -6 animals) 4 weeks after the last exposure day:

The highest concentrations of Cerium were found in the lung (~ 67 µg/g), followed by much lower concentrations in the heart (~2.2 µg/g), spleen (~1.1 µg/g), kidneys (~0.3 µg/g) and liver (~ 0.2 µg/g), respectively. As would be expected, significantly higher levels of Ce were observed in the lungs of exposed mice compared to the controls (~1.1 µg/g in control lung). In most of the other organs, the cerium concentrations were not statistically signicantly different from the background concentrations measured in the control animals (heart (~2.8 µg/g), spleen (~1.0 µg/g), kidneys (~1.3 µg/g) and liver (~ 0.05 µg/g)).

Tha authors indicated that background levels of Ce detected in various organs of animals exposed to clean air (controls) might be caused by Ce contamination of the drinking water, food and/or bedding of the animals as previously found by others (Yokel et al. 2012, 2013, see the section 7.1 Toxicokinetics, metabolism and distribution of this dossier)).

The authors said that, based on the estimated retained dose of nano CeO2 in the lung (16.2 µg, see Table 3) using MPPD model and the average lung weight (158 mg), the expected Ce concentration in the lung was approximately 103 µg/g while the measured Ce concentration in the lungs of the nano CeO2 exposed mice was slightly lower (67 µg/g) but in the same order of magnitude. They concluded that the difference between the predicted and measured concentrations may reflect the lung clearance rate used by the MPPD model compared to the actual lung clearance rate in vivo.

Table 1: Histopathological findings in lung of C57BL/6J, ApoE-/- and 5xFAD mice.

Histopathological finding →		Chronic broncho-alveolar or alveolar inflammation		Alveolar macrophages	Particle loaded alveolar macrophages
Strain¯	Treatment¯	Minimal	Slight	Minimal	Minimal	Slight
C57BL/6J	Control	60% (3/5)a	-	-	-	-
	Nano CeO2	80% (4/5)	-	20% (1/5)	-	-
ApoE-/-	Control	25% (2/8)	-	-	-	-
	Nano CeO2	38% (3/8)	-	-	13% (1/8)	75% (6/8)
5xFAD	Control	69% (11/16)	-	6% (1/16)	-	-
	Nano CeO2	83% (5/6)	-	17% (1/6)	-	-

A: Percentage of animals affected. Within brackets the number of animals with histopathological findings versus number of animals evaluated.

Conclusions:

Executive summary:

In this subacute inhalation study, Dekkers et al. (2015) explored the (patho)physiological effects of nano CeO2 exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. As this study was performed using a test procedure in agreement with generally accepted scientific standards but with some limitations (incomplete data on substance characterisation and on systemic toxicity), the study was awarded a reliability code of 2 according to Klimisch criteria and was used as weight of evidence.

Female mice from each strain were exposed nose-only to 0 (controls, exposed to filtered air) and 4 mg/m3 nano CeO2 (primary particle size of 4.7 nm) for 3h/day, 5 days/week for 4 weeks.

Four weeks after the last exposure day (i.e day 56 of the study), the animals of each strain were sacrificed, organs weights were measured and the effects of the treatment on the blood (total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cell counts) and on the lungs (analyses of the differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytes) and protein levels (total proteins, ALP, LDH and GGT) in BALF) were evaluated. Furthermore, histopathological analysis of the lungs were done and the tissue distribution of cerium was measured using ICP-MS in the lung, heart, kidney, spleen and liver of all animals. The effects of nano-CeO2 on the atherosclerosis burden in the brachiocephalic artery of ApoE-/- mice was also determined in this study to evaluate cardiovascular effects of treatment with nanoparticles by measuring the mean plaque size and of the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

According to the authors, no statistically significant differences were observed in the total or differential white blood cell counts in the blood and in the BALF of the exposed groups compared to the controls in all strains of mice. Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels in BALF between the exposed and control groups. Furthermore, no treatment related effects on organ weights were observed and no macroscopic findings related to treatment were observed. In lungs, the histopathological analyses showed an increased incidence, described as modest by the authors, in minimal chronic bronchoalveolar or alveolar inflammation observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in most of the ApoE-/- mice exposed to nano CeO2 NP but not in the other stains. In the bronchoalveolar lavage fluid, particle loaded macrophages were seen in all the exposed animals but not in the control groups. In tissues, background Ce concentrations were measured in the control animals and significantly higher levels of Ce were only observed in the lungs and the liver of exposed mice as compared to the controls but not in the other organs.

In ApoE-/- mice, exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries and in the proportion of plaque staining positive for mac-2. The evaluation of potential neurological effects of treatment on the 5xFAD mice were not reported in this article.

The authors concluded that in this subacute inhalation study, nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

No NOAEL was derived in this study by the authors. However, according to the results and the conclusion of the authors (nano CeO2 has a low biological activity in their mouse models), it can be concluded that the NOAEL for lung toxicity is > 4 mg/m3, the only dose tested in this study.

Reference 4

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

standard short-term inhalation study (STIS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

The study was performed neither according to an OECD guideline, nor in compliance with GLP, but it was well described and met generally accepted scientific principles. However, this study should be viewed as preliminary work since the study was designed to evaluate the hazard of 13 metal oxide nanomaterials and one micro-scale zinc oxide using a short-term (5 days) inhalation study (STIS).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Repeated exposure of rats to an aerosol of 13 metal oxide nanomaterials and one micro-scale zinc oxide for 5 consecutive days through nose-only inhalation route followed by a recovery period of 3 weeks.
- Pulmonary inflammation: Analysis of the bronchoalveolar lavage fluid, lung tissue homogenates and blood samples (i.e., cytokine profiles)
- Pulmonary tissue injury: Histopathological examination of all respiratory tract tissues using transmission electron microscopy (TEM)
- Zirconium (Zr) content in the lung, lung-associated mediastinal lymph nodes and extrapulmonary organs
- Epithelial hypertrophy: Cell proliferation rates
- Apoptosis in the terminal bronchi and alveoli
- Only results on nanometric zirconium dioxide (nano-ZrO2) will be presented in this summary.

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physicochemical characterisation of ZrO2 nanoparticles are presented below in "any other information on materials and methods incl. tables".

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: 1.3 - 2.0 µm / 1.8 - 2.3

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The atmospheric concentration of the aerosol (cascade impactor analysis) was determined by gravimetric measurement using a sampling probe with a diameter of 7 mm.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-ZrO2 conc.

Dose / conc.:

2.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-ZrO2 conc.

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

basis: nominal nano-ZrO2 conc.

No. of animals per sex per dose:

6 per group

Control animals:

yes, concurrent vehicle

other: negative control exposed to nano-BaSO4 at 50 mg/m3

Details on study design:

Positive control:

Coated nano-TiO2 (T-Lite SF™) at 0.5, 2 and 10 mg/m³

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (according to OECD 412)
- Time schedule for collection of blood: Shortly after the final exposure or after the 3-week post-exposure period (for recovery groups), the blood was obtained by retro-orbital plexus puncture from the same animals used for BALF collection.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight food withdrawal)
- How many animals: 5 animals/test group
- Parameters checked (hematology analyser): red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell and differential blood cell counts, haptoglobin (photometric assay), C-reactive protein (ELISA), α2-macroglobulin (ELISA) and other cell mediators (see in the BALF paragraph).

CLINICAL CHEMISTRY: Yes (according to OECD 412)
- Time schedule for collection of blood: Shortly after the final exposure or after the 3-week post-exposure period (for recovery groups), the blood was obtained by retro-orbital plexus puncture from the same animals used for BALF collection.
- Animals fasted: Yes (overnight food withdrawal)
- How many animals: 5 animals/test group
- Parameters checked: see in the BALF paragraph

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* LUNG INFLAMMATION AND INJURY (see in Table 3)
Bronchoalveloar lavage fluid (BALF) of 5 rats/test groups was analysed, determining BALF cytology, total protein, enzyme activities and (apart from test groups treated with coated nano-TiO2) BALF cytokines and chemokines. Cytokines and chemokines were additionally assessed in tissue homogenates of the lavaged lungs (n = 5). Animals were anesthetised and exsanguinated, and the exposed lungs were washed by 2 instillations of physiologic saline solution. Aliquots of combined BALF were used to determine:
- total cell counts (hematology analyser),
- differential cell counts = macrophages, polymorphonuclear (PMN) neutrophils, lymphocytes, eosinophils, monocytes and atypical cells (manual evaluation by counting at least 400 cells per sample from cytocentrifuge slide preparations),
- total protein concentration (turbidimetric method),
- lactate dehydrogenase (LDH) (kinetic UV test, 340 nm),
- alkaline phosphatase (ALP) (kinetic color test, 450 nm),
- N-acetyl-β-D-glucosaminidase (NAG) (color test, 580 nm),
- γ-glutamyltransferase (GGT) (Szasz method, kinetic color test, 415 nm).
Moreover, the following markers of pulmonary inflammation were assessed in the BALF and tissue homogenates of the lavaged lungs from test groups exposed to nano-ZrO2:
- MCP-1,
- CINC-1,
- M-CSF,
- OPN,
- IL-1α,
- TNF-α.

* CELL PROLIFERATION AND APOPTOSIS RATES
From the animals used for necropsy and histopathological evaluation (n = 6), cell proliferation and apoptosis rates were determined in the large and medium bronchi, terminal bronchioli and alveoli. Three days prior to necropsy, rats were subcutaneously implanted with osmotic pumps containing 5-bromo-2-deoxyuridine (BrdU). Making use of histopathological sections that were dewaxed to remove paraffin, cell proliferation was determined after immunostaining with mouse anti-BrdU antibody. Labelling indices (i.e., percentage of nuclei counted undergoing replicative DNA synthesis indicating cells present in the S-phase) were determined for the three pulmonary compartments abovementioned. In each compartment, a minimum of 1,000 cells was evaluated. Apoptosis in lung sections was determined with a TUNEL kit. Counts of apoptotic cells were performed in the three pulmonary compartments abovementioned.

Sacrifice and pathology:

Other examinations:

ORGAN BURDEN
In those animals submitted to necropsy, organ burden was determined in 3 animals/test group, i.e., the content of ZrO2 in the lung, mediastinal lymph nodes, and extra-pulmonary organs (i.e., liver). At necropsy, the lungs, the mediastinal lymph nodes and livers were excised. For organ burden analysis, the organs and tissues were digested, and their ZrO2 content was analysed either by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) or by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).
In addition to the analytically determined lung burden, the fraction of potential pulmonary deposition was calculated using the MPPD software.

Statistics:

Nano-ZrO2 induced effects on body weight were assessed using Dunnett’s test by comparing each dose group with the corresponding control group. All BALF data and the serum mediator data were subjected to non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p value was 0.05 or below, a pairwise comparison of each dose group with the corresponding control group was performed using the two-sided Wilcoxon test or the two-sided Mann–Whitney U-test. Cell proliferation and apoptosis data were analysed by pairwise comparison of each dose group with the corresponding control group using the one-sided Wilcoxon test.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Histopathological findings: neoplastic:

no effects observed

Details on results:

HAEMATOLOGY AND CLINICAL CHEMISTRY
The hematological parameters and acute phase protein levels in the blood remained unchanged. in rats treated with nano-ZrO2 (data not shown).

HISTOPATHOLOGY
There were no histopathological changes of the respiratory tract.

LUNG INFLAMMATION AND INJURY
Inhalation exposure to aerosol concentrations of up to 10 mg/m³ nano-ZrO2 did not induce any treatment-related effects in cytological, protein, enzyme, cytokine or chemokine levels in the BALF or in cytokine levels in the lung tissue, even though the comprehensive panel of 68 cell mediators was assessed both in the BALF and lung tissue.

CELL PROLIFERATION AND APOPTOSIS RATES
Cell proliferation rates and apoptotic reactions in lungs cells were comparable to those from the control groups (data not shown).

ORGAN BURDEN (see Table 4)
For nano-ZrO2, the recorded pulmonary deposition was consistent with the expected deposition calculated making use of the Mulltiple Path Particle Dosimetry Model (MPPD software, version 2.11). At the end of the 3-week recovery period, the decrease in lung burden was significant for nano-ZrO2 (up to 75%). No data were available for mediastinal lymph nodes. In the lung-draining lymph nodes of animals exposed to either of the ZrO2 materials (data not shown), zirconium was not detectable at any time point in any of the examined animals

Dose descriptor:

other: NOEC/NOAEC (systemic)

Based on:

other: nano-ZrO2 (test mat.)

Sex:

male

Basis for effect level:

other: No NOEC/NOAEC was determined in the publication and no NOEC/NOAEC could be set by the registrant since the study evaluated only lung toxicity.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Dose descriptor:

other: NOAEC (local)

Effect level:

>= 10 mg/m³ air (nominal)

Based on:

other: nano-ZrO2 (test mat.)

Sex:

male

Basis for effect level:

other: Basis: absence of loco-regional effects on the rat lung.

Critical effects observed:

not specified

Table 4: Measured test substance deposition in the lung, expected deposition calculated by the Multiple Path Particle Dosimetry (MPPD) model, measured (absolute and relative) decrease of lung burden (clearance) after the recovery period

Assumed 100% nano-ZrO2 deposition (mg)	0.18	0.936	3.456
(Lung burden) Measured nano-ZrO2 on study day 5 (µg/lung)	18.0 ± 5.6	20.3 ± 117.5	270.6 ± 28.9
Measured deposition (%)	10.0%	2.2% 12.6%	7.8%
Calculated deposition (%)	7.3%	8.2%	6.7%
Measured lung burden after recovery period (µg/lung)	6.8 ± 3.6	29.3 ± 6.2	157.6 ± 19.0
% Decrease of lung burden after recovery	-62%	-75%	-42%

Conclusions:

Overall, inhaled nano-ZrO2 did noy elicit any adverse effects in the rat lung at any of the concentrations tested.

Executive summary:

Landsiedel et al. (2014) assessed the hazard of nanometric zirconium dioxide (nano-ZrO2) by performing a standard short-term inhalation study (STIS).

Nano-ZrO2 of 25 to 60 nm was used in this study. In dry state, the globular nanoparticles agglomerated (1,000 - 5,000 nm). Moreover, the crystalline nano-ZrO2 displayed a specific surface area of 24.9 m²/g, a zeta potential of -12 mV and an extremely low solubility (Zr < 0.1 ppm). The nano-ZrO2 MMAD ranged between 1.3 and 2.0 µm depending on the concentration tested. At last, although the tested nano-ZrO2 was described as pure and uncoated, the surface chemistry showed that Al and N were present at 1 and 3%, respectively, on the nano-ZrO2 surface.

Male Wistar rats (6/group) were exposed to a nano-ZrO2 aerosol at 0 (control), 0.5, 2.5 and 10 mg/m³ for 6 h/day on 5 consecutive days with a 21-day post-exposure period of observation. Control animals were exposed to conditioned air. Blood was collected at the end of exposure and recovery periods; haematology and clinical chemistry were then performed. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined to determine the lung inflammation and injury. Pulmonary deposition and clearance of nano-ZrO2 were assessed.

Inhaled nano-ZrO2 was found in the lungs but was not detectable in the lung-draining lymph nodes at any time in any of the examined animals and not found in extrapulmonary organs according to the authors. Nano-ZrO2 did not elicit local effects in the rat lung. Based on the absence of effects noted, the authors derived a NOAEC >= 10 mg/m³. Therefore, Landsiedel et al. considered nano-ZrO2 in a group of materials causing no lung effects up to the highest tested concentration.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Some parameters for characterisation of the Zr-doped nano CeO2 are missing and limited information on systemic toxicity are reported in this article.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Systemic toxicity was not deeply evaluated in this study which focused on the lung effect of Zr-doped nano-CeO2 using 3 different mouse models.

Principles of method if other than guideline:

This study was performed in order to assess the influence of redox activity by modifications of cerium dioxide nanoparticles (nano-CeO2) via zirconium doping on the distribution, pulmonary and cardiovascular effects in mice following sub-acute inhalation. This study further explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. Only results observed with Zr-doped nano CeO2 are reported in this summary; effects observed with nano CeO2 are reported in a separate entry. Mice from each strain were exposed to the nanoparticles during 4 weeks and the effects were evaluated 4 weeks after the last exposure day (i.e day 56).
Experiments were conducted at Intravacc (Bilthoven, The Netherlands) under a protocol approved by the Ethics Committee for Animal Experiments of the RIVM and performed according to applicable local and EU regulations.

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physico-chemical characterisation of Zr-doped nano CeO2 are presented below in "any other information on materials and methods incl. tables".

Species:

mouse

Strain:

other:

Remarks:

See below: in Details on species / strain selection

Details on species / strain selection:

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain and source:
* Specific Pathogen free (SPF) ApoE−/− mice come from Taconic, Denmark
* 5xFAD and wild type (WT) cross bread C57BL/6J littermates mice come from Jackson Laboratories.
- Age at study initiation:
* ApoE−/− mice: 10-12 weeks
* 5xFAD and C57BL/6J mice: 8-11 weeks
- Weight at study initiation, fasting period before study: No data available
- Housing: macrolon cages
- Diet: Ad libitum, The ApoE-/- mice were fed a commercially available rodent Western (high fat) diet (Purified Diet Western 4021.06, ABdiets, Woerden, The Netherlands), starting at the first day of the exposure period until the end of the experiment. The other mice were fed a standard commercially available rodent diet (SMR-A, ABdiets, Woerden, The Netherlands).
- Water: Ad libitum, except during the exposure periods
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-2 °C
- Humidity: 40 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 288 - <= 316 nm

Remarks on MMAD:

The Count Median Diameter (CMD) and the Mass Median Diameter (MMD) were estimated using the Aerosol Instrument Manager Software (Release Version 9.0.0.0, 15:32:53, Nov 11 2010 from TSI Inc., St Pauls, MN, USA).

27% Zr-doped CeO2: MMD = 288 ± 9; GSD 1.56 assuming spherical aggregation around primary particles of 4.6 ± 1.4 nm.
78% Zr-doped CeO2: MMD = 316 ± 14; GSD 1.57 assuming spherical aggregation around primary particles of 4.7 ± 1.4 nm.

Details on inhalation exposure:

Approximately one week before the 4-week exposure period, 20 samples of each nanoparticle (one for each day) with a concentration of 1 mg/mL were prepared from the stock dispersions (20 or 29 mg/mL for 27% and 78% Zr-doped CeO2, respectively), by diluting with ultrapure water to the desired concentration. Stock and sample dispersions were sonicated for 5 minutes in an ultrasonic bath (Branson CPX2800, 40 kHz, 110W) before use to re-disperse any possible agglomerates.
Freshly generated aerosols of nanoparticles were generated using a spray nozzle technique, diluted with pressurszed clean particle-free air, and heated to 24-25°C.
Exposure was controlled based on stable particle number counts, mass concentrations, temperature and relative humidity, measured continuously using a condensation particle counter (CPC 3022A from TSI Inc., St. Paul, MN, USA), a tempered element oscillating microbalance (TEOM series1400A from Rupprecht & Patashnick, NY, USA) and M-170 Measurement Indicator (Vaisala M170, Vaisala Oyj, Helsinki, Sweden), respectively, during each exposure period.
During the 3 hour exposure periods to the nanoparticles, the control groups were exposed to filtered air under the same conditions (nose-only tubes) for the same amount of time.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmosphere was characterised at least twice during each exposure session using an optical particle sizer (OPS 3330, TSI Inc., St. Paul, MN, USA) and a scanning mobility particle sizer (SMPS 3936 from TSI Inc.,St. Paul, MN, USA). In addition, aerosols were collected on polycarbonate filters for scanning electron microscopy (SEM) analysis. The SEM samples were prepared by placing a small piece of the filter on the SEM stub and coating it with platinum, and visualising with an XL30 Environmental SEM-FEG microscope (Philips XL30 ESEMFEG).
The total mass concentration generated over the 3-hour exposure period was determined by gravimetric analysis of pre-weighed and post-weighed polytetrafluoroethylene (PTFE) filters (Teflo R2PJO47, Pall corporation, Port Washington, New York, USA) using a micro-balance (Mettler MC or ME-5 microbalance, Mettler-Toledo LLC, Columbus, OH, USA).

Duration of treatment / exposure:

4 weeks of exposure. The effects were assessed 4 weeks post the final exposure day (56 days after the initial exposure).

Frequency of treatment:

5 days/week for 3 hours/day

Dose / conc.:

4 mg/m³ air (nominal)

Remarks:

CeO2 nanoparticles with 27% Zr

Dose / conc.:

4 mg/m³ air (nominal)

Remarks:

CeO2 nanoparticles with 78% Zr

No. of animals per sex per dose:

- 8 ApoE-/- mice per group
- 16 5xFAD mice per group
- 10 C57BL/6J mice per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

To explore the (patho)physiological effects of nanoparticles on multiple organ systems, three different mouse models were exposed:
- Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice are a well established model for the study of the vascular disease atherosclerosis (Coleman et al., 2006), a disease characterised by the build-up of lipid- and inflammatory cell-rich plaques within arteries, which underlies the majority of cardiovascular diseases. The 4-week exposure protocol was integrated into an 8-week high-fat feeding regime that has been shown to generate complex atherosclerotic plaques with many of the hallmarks of the human disease in specific arterial locations (Cassee et al., 2012, Miller et al., 2013).
ApoE−/− mice were used to study hematology, pulmonary and cardiovascular effects.
- The 5xFAD mice are an Alzheimer’s disease mouse model. Although these mice were included to study neurological effects that will be published in a separate paper, the hematology and pulmonary effects were also studied within the same animals and reported in this paper.
- C57BL/6J mice were used as the background (non-genetically modified) strain of the disease mouse models and used to study biodistribution, hematology and pulmonary effects.
Hematology, pulmonary and cardiovascular effects were assessed 4 weeks post exposure. This period was included, firstly, to provide the extra four weeks necessary for mice to develop sufficient plaque formation in arteries and secondly, to investigate the persistency of the pulmonary and cardiovascular effects.
One group of each mouse model was exposed nose-only for four weeks to 4 mg/m³ Zr-doped nano-CeO2 for 5 days/week for 3 hours/day and one control group to clean air under the same experimental conditions (nose-only tube, 3h). Effects were assessed 4 weeks post the final exposure (56 days after the initial exposure).
The number of animals per group was different for each of the mouse models, to provide sufficient statistical power to detect differences between exposed and control animals in the most important effect parameter of each mouse model. Based on previous experiments, 8 ApoE-/- mice per group were expected to be sufficient to detect statistically significant differences in atherosclerotic plaque size and 5 C57BL/6J mice per group were expected to be sufficient to detect statistically significant differences in the number of neutrophils in the bronchoalveolar lavage fluid (BALF). However, 10 C57BL/6J mice per group were included, since 10 C57BL/6J mice per group and 16 5xFAD mice per group were needed to provide sufficient statistical power in the neurological study.

Positive control:

Observations and examinations performed and frequency:

Animals were monitored by cage-side observations and, if necessary, handled to detect signs of compromised health. The body weight of each animal was recorded one day before the start of exposure (day -1), prior to exposure on the first day and weekly thereafter.

Hematology, neurological (results published in a separate article which was not avaible at the time of the dossier preparation), pulmonary and cardiovascular effects were assessed 4 weeks after the last exposure of the mice (i.e. 56 days after the initial exposure).

- QUANTIFICATION OF CERIUM AND ZIRCONIUM IN TISSUES
During necropsy, organs from half of the C57BL/6J mice per group were obtained to evaluate the distribution of the Zr-doped nano CeO2 throughout the body. Liver, spleen, kidneys, heart and right (exposed mice) or left (control mice) lung, were weighed and immediately frozen in liquid nitrogen for determination of the Ce and Zr concentrations. To allow measurement of multiple parameters within the same animal, different parts of the lungs were selected for the exposed compared to the control animals. From the exposed groups the right lung was used for quantification of cerium and zirconium, because the left lung was needed for histopathological examination. From the control group the left lung was used for quantification of cerium and zirconium, because the right lung was needed for bronchoalveolar lavage. The organs were digested by acidification of each sample with 2 mL nitric acid for 12h. Hydrogen fluoride (0.2 mL) was added, followed by microwave heating for 45 min up to 185°C, and maintained for a further 20 min. Boric acid (2 mL) was added to neutralise the hydrogen fluoride, and the samples were re-heated for 20 min to 160 °C, and maintained for 10 min. Once cooled, samples were filtered with a 450 nm syringe filter, diluted with 10 mL deionised water and stored at room temperature (RT). The presence of cerium and zirconium in the lungs, liver, spleen, kidneys and heart was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer NexION 300X instrument operated in standard mode for cerium. The isotope measured was 140Ce using 115In and 159Tb as internal standards. Calibration standards (0 – 100 μg/L) were prepared from VWR 1000 mg/L stock solutions. Quantities are expressed as µg/g organ tissue.

- HEMATOLOGY
Animals were anaesthetised with a mixture of ketamine and xylazine. Two blood samples were taken by eye extraction. The first sample was collected in a K3-EDTA tube (Minicollect K3EDTA, 1 mL, 450474 Greiner Bio-One) for hematological parameters as determined in a blood auto analyser (ADVIA 2120 Hematology System, Siemens Healthineers) within 3 hours after collection. The second sample was collected in a serum tube and stored at -20°C for further analysis. Total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cells counts were meaured.

- BRONCHOALVEOLAR LAVAGE
Lung lavage was performed at necropsy. A cannula was placed in the trachea and the diaphragm opened to decrease the amount of air inside the lungs. For the control animals and all ApoE-/- animals, the right lung half was rinsed twice with approximately 0.5 mL (26.7 mL per kg body weight) of physiological saline solution, after ligation of the left lung. The injected volume was inserted and recovered 3 times, after which the lavage liquid was collected and stored on ice for less than 2 hours. To allow necropsy of the planned number of animals within one day, both lungs were lavaged for 10 of the 16 exposed 5xFAD mice and 5 of the 10 exposed C57BL/6J mice, using the same procedure, but approximately 0.8 mL (40 mL per kg body weight) of physiological saline solution.
BALF was centrifuged at 400g for 10 minutes at 4°C. The supernatant was divided into two separate aliquots of 125 µL for total protein (TP; an indicator for acute lung injury), lactate dehydrogenase (LDH; an indicator of cytotoxicity), gammaglutamyl transferase (GGT; an indicator of lung cell damage) and alkaline phosphatase (ALP; an indicator of type II cell damage) measurements using an autoanalyser (LX20- Pro, Beckman-Coulter, Woerden, the Netherlands). The cell pellet was scored for the presence of erythrocytes, re-suspended in 500 μL phosphate-buffered saline (PBS) and kept on ice. Cell counts were determined in the re-suspended pellet using a Coulter counter (Beckman-Coulter, Live Sciences). Cell concentrations were determined using a single sample using at least 150 µL of the re-suspended cells. Cytospins (Cytospin 3, Thermo-Shandon) were prepared and stained using May-Grunwald and Giemsa stain, and cell differentiation was performed by counting 400 cells per slide.

Sacrifice and pathology:

HISTOPATHOLOGY
- Lung tissue:
For the majority of the animals, the right lung was removed after the collection of the BALF and immediately frozen in liquid nitrogen and stored at - 80°C for further analysis. The left lung was removed and, after weighing, cannulated and infused with formaldehyde for 1 hour at a pressure of 20 cm H2O. Lungs were processed for histopathology; embedded in paraffin wax, sectioned at 2-4 µm intervals and stained with hematoxylin and eosin for histopathological examination.
Histopathological changes were described according to distribution, severity and morphological characteristics. The morphological characteristics of chronic inflammation include for example the presence of lymphocytes and macrophages in the lung tissue, while acute inflammation is characterised by the presence of polymorphonuclear neutrophils (PMNs).
Severity scores were assigned as follows: Grade 1 Minimal/very few/very small; Grade 2 Slight/few/small; Grade 3 Moderate/moderate number/moderate size; Grade 4 Marked/many/large; Grade 5 Massive/extensive number/extensive size.

- Assessment of atherosclerosis:
Arteries (brachiocephalic, aortic arch, thoracic aorta) were isolated from ApoE-/- mice. Atherosclerosis was quantified, as previously published (Miller et al., 2013). Briefly, brachiocephalic arteries were fixed in formalin and histological sections were taken in triplicate at 100 µm intervals, beginning at the first section of the artery with a fully intact media. Sections were stained with Masson’s Trichrome. The cross-sectional area of the plaque was measured and standardised to the medial area. A single mean value for atherosclerotic burden for each animal was calculated from the plaque size from each complete serial section throughout the brachiocephalic artery. A single section from each artery (the section exhibiting the largest plaque in cross-section) was chosen for mac-2 immunohistochemistry for macrophage-derived cells. A rat anti-mouse primary antibody was used (1/12000; CL8942AP, VH Bio, Gateshead, UK) with rat IgG (1/12000; I-400, Vector Labs, Peterborough, UK) as a negative control, followed by a goat anti-rat IgG biotinylated secondary antibody (BA-9400, Vector Labs). The area of positive staining was expressed as a proportion of the total plaque area.

- Other organs:
The spleen, liver, heart and kidneys were removed, weighed and stored in 4% formaldehyde for pathological analysis if required based on macroscopic findings.

Statistics:

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Total and differential white blood cell (lymphocytes, neutrophils, eosinophils and monocytes) counts were measured 4 weeks after the last day of exposure (day 56). Results are the mean of 8 to 16 animals. ApoE-/- mice had more neutrophils compared to exposed and control C57BL/6J and 5xFAD mice (p<0.05 in Tukey’s post-hoc test following one-way ANOVA). No statistically significant differences were observed in the total white blood cell counts or differential white blood cell counts of the exposed groups compared to the controls in blood from all strains of mice, except for a small decrease in neutrophils in ApoE-/- mice exposed to 78%-doped CeO2 nanoparticles (Tukey’s post-hoc test following one-way ANOVA with p=0.02). In addition, there was a decrease (post-hoc test for linear trend following one-way ANOVA with p=0.002) in neutrophils in ApoE-/- mice with increasing amounts of Zr-doping.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related effects on organ weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings related to treatment (data not shown) were observed and thus no histopathological analysis was performed on the organs other than lungs.

Neuropathological findings:

not examined

Description (incidence and severity):

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- LUNG: While modest, an increased incidence in minimal chronic bronchoalveolar or alveolar inflammation was observed in the exposed animals compared to the control mice of the 3 strains. The increase did not appear to be related to the percentage Zr-doping.
Particle loaded alveolar macrophages were observed in seven of the eight ApoE-/- mice exposed to nano CeO2 NP. This effect was not observed in ApoE-/- mice exposed to 27% or 78% Zr-doped CeO2 NPs or any other exposure or the control group of the other mouse models (see table 1 in "Any information on results incl. tables"), indicating that Zr-doping may influence particle loading of alveolar macrophages in ApoE-/- mice.
- BALF: In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the nanoparticle-exposed animals but not in the control groups.

Other effects:

no effects observed

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE (BALF)
- Total and differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytesin) from BALF measured 4 weeks after the last exposure day (mean of 5 to 16 animals) in the 3 mice strains:
No statistically significant differences were observed in the total cell counts or differential cell counts in BALF of the exposed groups compared to the controls for any strain of mouse, except for the total cell count in 5xFAD mice exposed to 27% Zr-doped CeO2 nanoparticles, which was lower than that of the control 5xFAD mice. There was a decrease (p<0.1; post-hoc test for linear trend following one-way ANOVA) in total cell counts (p=0.075), % macrophages (p=0.014), and % neutrophils (p=0.05) in ApoE-/- mice with increasing amounts of Zr-doping. However, no such trends were observed in C57 BL/6J or 5xFAD mice.

- Total protein, Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP) and Gamma-Glutamyl Transpeptidase (GGT) levels in bronchoalveolar lavage fluid (BALF) 4 weeks after exposure (mean of 5 to 16 mice from the each tested strains):
Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels between the exposed and control groups.

No constitutive differences in differential cell counts or protein levels were observed between the different mice strains.

EFFECTS ON ATHEROSCLEROSIS IN THE BRACHIOCEPHALIC ARTERY OFApoE-/- MICE AFTER INHALATION
ApoE-/- mice exhibited regions of dense plaques in the aortic arch and branch points of large arteries. Plaques were composed of fibroblastic matrix, smooth muscle cells, lipid cavities and cholesterol crystals. Atherosclerotic burden was quantified in the brachiocephalic artery, with control (air-exposed) mice having a mean plaque size of 94 ± 9% (standardised to the area of the vascular media). Exposure to Zr-doped nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries (p=0.62; One-way ANOVA). However, there was an increase (post-hoc test for linear trend following one-way ANOVA with p=0.09) in the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells) with increasing Zr content of the CeO2 nanoparticles.

Dose descriptor:

NOAEL

Effect level:

> 4 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: Zr-doped nano CeO2 had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

Critical effects observed:

Estimated deposition of inhaled Zr-doped nano CeO2 in different regions of the lungs:

The total inhaled dose, estimated as tidal volume (0.203 mL) x breathing frequency (353 min-1) x exposure concentration (≈ 4 x 10-3 μg/mL) x exposure duration (3600 min), was calculated to be 1043 µg and 1056 µg for 27% and 78% Zr-doped nano CeO2, respectively. The total deposited doses (estimated using the MPPD model) were found to be 625 µg for the 27% Zr-doped nano CeO2, and 639 µg for the 78% Zr-doped nano CeO2. The deposited fractions per region, estimated using the MPPD model, were found to be 48.8% and 49.8% (27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively) for the head, 3.4% and 3.3% (27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively) for the tracheobronchial region and 7.9% and 7.5% (27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively) for the alveolar region of the inhaled mass dose. The retained doses in lung, corresponding to the retained dose in the tracheobronchial and alveolar region 4 weeks post-exposure, estimated using the MPPD model, were found to be 16.2 µg and 15.6 µg, for the 27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively.

Quantification of cerium and zirconium in tissues (in µg/g of tissues, mean of 4 -6 animals) 4 weeks after the last exposure day:

The highest concentrations of cerium were found in the lung (~ 20 µg/g for the 27% Zr-doped nano CeO2 and ~ 61 µg/g for the 78% Zr-doped nano CeO2) followed by much lower concentrations in the heart (~ 1.8 µg/g for the 27% Zr-doped nano CeO2 and ~ 6 µg/g for the 78% Zr-doped nano CeO2), spleen (~ 1.0 µg/g for the 27% Zr-doped nano CeO2 and ~ 1.1 µg/g for the 78% Zr-doped nano CeO2), kidneys (~ 4.5 µg/g for the 27% Zr-doped nano CeO2 and ~ 4.4 µg/g for the 78% Zr-doped nano CeO2), and liver (~ 0.1 µg/g for the 27% Zr-doped nano CeO2 and ~ 0.2 µg/g for the 78% Zr-doped nano CeO2), respectively. As would be expected, significantly higher levels of cerium were observed in the lungs of exposed mice compared to the controls (~1.1 µg/g in control lung).

The highest concentrations of zirconium were also found in the lung (~ 63 µg/g for the 27% Zr-doped nano CeO2 and ~ 82 µg/g for the 78% Zr-doped nano CeO2) followed by much lower concentrations in the heart (~ 5 µg/g for the 27% Zr-doped nano CeO2 and ~ 14 µg/g for the 78% Zr-doped nano CeO2), spleen (~ 2.3 µg/g for the 27% Zr-doped nano CeO2 and ~ 3.1 µg/g for the 78% Zr-doped nano CeO2), kidneys (~ 4.6 µg/g for the 27% Zr-doped nano CeO2 and ~ 4.4 µg/g for the 78% Zr-doped nano CeO2), and liver (~ 0.6 µg/g for the 27% Zr-doped nano CeO2 and ~ 0.5 µg/g for the 78% Zr-doped nano CeO2), respectively. As would be expected, significantly higher levels of zirconium were observed in the lungs of exposed mice compared to the controls (≤ 1µg/g in control lung).

Surprisingly, increasing amounts of Zr-doping in the nanoparticles did not result in decreasing Ce concentrations or increasing Zr concentrations in the lung. In most of the other organs, the cerium and zirconium concentrations were not statistically significantly different from the background concentrations measured in the control animals (heart (~ 2.8 µg/g for Ce, ~ 8 µg/g for Zr), spleen (~ 1.0 µg/g for Ce, ~ 2.5 µg/g for Zr), kidneys (~ 1.3 µg/g for Ce, ~ 2.1 µg/g for Zr) and liver (~ 0.05 µg/g for Ce, ~ 0.5 µg/g for Zr)). A few exceptions were observed, for example, the Ce concentration in the hearts of mice exposed to 78% Zr-doped CeO2.

The authors indicated that background levels of Ce and Zr detected in various organs of animals exposed to clean air (controls) might be caused by Ce and Zr contamination of the drinking water, food and/or bedding of the animals as previously found by others (Yokel et al. 2012, 2013).

Table 1: Histopathological findings in lung of C57BL/6J, ApoE-/- and 5xFAD mice.

Histopathological finding →		Chronic broncho-alveolar or alveolar inflammation		Alveolar macrophages
Strain¯	Treatment¯	Minimal	Slight	Minimal
C57BL/6J	Control	60% (3/5)a	-	-
	27% Zr-doped nano CeO₂	40% (2/5)	-	20% (1/5)
	78% Zr-doped nano CeO₂	80% (4/5) -	-	-
ApoE-/-	Control	25% (2/8)	-	-
	27% Zr-doped nano CeO₂	38% (3/8)	13% (1/8)	-
	78% Zr-doped nano CeO₂	50% (4/8)	-	-
5xFAD	Control	69% (11/16)	-	6% (1/16)
	27% Zr-doped nano CeO₂	67% (4/6)	-	-
	78% Zr-doped nano CeO₂	67% (4/6)	-	33% (2/6)

A: Percentage of animals affected. Within brackets the number of animals with histopathological findings versus number of animals evaluated.

Conclusions:

The authors concluded that nano CeO2 had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease. Zr-doping of CeO2 nanoparticles had limited effects on these responses, although indications that Zr-doping could potentially alter particle loading of alveolar macrophages and increase the inflammatory cell content in atherosclerosis plaques merit further investigation.

Executive summary:

In this subacute inhalation study, Dekkers et al. (2015) explored the (patho)physiological effects of Zr-doped nano CeO2 exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. As this study was performed using a test procedure in agreement with generally accepted scientific standards but with some limitations (incomplete data on substance characterisation and on systemic toxicity), the study was awarded a reliability score of 2 according to Klimisch criteria and was used as weight of evidence.

Female mice from each strain were exposed nose-only to 0 (controls, exposed to filtered air) and 4 mg/m³ of 27% or 78% Zr-doped nano CeO2 (primary particle size of 4.6 (27% Zr-doped nano CeO2) or 4.7 nm (78% Zr-doped nano CeO2)) for 3h/day, 5 days/week for 4 weeks.

Four weeks after the last exposure day (i.e day 56 of the study), the animals of each strain were sacrificed, organs weights were measured and the effects of the treatment on the blood (total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cell counts) and on the lungs (analyses of the differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytes) and protein levels (total proteins, ALP, LDH and GGT) in BALF) were evaluated. Furthermore, histopathological analysis of the lungs was done and the tissue distribution of cerium and zirconium was measured using ICP-MS in the lung, heart, kidney, spleen and liver of all animals. The effects of Zr-doped nano-CeO2 on the artherosclerosis burden in the brachiocephalic artery of ApoE-/- mice was also determined in this study to evaluate cardiovascular effects of treatment with nanoparticles by measuring the mean plaque size and of the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels in BALF between the exposed and control groups. Furthermore, no treatment related effects on organ weights were observed and no macroscopic findings related to treatment were observed. In lungs, the histopathological analyses showed an increased incidence, described as modest by the authors, in minimal chronic bronchoalveolar or alveolar inflammation observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were not observed in mice exposed to 27% or 78% Zr-doped CeO2, indicating Zr-doping may influence particle loading of alveolar macrophages in ApoE-/- mice (in these mice, particle-loaded alveolar macrophages were observed in almost all test animals exposed to pure nano cerium dioxide). In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the nanoparticle-exposed animals but not in the control groups.

In tissues, background Ce and Zr concentrations were measured in the control animals and significantly higher levels of Ce and Zr were only observed in the lungs and the liver of exposed mice as compared to the controls but not in the other organs.

In ApoE-/- mice, exposure to nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries. However, there was an increase in the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells) with increasing Zr content of the nano CeO2 exposure.

The evaluation of potential neurological effects of treatment on the 5xFAD mice were not reported in this article.

The authors concuded that in this subacute inhalation study, Zr-doped nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

No NOAEL was derived in this study by the authors. However, according to the results and the conclusion of the authors (Zr-doped nano CeO2 has a low biological activity in their mouse models), it can be concluded that the NOAEL for lung toxicity is > 4 mg/m³, the only dose tested in this study.

Reference 6

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Well documented, scientifically sound study that is similar to the OECD 412 "Repeated Dose Inhalation Toxicity: 28-day or 14-day study" guideline with some deviations.
Remarks:: (1) only one dose was used rather than 3; (2) 2 dogs, 6 rabbits, and 20 rats (sex not provided) were exposed rather than 5 animals/sex/dose; (3) complete list of tissues examined for gross and histopathology were not provided; (4) no urinalysis or clinical chemistry parameters were examined.
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:: yes
Remarks:: see rationale for reliability
GLP compliance:: no
Limit test:: no
Species:: other: dog, rabbit, rat
Strain:: not specified
Sex:: not specified
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Vehicle:: other: no data
Remarks on MMAD:: MMAD / GSD: Mass median particle diameter: 1.5 microns
Details on inhalation exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: copper-lined chamber constructed of wood with observation windows on three sides, 6 x 8 x 6 ft high and volume of 288 cubic feet.
- Method of conditioning air: A centrally located duct in the ceiling of the chamber served as the inlet for the test substance. Baffles below the inlet and two fans near the ceiling dispersed the test substance and distributed the test substance uniformly throughout the chamber. In the four bottom corners were outlets connected to an exhaust system. Air turnover during exposure was approximately 140 cfm or one change every two minutes with no recycling.
- System of generating particulates/aerosols: Wright dust feed.
- Temperature, humidity, pressure in air chamber: 74 +/- 3 degrees F; 47% +/- 6%; few hundredths of an inch of water less than atmospheric pressure.
- Air flow rate: 140 cfm or one change every two minutes.
- Method of particle size determination: test substance was twice ground in a Mikropulverizer to a mean bulk particle size.
- Treatment of exhaust air: not recycled

TEST ATMOSPHERE
- Brief description of analytical method used: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
- Samples taken from breathing zone: yes, hourly
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
Duration of treatment / exposure:: 30 days
Frequency of treatment:: 6 hours/day 5 days/week
Dose / conc.:: 100.8 mg/m³ air
Remarks:: Basis: analytical conc. (ZrO2)
No. of animals per sex per dose:: Dog 2 (sex not known)
Rabbit 6 (sex not known)
Rat 20 (sex not known)
Control animals:: other: yes, only control rabbits and rats were used
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weekly
- Animals fasted: No data
- How many animals: 2 dogs
- Parameters: Red blood cell and differential white cell counts, as well as determinations of hemoglobin, mean corpuscular cell volume and clotting time

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: HISTOPATHOLOGY: Yes, lung, kidney, liver. Other tissues were examined, but the specific list of other tissues was not provided
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: not specified
Gross pathological findings:: not specified
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Details on results:: CLINICAL SIGNS AND MORTALITY: One rabbit died during exposure to the test substances, however this matched the control group.

BODY WEIGHT AND WEIGHT GAIN: Throughout the exposure periods the weekly weight responses were identical to those of normal animals.

HAEMATOLOGY: There were no reported hematological changes in the two dogs that were selected for this parameter during the exposure period.

HISTOPATHOLOGY: NON-NEOPLASTIC: There are no reported histological changes that could be attributed to the test substance. The abnormalities that were reported were similar for all species regardless of dose, duration of exposure or test substance inhaled. In all exposed animals, an apparently granular material, brownish-black and doubly refracting, was found in alveolar walls and in phagocytes. Occasionally, this dust was also seen in bronchi and lymph nodes.
Dose descriptor:: NOAEC
Effect level:: >= 100.8 mg/m³ air
Based on:: test mat.
Sex:: not specified
Basis for effect level:: other: no effects were reported in any species tested
Critical effects observed:: no
Conclusions:: Inhalation of 100.8 mg/m3 zirconium dioxide for 30 days produced no significant changes in animals in mortality rate, growth, hematology values or histopathology. The NOAEC was deemed to be greater than (or equal to) 100.8 mg/m3.

Reference 7

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Well documented, scientifically sound study that is similar to the OECD 413 "Subchronic Inhalation Toxicity: 90-day study" guideline with some deviations.
Remarks:: 1) only 1 dose tested rather than 3; (2) exposure was for 60 days rather than 90 days; (3) complete list of tissues examined for histopathology was not provided; (4) limited clinical chemistry and urinalysis; (5) 4 cats, 4 dogs, 20 guinea pigs, 19 rabbits and 72 rats (sex not provided) were exposed rather than 10 rodents per sex per dose.
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:: yes
Remarks:: see rationale for reliability
GLP compliance:: no
Limit test:: no
Species:: other: cat, dog, guinea pig, rabbit, rat
Strain:: not specified
Sex:: not specified
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Vehicle:: other: no data
Remarks on MMAD:: MMAD / GSD: Mass median particle diameter: 1.6 microns
Details on inhalation exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: copper-lined chamber constructed of wood with observation windows on three sides, 6 x 8 x 6 ft high and volume of 288 cubic feet.
- Method of conditioning air: A centrally located duct in the ceiling of the chamber served as the inlet for the test substance. Baffles below the inlet and two fans near the ceiling dispersed the test substance and distributed the test substance uniformly throughout the chamber. In the four bottom corners were outlets connected to an exhaust system. Air turnover during exposure was approximately 140 cfm or one change every two minutes with no recycling.
- System of generating particulates/aerosols: Wright dust feed.
- Temperature, humidity, pressure in air chamber: 74 +/- 3 degrees F; 47% +/- 6%; few hundredths of an inch of water less than atmospheric pressure.
- Air flow rate: 140 cfm or one change every two minutes.
- Method of particle size determination: test substance was twice ground in a Mikropulverizer to a mean bulk particle size.
- Treatment of exhaust air: not recycled

TEST ATMOSPHERE
- Brief description of analytical method used: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
- Samples taken from breathing zone: yes, hourly
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
Duration of treatment / exposure:: 60 days
Frequency of treatment:: 6 hours/day, 5 days/week
Dose / conc.:: 15.4 mg/m³ air
Remarks:: Basis: analytical conc. (ZrO2)
No. of animals per sex per dose:: Cat- 4
Dog - 4
Guinea Pig - 20
Rabbit - 19
Rat - 72
Control animals:: other: yes, only control rabbits and rats were used
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weekly
- Animals fasted: No data
- How many animals: 4 dogs
- Parameters: Red blood cell and differential white cell counts, as well as determinations of hemoglobin, mean corpuscular cell volume and clotting time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: semi-monthly
- Animals fasted: No data
- How many animals: 4 dogs and 4 rabbits
- Parameters: Blood nonprotein nitrogen

URINALYSIS: Yes
- Time schedule for collection of blood: semi-monthly
- Animals fasted: No data
- How many animals: 4 dogs and 4 rabbits
- Parameters: urinary protein

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: HISTOPATHOLOGY: Yes, lung, kidney, liver. Other tissues were evaluated, but a complete list of other tissues was not provided
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: not specified
Gross pathological findings:: not specified
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Details on results:: CLINICAL SIGNS AND MORTALITY: One rabbit and one guinea pig in the 15.4 mg ZrO2/m3 dose group died during exposure to the test substances; however, this matched the deaths observed in the the control group.

BODY WEIGHT AND WEIGHT GAIN: Throughout the exposure periods the weekly weight responses were identical to those of normal animals. Adult cats, dogs, guinea pigs, and rabbits maintained their weights with only slight random fluctuations; both male and female rats showed small gains consistent with their ages.

HAEMATOLOGY: Four dogs exposed to 15.4 mg ZrO2/m3 showed no hematologic changes during semi-monthly intervals of analysis.

CLINICAL CHEMISTRY: No significant blood changes were found among the criteria studied. Blood fibrinogen levels also remained constant during exposure of animals to the test substance.

URINALYSIS: No significant urine changes were found among the criteria studied.

HISTOPATHOLOGY: NON-NEOPLASTIC: There are no reported histological changes that could be attributed to the test substance. The abnormalities that were reported were similar for all species regardless of dose, duration of exposure or test substance inhaled. The lungs showed varying, but small, amounts of congestion, edema and hemorrhage, approximately half of all animals having histological lesions. Control animals, however, exhibited a similar rate of incidence. In all exposed animals, an apparently granular material, brownish-black and doubly refracting, was found in alveolar walls and in phagocytes. Occasionally, this dust was also seen in bronchi and lymph nodes. Among the other tissues, only the kidney consistently showed damage. These renal lesions were a low-grade interstitial nephritis of parasitic origin. Occasionally, parasitic granulomata were found, and two instances of testicular atrophy were noted among 160 animals.
Dose descriptor:: NOAEC
Effect level:: >= 15.4 mg/m³ air
Based on:: test mat.
Sex:: not specified
Basis for effect level:: other: no effects were reported in any of the animals studied
Critical effects observed:: no
Conclusions:: Inhalation of 15.4 mg/m3 zirconium dioxide for 60 days produced no significant changes in mortality rate, growth, biochemistry, hematology values or histopathology. The NOAEC was deemed to be greater than (or equal to) 15.4 mg/m3.

Reference 8

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Justification for type of information:: Read across from studies performed with cerium dioxide and zirconium dioxide. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Dose descriptor:: other: read across conclusion
Remarks on result:: other: The reaction mass of cerium dioxide and zirconium dioxide is concluded not to present any systemic toxicity after repeated inhalation exposure.
Remarks:: Conclusion based on the results of the various read across studies performed with bulk and nano zirconium dioxide and bulk and nano cerium dioxide, in addition to the results from a study performed with various Zr-doped nano cerium dioxide forms. In none of these studies, systemic adverse effects have been observed. Further, the observation of loco-regional effects in the lungs seems to be related to cerium dioxide rather than zirconium dioxide, but also appeared to be species-dependent, as such effects have been observed in rats but not in mouse. Moreover, the observed loco-regional effects of cerium dioxide in rats are considered to represent rather a non-specific adaptive response to particle overload of the lung and indicate the sensitivity of rats to this type of condition. Taking all evidence together, it is not considered necessary to perform further repeated dose inhalation toxicity studies with the reaction mass of cerium dioxide and zirconium dioxide, as this is not expected to bring further insights, and it is therefore not considered justifiable taking into account the obligation to avoid animal testing where necessary.
Critical effects observed:: no

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 1993 - December 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Filters from 2nd day of exposure and monthly thereafter used for chemical analysis by Sponsor.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Dose / conc.:

0.005 mg/L air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

0.051 mg/L air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

0.507 mg/L air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

5 mg/m³ air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

50.5 mg/m³ air

Remarks:

Basis: mean achieved chamber concentrations

Dose / conc.:

507.5 mg/m³ air

Remarks:

Basis: mean achieved chamber concentrations

No. of animals per sex per dose:

Control animals:

yes

Details on study design:

Positive control:

Not applicable

Observations and examinations performed and frequency:

Sacrifice and pathology:

Other examinations:

Not applicable

Statistics:

Clinical signs:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Mortality:

no mortality observed

Description (incidence):

No relevant (treatment-related) effects were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- No relevant changes in females
- Food consumption of high-dose males marginally lower than that of controls although without statistical significance

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No relevant (treatment-related) effects were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOEC

Effect level:

0.507 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical biochemistry

clinical signs

mortality

ophthalmological examination

urinalysis

Dose descriptor:

NOEC

Effect level:

0.507 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOEC

Effect level:

0.051 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male

Basis for effect level:

haematology

Dose descriptor:

LOAEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

female

Basis for effect level:

haematology

Dose descriptor:

NOAEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

LOAEC

Effect level:

0.005 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

LOAEC

Effect level:

0.051 mg/L air (analytical)

Based on:

test mat.

Remarks:

Gravimetric concentration

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Tissue/Lesion	Incidence of microscopic findings (/No. of animals examined)
	Males - 0 mg/L	Females - 0 mg/L	Males - 0.005 mg/L	Females - 0.005 mg/L	Males - 0.0505 mg/L	Females - 0.0505 mg/L	Males - 0.5075 mg/L	Females - 0.5075 mg/L
Bronchi: pigment accumulation	0/15	0/15	1/15	0/15	5/15*	4/15*	15/15*	15/15*
Nasal cavity:
- Goblet cell hypertrophy and/or hyperplasia	0/15	0/15	0/15	1/15	0/15	0/15	1/15	2/15
- Pigment accumulation	0/15	0/15	12/15*	3/15	15/15*	11/15*	15/15*	15/15*
Larynx:
- Metaplasia	0/15	0/15	3/15	3/15	9/15*	6/15*	13/15*	9/15*
- Pigment accumulation	0/15	0/15	6/15*	0/15	9/15*	7/15*	12/15*	9/15*
Liver: pigment accumulation	0/15	0/15	0/9	0/1	0/7	0/5	6/15*	5/15*
Lungs:
- Pigment accumulation	0/15	0/15	15/15*	15/15*	15/15*	15/15*	15/15*	15/15*
- Alveolar epithelial hyperplasia (with severity)	0/15	0/15	1/15(+)	0/15	11/15* (+ to ++)	5/15* (+ to ++)	14/15* (+ to +++)	15/15* (+ to ++)
- Lymphoid hyperplasia	0/15	0/15	0/15	0/15	0/15	1/15	12/15*	7/15*
Mandibular lymph nodes: pigment accumulation	0/15	0/15	0/3	0/5	0/5	0/3	6/15*	6/15*
Spleen: pigment accumulation	0/15	0/15	0/1	0/0	0/0	0/0	6/15*	3/15
Trachea: pigment accumulation	0/15	0/15	0/15	0/15	1/15	1/15	14/15*	14/15*
Bronchial lymph nodes:
- Pigment accumulation	0/15	0/15	13/13*	14/15*	15/15*	15/15*	15/15*	15/15*
- Lymphoid hyperplasia	0/15	0/15	11/13*	13/15*	15/15*	15/15*	15/15*	15/15*
Mediastinal lymph nodes:
- Pigment accumulation	0/0	0/1	2/2	10/10	8/10	9/9	9/9	9/10
- Lymphoid hyperplasia	0/0	0/1	2/2	10/10	9/10	9/9	9/9	9/10
Pancreatic lymph nodes:
- Pigment accumulation	-	0/2	-	0/0	-	1/1	-	1/1
- Lymphoid hyperplasia	-	0/2	-	0/0	-	1/1	-	0/1

* p < 0.05

+: slight

++: mild

+++: moderate

Conclusions:

Executive summary:

Treatment-related changes included changes in hematology, lung and spleen weights, macroscopic observations at necropsy and histopathology of the respiratory tract and lymphoreticular system.

This study is classified as acceptable. It satisfies the OECD 413 guideline requirements on subchronic inhalation toxicity.

Reference 2

Endpoint:: short-term repeated dose toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Well documented, scientifically sound study that is similar to the OECD 412 "Repeated Dose Inhalation Toxicity: 28-day or 14-day study" guideline with some deviations.
Remarks:: (1) only one dose was used rather than 3; (2) 2 dogs, 6 rabbits, and 20 rats (sex not provided) were exposed rather than 5 animals/sex/dose; (3) complete list of tissues examined for gross and histopathology were not provided; (4) no urinalysis or clinical chemistry parameters were examined.
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:: yes
Remarks:: see rationale for reliability
GLP compliance:: no
Limit test:: no
Species:: other: dog, rabbit, rat
Strain:: not specified
Sex:: not specified
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Vehicle:: other: no data
Remarks on MMAD:: MMAD / GSD: Mass median particle diameter: 1.5 microns
Details on inhalation exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: copper-lined chamber constructed of wood with observation windows on three sides, 6 x 8 x 6 ft high and volume of 288 cubic feet.
- Method of conditioning air: A centrally located duct in the ceiling of the chamber served as the inlet for the test substance. Baffles below the inlet and two fans near the ceiling dispersed the test substance and distributed the test substance uniformly throughout the chamber. In the four bottom corners were outlets connected to an exhaust system. Air turnover during exposure was approximately 140 cfm or one change every two minutes with no recycling.
- System of generating particulates/aerosols: Wright dust feed.
- Temperature, humidity, pressure in air chamber: 74 +/- 3 degrees F; 47% +/- 6%; few hundredths of an inch of water less than atmospheric pressure.
- Air flow rate: 140 cfm or one change every two minutes.
- Method of particle size determination: test substance was twice ground in a Mikropulverizer to a mean bulk particle size.
- Treatment of exhaust air: not recycled

TEST ATMOSPHERE
- Brief description of analytical method used: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
- Samples taken from breathing zone: yes, hourly
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
Duration of treatment / exposure:: 30 days
Frequency of treatment:: 6 hours/day 5 days/week
Dose / conc.:: 100.8 mg/m³ air
Remarks:: Basis: analytical conc. (ZrO2)
No. of animals per sex per dose:: Dog 2 (sex not known)
Rabbit 6 (sex not known)
Rat 20 (sex not known)
Control animals:: other: yes, only control rabbits and rats were used
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weekly
- Animals fasted: No data
- How many animals: 2 dogs
- Parameters: Red blood cell and differential white cell counts, as well as determinations of hemoglobin, mean corpuscular cell volume and clotting time

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: HISTOPATHOLOGY: Yes, lung, kidney, liver. Other tissues were examined, but the specific list of other tissues was not provided
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: not specified
Gross pathological findings:: not specified
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Details on results:: CLINICAL SIGNS AND MORTALITY: One rabbit died during exposure to the test substances, however this matched the control group.

BODY WEIGHT AND WEIGHT GAIN: Throughout the exposure periods the weekly weight responses were identical to those of normal animals.

HAEMATOLOGY: There were no reported hematological changes in the two dogs that were selected for this parameter during the exposure period.

HISTOPATHOLOGY: NON-NEOPLASTIC: There are no reported histological changes that could be attributed to the test substance. The abnormalities that were reported were similar for all species regardless of dose, duration of exposure or test substance inhaled. In all exposed animals, an apparently granular material, brownish-black and doubly refracting, was found in alveolar walls and in phagocytes. Occasionally, this dust was also seen in bronchi and lymph nodes.
Dose descriptor:: NOAEC
Effect level:: >= 100.8 mg/m³ air
Based on:: test mat.
Sex:: not specified
Basis for effect level:: other: no effects were reported in any species tested
Critical effects observed:: no
Conclusions:: Inhalation of 100.8 mg/m3 zirconium dioxide for 30 days produced no significant changes in animals in mortality rate, growth, hematology values or histopathology. The NOAEC was deemed to be greater than (or equal to) 100.8 mg/m3.

Reference 3

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Well documented, scientifically sound study that is similar to the OECD 413 "Subchronic Inhalation Toxicity: 90-day study" guideline with some deviations.
Remarks:: 1) only 1 dose tested rather than 3; (2) exposure was for 60 days rather than 90 days; (3) complete list of tissues examined for histopathology was not provided; (4) limited clinical chemistry and urinalysis; (5) 4 cats, 4 dogs, 20 guinea pigs, 19 rabbits and 72 rats (sex not provided) were exposed rather than 10 rodents per sex per dose.
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:: yes
Remarks:: see rationale for reliability
GLP compliance:: no
Limit test:: no
Species:: other: cat, dog, guinea pig, rabbit, rat
Strain:: not specified
Sex:: not specified
Route of administration:: inhalation: dust
Type of inhalation exposure:: whole body
Vehicle:: other: no data
Remarks on MMAD:: MMAD / GSD: Mass median particle diameter: 1.6 microns
Details on inhalation exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: copper-lined chamber constructed of wood with observation windows on three sides, 6 x 8 x 6 ft high and volume of 288 cubic feet.
- Method of conditioning air: A centrally located duct in the ceiling of the chamber served as the inlet for the test substance. Baffles below the inlet and two fans near the ceiling dispersed the test substance and distributed the test substance uniformly throughout the chamber. In the four bottom corners were outlets connected to an exhaust system. Air turnover during exposure was approximately 140 cfm or one change every two minutes with no recycling.
- System of generating particulates/aerosols: Wright dust feed.
- Temperature, humidity, pressure in air chamber: 74 +/- 3 degrees F; 47% +/- 6%; few hundredths of an inch of water less than atmospheric pressure.
- Air flow rate: 140 cfm or one change every two minutes.
- Method of particle size determination: test substance was twice ground in a Mikropulverizer to a mean bulk particle size.
- Treatment of exhaust air: not recycled

TEST ATMOSPHERE
- Brief description of analytical method used: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
- Samples taken from breathing zone: yes, hourly
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Hourly samples were taken with a filter paper sampler and weighed on an analytical balance, spectrographic analysis of each day's accumulation of filter paper samples was used to verify the weight-samples and to make such slight adjustments in concentration results as might occasionally arise from varying amounts of nuisance dust.
Duration of treatment / exposure:: 60 days
Frequency of treatment:: 6 hours/day, 5 days/week
Dose / conc.:: 15.4 mg/m³ air
Remarks:: Basis: analytical conc. (ZrO2)
No. of animals per sex per dose:: Cat- 4
Dog - 4
Guinea Pig - 20
Rabbit - 19
Rat - 72
Control animals:: other: yes, only control rabbits and rats were used
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weekly
- Animals fasted: No data
- How many animals: 4 dogs
- Parameters: Red blood cell and differential white cell counts, as well as determinations of hemoglobin, mean corpuscular cell volume and clotting time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: semi-monthly
- Animals fasted: No data
- How many animals: 4 dogs and 4 rabbits
- Parameters: Blood nonprotein nitrogen

URINALYSIS: Yes
- Time schedule for collection of blood: semi-monthly
- Animals fasted: No data
- How many animals: 4 dogs and 4 rabbits
- Parameters: urinary protein

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: HISTOPATHOLOGY: Yes, lung, kidney, liver. Other tissues were evaluated, but a complete list of other tissues was not provided
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: not specified
Gross pathological findings:: not specified
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Details on results:: CLINICAL SIGNS AND MORTALITY: One rabbit and one guinea pig in the 15.4 mg ZrO2/m3 dose group died during exposure to the test substances; however, this matched the deaths observed in the the control group.

BODY WEIGHT AND WEIGHT GAIN: Throughout the exposure periods the weekly weight responses were identical to those of normal animals. Adult cats, dogs, guinea pigs, and rabbits maintained their weights with only slight random fluctuations; both male and female rats showed small gains consistent with their ages.

HAEMATOLOGY: Four dogs exposed to 15.4 mg ZrO2/m3 showed no hematologic changes during semi-monthly intervals of analysis.

CLINICAL CHEMISTRY: No significant blood changes were found among the criteria studied. Blood fibrinogen levels also remained constant during exposure of animals to the test substance.

URINALYSIS: No significant urine changes were found among the criteria studied.

HISTOPATHOLOGY: NON-NEOPLASTIC: There are no reported histological changes that could be attributed to the test substance. The abnormalities that were reported were similar for all species regardless of dose, duration of exposure or test substance inhaled. The lungs showed varying, but small, amounts of congestion, edema and hemorrhage, approximately half of all animals having histological lesions. Control animals, however, exhibited a similar rate of incidence. In all exposed animals, an apparently granular material, brownish-black and doubly refracting, was found in alveolar walls and in phagocytes. Occasionally, this dust was also seen in bronchi and lymph nodes. Among the other tissues, only the kidney consistently showed damage. These renal lesions were a low-grade interstitial nephritis of parasitic origin. Occasionally, parasitic granulomata were found, and two instances of testicular atrophy were noted among 160 animals.
Dose descriptor:: NOAEC
Effect level:: >= 15.4 mg/m³ air
Based on:: test mat.
Sex:: not specified
Basis for effect level:: other: no effects were reported in any of the animals studied
Critical effects observed:: no
Conclusions:: Inhalation of 15.4 mg/m3 zirconium dioxide for 60 days produced no significant changes in mortality rate, growth, biochemistry, hematology values or histopathology. The NOAEC was deemed to be greater than (or equal to) 15.4 mg/m3.

Reference 4

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

standard short-term inhalation study (STIS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physicochemical characterisation of CeO2 nanoparticles are presented below in "any other information on materials and methods incl. tables".

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: 0.6 - 0.9 µm / 2.3 - 2.9

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The atmospheric concentration of the aerosol (cascade impactor analysis) was determined by gravimetric measurement using a sampling probe with a diameter of 7 mm.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

2.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

basis: nominal nano-CeO2 conc.

No. of animals per sex per dose:

3 to 6 per group

Control animals:

yes, concurrent vehicle

other: negative control exposed to nano-BaSO4 at 50 mg/m3

Details on study design:

Positive control:

Coated nano-TiO2 (T-Lite SF™) at 0.5, 2 and 10 mg/m3

Observations and examinations performed and frequency:

Sacrifice and pathology:

Other examinations:

Statistics:

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see in "Details on results" below

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see in "Details on results" below

Histopathological findings: neoplastic:

no effects observed

Details on results:

Dose descriptor:

other: NOEC/NOAEC (systemic)

Based on:

other: nano-CeO2 (test mat.)

Sex:

male

Basis for effect level:

other: No NOEC/NOAEC was determined in the publication and no NOEC/NOAEC could be set by the registrant since the study evaluated only lung toxicity.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Dose descriptor:

other: NOAEC (local)

Effect level:

<= 0.5 mg/m³ air (nominal)

Based on:

other: nano-CeO2 (test mat.)

Sex:

male

Basis for effect level:

Critical effects observed:

not specified

Table 4: Effects induced by nano-CeO2 in the BALF

	Exposure groups			Recovery groups
Concentration (mg/m3)	0.5	2.5	10	0.5	2.5	10
Protein/Enzymes
Total protein	0.6*	1.4	2.8**	1.1	1.2	1.4
GGT	2.1	6.4**	6.6**	4	4.1	5.4
LDH	1.1	2.2**	4.3**	1.2	1.8*	2.3**
ALP	1.0	2.9*	4.1**	1.7	2.0**	2.4**
NAG	0.4	1.4	3.3**	1.2	1.6*	1.7**
Cell counts
Total cells	1.3	1.3	2.6	1.4	1.2	2.6
Macrophages	1.3	0.4*	0.4**	1.4	1.1	0.9
Lymphocytes	2.7*	10.3**	26.2**	0.8	3.3*	5.5*
PMN neutrophils	4.6*	164.1**	386.6**	0.9	8.0**	46.7**
Mediators
β2-Microglobulin	1.2	2.5**	3.5**	1.0	1.4	1.8*
CINC-1/IL-8	1.1	3.9*	5.3**	1.2	1.7*	2.1**
Clusterin	0.6	1.8	3.6**	1.0	1.4	1.1
Cystatin C	0.9	1.8*	3.8**	2.7**	3.3**	2.6**
Eotaxin	1.2	1.5*	2.3**	1.0	1.1*	1.1
Fibrinogen	1.0	1.4	2.7**	1.1	1.4	0.9
GCP-2	0.7	10.8**	15.4**	1.4*	2.8**	5.8**
IP-10	1.1	1.6	2.3*	0.9	0.9	0.7
MCP-1	1.3	5.2**	21.9**	0.7	1.4**	3.3**
MCP-3	1.3	6.6**	24.8**	1.0	1.3	1.9**
M-CSF	1.3*	3.4**	9.5**	1.0	1.2*	1.4**
MDC	1.7	79.0**	362.5**	1.1	1.2	3.3
MIP-1β	0.8	1.4	2.8**	0.9	1.0	1.0
MIP-2	0.8	2.8*	2.9*	1.0	1.0	0.9
MPO	1.1	55.5**	115.0**	2.9	6.2*	21.3**
VEGF	1.2	3.1**	15.7**	1.7*	1.6*	1.9*

Table 5: Effects induced by nano-CeO2 in the lung tissue homogenates

	Exposure groups			Recovery groups
Concentration (mg/m3)	0.5	2.5	10	0.5	2.5	10
Mediators
CINC-1/IL-8	1.2	3.2**	4.0**	1.1	1.2*	1.7**
KC/GROα	1.0	4.2**	4.5**	1.0	1.3*	2.0**
MCP-1	0.8	1.8**	2.5**	1.1	1.2	1.3*
MCP-3	1.0	1.7**	2.0**	1.1	1.3*	1.3
M-CSF	1.1	1.4**	2.1**	0.9	1.0	1.1
MDC	0.9	3.0**	7.6**	0.8	1.5	1.8*
MIP-1α	1.5*	1.7*	2.3**	1.2	1.0	1.4
MIP-2	0.9	3.3**	3.0**	1.1	1.2	1.6*
NGAL	1.0	2.0*	2.6**	1.0	1.0	1.4**

Table 6: Summary of incidences and severities of main histopathological pulmonary observations in male Wistar rats exposed to nano-CeO2

	Exposure groups				Recovery groups
Concentration (mg/m3)	0	0.5	2.5	10	0	0.5	2.5	10
No. of animals examined	6	6	6	6		6	6	6
Histiocytosis, diffuse Grade 1								2 2
Histiocytosis, (multi)focal Grade 1 Grade 2						2 2 2	2 1 1	2 2 2
Particles in macrophage Present		6 6	6 6	6 6		6 6	6 6	6 6

Assumed 100% nano-CeO2 deposition (mg)	0.277	1.069	4.176
(Lung burden) Measured nano-CeO2 on study day 5 (µg/lung)	52.0 ± 5.0	165.8 ± 18.4	417.6 ± 44.3
Measured deposition (%)	18.8%	15.5%	10.0%
Calculated deposition (%)	16.6%	14.3%	15.4%
Measured lung burden after recovery period (µg/lung)	44.2 ± 4.4	157.6 ± 18.7	470.9 ± 51.2
% Decrease of lung burden after recovery	-15%	-5%	+13%

Conclusions:

Executive summary:

Landsiedel et al. (2014) assessed the hazard of nanometric cerium dioxide (nano-CeO2) by performing a standard short-term inhalation study (STIS).

In conclusion, inhaled nano-CeO2 induced only loco-regional effects manifested by a transient pulmonary inflammation. However, no systemic toxicity occurred.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Some parameters for characterisation of the nano CeO2 are missing and limited information on systemic toxicity are reported in this article.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Systemic toxicity was not deeply evaluated in this study which focussed on the lung effect of nano-CeO2 using 3 different mouse models.

Principles of method if other than guideline:

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physicochemical characterisation of nano CeO2 are presented below in "any other information on materials and methods incl. tables".

Species:

mouse

Strain:

other:

Remarks:

See below: in Details on species / strain selection

Details on species / strain selection:

Sex:

female

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 280 nm

Geometric standard deviation (GSD):

1.55

Remarks on MMAD:

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

4 weeks of exposure. The effects were assessed 4 weeks post the final exposure day (56 days after the initial exposure).

Frequency of treatment:

5 days/week for 3 hours/day

Dose / conc.:

4 mg/m³ air (nominal)

No. of animals per sex per dose:

- 8 ApoE-/- mice per group
- 16 5xFAD mice per group
- 10 C57BL/6J mice per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

Positive control:

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related effects on organ weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings related to treatment (data not shown) were observed and thus no histopathological analysis was performed on the organs other than lungs.

Neuropathological findings:

not examined

Description (incidence and severity):

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- LUNG: While modest, an increased incidence in minimal chronic bronchoalveolar or alveolar inflammation was observed in the exposed animals compared to the control mice of the 3 strains. Particle loaded alveolar macrophages were observed in seven of the eight ApoE-/- mice exposed to nano CeO2 NP but not in the other strains (see table 1 in "Any information on results incl. tables").
- BALF: In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the NP exposed animals but not in the control groups

Other effects:

no effects observed

Description (incidence and severity):

Dose descriptor:

NOAEL

Effect level:

> 4 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: NanoCeO2 had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

Critical effects observed:

Estimated deposition of inhaled nano-CeO2 in different regions of the lungs:

The total inhaled dose, estimated as tidal volume (0.203 mL) x breathing frequency (353 min-1) x exposure concentration (≈ 4 x 10-3 μg/mL) x exposure duration (3600 min), was calculated to be 1020 µg. The total deposited dose (estimated using the MPPD model) was found to be 618 µg. The deposited fraction per region, estimated using the MPPD model, was found to be 48.8 % for the head, 3.5 % for the tracheobronchial region and 8.0 % for the alveolar region of the inhaled mass dose. The retained dose in lung, corresponding to the retained dose in the tracheobronchial and alveolar region 4 weeks post-exposure, estimated using the MPPD model, was found to be 16.2 µg.

Quantification of cerium in tissues (en µg/g of tissues, mean of 4 -6 animals) 4 weeks after the last exposure day:

Table 1: Histopathological findings in lung of C57BL/6J, ApoE-/- and 5xFAD mice.

Histopathological finding →		Chronic broncho-alveolar or alveolar inflammation		Alveolar macrophages	Particle loaded alveolar macrophages
Strain¯	Treatment¯	Minimal	Slight	Minimal	Minimal	Slight
C57BL/6J	Control	60% (3/5)a	-	-	-	-
	Nano CeO2	80% (4/5)	-	20% (1/5)	-	-
ApoE-/-	Control	25% (2/8)	-	-	-	-
	Nano CeO2	38% (3/8)	-	-	13% (1/8)	75% (6/8)
5xFAD	Control	69% (11/16)	-	6% (1/16)	-	-
	Nano CeO2	83% (5/6)	-	17% (1/6)	-	-

A: Percentage of animals affected. Within brackets the number of animals with histopathological findings versus number of animals evaluated.

Conclusions:

Executive summary:

Female mice from each strain were exposed nose-only to 0 (controls, exposed to filtered air) and 4 mg/m3 nano CeO2 (primary particle size of 4.7 nm) for 3h/day, 5 days/week for 4 weeks.

The authors concluded that in this subacute inhalation study, nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

Reference 6

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

standard short-term inhalation study (STIS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Repeated exposure of rats to an aerosol of 13 metal oxide nanomaterials and one micro-scale zinc oxide for 5 consecutive days through nose-only inhalation route followed by a recovery period of 3 weeks.
- Pulmonary inflammation: Analysis of the bronchoalveolar lavage fluid, lung tissue homogenates and blood samples (i.e., cytokine profiles)
- Pulmonary tissue injury: Histopathological examination of all respiratory tract tissues using transmission electron microscopy (TEM)
- Zirconium (Zr) content in the lung, lung-associated mediastinal lymph nodes and extrapulmonary organs
- Epithelial hypertrophy: Cell proliferation rates
- Apoptosis in the terminal bronchi and alveoli
- Only results on nanometric zirconium dioxide (nano-ZrO2) will be presented in this summary.

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physicochemical characterisation of ZrO2 nanoparticles are presented below in "any other information on materials and methods incl. tables".

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: conditioned air

Remarks on MMAD:

MMAD / GSD: 1.3 - 2.0 µm / 1.8 - 2.3

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The atmospheric concentration of the aerosol (cascade impactor analysis) was determined by gravimetric measurement using a sampling probe with a diameter of 7 mm.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

(control)

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-ZrO2 conc.

Dose / conc.:

2.5 mg/m³ air (nominal)

Remarks:

basis: nominal nano-ZrO2 conc.

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

basis: nominal nano-ZrO2 conc.

No. of animals per sex per dose:

6 per group

Control animals:

yes, concurrent vehicle

other: negative control exposed to nano-BaSO4 at 50 mg/m3

Details on study design:

Positive control:

Coated nano-TiO2 (T-Lite SF™) at 0.5, 2 and 10 mg/m³

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (according to OECD 412)
- Time schedule for collection of blood: Shortly after the final exposure or after the 3-week post-exposure period (for recovery groups), the blood was obtained by retro-orbital plexus puncture from the same animals used for BALF collection.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight food withdrawal)
- How many animals: 5 animals/test group
- Parameters checked (hematology analyser): red blood cell counts, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet counts, total white blood cell and differential blood cell counts, haptoglobin (photometric assay), C-reactive protein (ELISA), α2-macroglobulin (ELISA) and other cell mediators (see in the BALF paragraph).

CLINICAL CHEMISTRY: Yes (according to OECD 412)
- Time schedule for collection of blood: Shortly after the final exposure or after the 3-week post-exposure period (for recovery groups), the blood was obtained by retro-orbital plexus puncture from the same animals used for BALF collection.
- Animals fasted: Yes (overnight food withdrawal)
- How many animals: 5 animals/test group
- Parameters checked: see in the BALF paragraph

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
* LUNG INFLAMMATION AND INJURY (see in Table 3)
Bronchoalveloar lavage fluid (BALF) of 5 rats/test groups was analysed, determining BALF cytology, total protein, enzyme activities and (apart from test groups treated with coated nano-TiO2) BALF cytokines and chemokines. Cytokines and chemokines were additionally assessed in tissue homogenates of the lavaged lungs (n = 5). Animals were anesthetised and exsanguinated, and the exposed lungs were washed by 2 instillations of physiologic saline solution. Aliquots of combined BALF were used to determine:
- total cell counts (hematology analyser),
- differential cell counts = macrophages, polymorphonuclear (PMN) neutrophils, lymphocytes, eosinophils, monocytes and atypical cells (manual evaluation by counting at least 400 cells per sample from cytocentrifuge slide preparations),
- total protein concentration (turbidimetric method),
- lactate dehydrogenase (LDH) (kinetic UV test, 340 nm),
- alkaline phosphatase (ALP) (kinetic color test, 450 nm),
- N-acetyl-β-D-glucosaminidase (NAG) (color test, 580 nm),
- γ-glutamyltransferase (GGT) (Szasz method, kinetic color test, 415 nm).
Moreover, the following markers of pulmonary inflammation were assessed in the BALF and tissue homogenates of the lavaged lungs from test groups exposed to nano-ZrO2:
- MCP-1,
- CINC-1,
- M-CSF,
- OPN,
- IL-1α,
- TNF-α.

* CELL PROLIFERATION AND APOPTOSIS RATES
From the animals used for necropsy and histopathological evaluation (n = 6), cell proliferation and apoptosis rates were determined in the large and medium bronchi, terminal bronchioli and alveoli. Three days prior to necropsy, rats were subcutaneously implanted with osmotic pumps containing 5-bromo-2-deoxyuridine (BrdU). Making use of histopathological sections that were dewaxed to remove paraffin, cell proliferation was determined after immunostaining with mouse anti-BrdU antibody. Labelling indices (i.e., percentage of nuclei counted undergoing replicative DNA synthesis indicating cells present in the S-phase) were determined for the three pulmonary compartments abovementioned. In each compartment, a minimum of 1,000 cells was evaluated. Apoptosis in lung sections was determined with a TUNEL kit. Counts of apoptotic cells were performed in the three pulmonary compartments abovementioned.

Sacrifice and pathology:

Other examinations:

Statistics:

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

see in "Details on results" below

Histopathological findings: neoplastic:

no effects observed

Details on results:

Dose descriptor:

other: NOEC/NOAEC (systemic)

Based on:

other: nano-ZrO2 (test mat.)

Sex:

male

Basis for effect level:

other: No NOEC/NOAEC was determined in the publication and no NOEC/NOAEC could be set by the registrant since the study evaluated only lung toxicity.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Dose descriptor:

other: NOAEC (local)

Effect level:

>= 10 mg/m³ air (nominal)

Based on:

other: nano-ZrO2 (test mat.)

Sex:

male

Basis for effect level:

other: Basis: absence of loco-regional effects on the rat lung.

Critical effects observed:

not specified

Assumed 100% nano-ZrO2 deposition (mg)	0.18	0.936	3.456
(Lung burden) Measured nano-ZrO2 on study day 5 (µg/lung)	18.0 ± 5.6	20.3 ± 117.5	270.6 ± 28.9
Measured deposition (%)	10.0%	2.2% 12.6%	7.8%
Calculated deposition (%)	7.3%	8.2%	6.7%
Measured lung burden after recovery period (µg/lung)	6.8 ± 3.6	29.3 ± 6.2	157.6 ± 19.0
% Decrease of lung burden after recovery	-62%	-75%	-42%

Conclusions:

Overall, inhaled nano-ZrO2 did noy elicit any adverse effects in the rat lung at any of the concentrations tested.

Executive summary:

Landsiedel et al. (2014) assessed the hazard of nanometric zirconium dioxide (nano-ZrO2) by performing a standard short-term inhalation study (STIS).

Reference 7

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Some parameters for characterisation of the Zr-doped nano CeO2 are missing and limited information on systemic toxicity are reported in this article.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Systemic toxicity was not deeply evaluated in this study which focused on the lung effect of Zr-doped nano-CeO2 using 3 different mouse models.

Principles of method if other than guideline:

This study was performed in order to assess the influence of redox activity by modifications of cerium dioxide nanoparticles (nano-CeO2) via zirconium doping on the distribution, pulmonary and cardiovascular effects in mice following sub-acute inhalation. This study further explored the (patho)physiological effects of nanoparticle exposure on multiple organ systems by using three different mouse models: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. Only results observed with Zr-doped nano CeO2 are reported in this summary; effects observed with nano CeO2 are reported in a separate entry. Mice from each strain were exposed to the nanoparticles during 4 weeks and the effects were evaluated 4 weeks after the last exposure day (i.e day 56).
Experiments were conducted at Intravacc (Bilthoven, The Netherlands) under a protocol approved by the Ethics Committee for Animal Experiments of the RIVM and performed according to applicable local and EU regulations.

GLP compliance:

not specified

Remarks:

The GLP status was not specified in this published study.

Limit test:

Specific details on test material used for the study:

Further explanations on the physico-chemical characterisation of Zr-doped nano CeO2 are presented below in "any other information on materials and methods incl. tables".

Species:

mouse

Strain:

other:

Remarks:

See below: in Details on species / strain selection

Details on species / strain selection:

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain and source:
* Specific Pathogen free (SPF) ApoE−/− mice come from Taconic, Denmark
* 5xFAD and wild type (WT) cross bread C57BL/6J littermates mice come from Jackson Laboratories.
- Age at study initiation:
* ApoE−/− mice: 10-12 weeks
* 5xFAD and C57BL/6J mice: 8-11 weeks
- Weight at study initiation, fasting period before study: No data available
- Housing: macrolon cages
- Diet: Ad libitum, The ApoE-/- mice were fed a commercially available rodent Western (high fat) diet (Purified Diet Western 4021.06, ABdiets, Woerden, The Netherlands), starting at the first day of the exposure period until the end of the experiment. The other mice were fed a standard commercially available rodent diet (SMR-A, ABdiets, Woerden, The Netherlands).
- Water: Ad libitum, except during the exposure periods
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-2 °C
- Humidity: 40 to 70%
- Air changes: 15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: No data available

Route of administration:

inhalation: mist

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 288 - <= 316 nm

Remarks on MMAD:

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmosphere was characterised at least twice during each exposure session using an optical particle sizer (OPS 3330, TSI Inc., St. Paul, MN, USA) and a scanning mobility particle sizer (SMPS 3936 from TSI Inc.,St. Paul, MN, USA). In addition, aerosols were collected on polycarbonate filters for scanning electron microscopy (SEM) analysis. The SEM samples were prepared by placing a small piece of the filter on the SEM stub and coating it with platinum, and visualising with an XL30 Environmental SEM-FEG microscope (Philips XL30 ESEMFEG).
The total mass concentration generated over the 3-hour exposure period was determined by gravimetric analysis of pre-weighed and post-weighed polytetrafluoroethylene (PTFE) filters (Teflo R2PJO47, Pall corporation, Port Washington, New York, USA) using a micro-balance (Mettler MC or ME-5 microbalance, Mettler-Toledo LLC, Columbus, OH, USA).

Duration of treatment / exposure:

4 weeks of exposure. The effects were assessed 4 weeks post the final exposure day (56 days after the initial exposure).

Frequency of treatment:

5 days/week for 3 hours/day

Dose / conc.:

4 mg/m³ air (nominal)

Remarks:

CeO2 nanoparticles with 27% Zr

Dose / conc.:

4 mg/m³ air (nominal)

Remarks:

CeO2 nanoparticles with 78% Zr

No. of animals per sex per dose:

- 8 ApoE-/- mice per group
- 16 5xFAD mice per group
- 10 C57BL/6J mice per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

Positive control:

Observations and examinations performed and frequency:

Animals were monitored by cage-side observations and, if necessary, handled to detect signs of compromised health. The body weight of each animal was recorded one day before the start of exposure (day -1), prior to exposure on the first day and weekly thereafter.

Hematology, neurological (results published in a separate article which was not avaible at the time of the dossier preparation), pulmonary and cardiovascular effects were assessed 4 weeks after the last exposure of the mice (i.e. 56 days after the initial exposure).

- QUANTIFICATION OF CERIUM AND ZIRCONIUM IN TISSUES
During necropsy, organs from half of the C57BL/6J mice per group were obtained to evaluate the distribution of the Zr-doped nano CeO2 throughout the body. Liver, spleen, kidneys, heart and right (exposed mice) or left (control mice) lung, were weighed and immediately frozen in liquid nitrogen for determination of the Ce and Zr concentrations. To allow measurement of multiple parameters within the same animal, different parts of the lungs were selected for the exposed compared to the control animals. From the exposed groups the right lung was used for quantification of cerium and zirconium, because the left lung was needed for histopathological examination. From the control group the left lung was used for quantification of cerium and zirconium, because the right lung was needed for bronchoalveolar lavage. The organs were digested by acidification of each sample with 2 mL nitric acid for 12h. Hydrogen fluoride (0.2 mL) was added, followed by microwave heating for 45 min up to 185°C, and maintained for a further 20 min. Boric acid (2 mL) was added to neutralise the hydrogen fluoride, and the samples were re-heated for 20 min to 160 °C, and maintained for 10 min. Once cooled, samples were filtered with a 450 nm syringe filter, diluted with 10 mL deionised water and stored at room temperature (RT). The presence of cerium and zirconium in the lungs, liver, spleen, kidneys and heart was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer NexION 300X instrument operated in standard mode for cerium. The isotope measured was 140Ce using 115In and 159Tb as internal standards. Calibration standards (0 – 100 μg/L) were prepared from VWR 1000 mg/L stock solutions. Quantities are expressed as µg/g organ tissue.

- HEMATOLOGY
Animals were anaesthetised with a mixture of ketamine and xylazine. Two blood samples were taken by eye extraction. The first sample was collected in a K3-EDTA tube (Minicollect K3EDTA, 1 mL, 450474 Greiner Bio-One) for hematological parameters as determined in a blood auto analyser (ADVIA 2120 Hematology System, Siemens Healthineers) within 3 hours after collection. The second sample was collected in a serum tube and stored at -20°C for further analysis. Total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cells counts were meaured.

- BRONCHOALVEOLAR LAVAGE
Lung lavage was performed at necropsy. A cannula was placed in the trachea and the diaphragm opened to decrease the amount of air inside the lungs. For the control animals and all ApoE-/- animals, the right lung half was rinsed twice with approximately 0.5 mL (26.7 mL per kg body weight) of physiological saline solution, after ligation of the left lung. The injected volume was inserted and recovered 3 times, after which the lavage liquid was collected and stored on ice for less than 2 hours. To allow necropsy of the planned number of animals within one day, both lungs were lavaged for 10 of the 16 exposed 5xFAD mice and 5 of the 10 exposed C57BL/6J mice, using the same procedure, but approximately 0.8 mL (40 mL per kg body weight) of physiological saline solution.
BALF was centrifuged at 400g for 10 minutes at 4°C. The supernatant was divided into two separate aliquots of 125 µL for total protein (TP; an indicator for acute lung injury), lactate dehydrogenase (LDH; an indicator of cytotoxicity), gammaglutamyl transferase (GGT; an indicator of lung cell damage) and alkaline phosphatase (ALP; an indicator of type II cell damage) measurements using an autoanalyser (LX20- Pro, Beckman-Coulter, Woerden, the Netherlands). The cell pellet was scored for the presence of erythrocytes, re-suspended in 500 μL phosphate-buffered saline (PBS) and kept on ice. Cell counts were determined in the re-suspended pellet using a Coulter counter (Beckman-Coulter, Live Sciences). Cell concentrations were determined using a single sample using at least 150 µL of the re-suspended cells. Cytospins (Cytospin 3, Thermo-Shandon) were prepared and stained using May-Grunwald and Giemsa stain, and cell differentiation was performed by counting 400 cells per slide.

Sacrifice and pathology:

HISTOPATHOLOGY
- Lung tissue:
For the majority of the animals, the right lung was removed after the collection of the BALF and immediately frozen in liquid nitrogen and stored at - 80°C for further analysis. The left lung was removed and, after weighing, cannulated and infused with formaldehyde for 1 hour at a pressure of 20 cm H2O. Lungs were processed for histopathology; embedded in paraffin wax, sectioned at 2-4 µm intervals and stained with hematoxylin and eosin for histopathological examination.
Histopathological changes were described according to distribution, severity and morphological characteristics. The morphological characteristics of chronic inflammation include for example the presence of lymphocytes and macrophages in the lung tissue, while acute inflammation is characterised by the presence of polymorphonuclear neutrophils (PMNs).
Severity scores were assigned as follows: Grade 1 Minimal/very few/very small; Grade 2 Slight/few/small; Grade 3 Moderate/moderate number/moderate size; Grade 4 Marked/many/large; Grade 5 Massive/extensive number/extensive size.

- Assessment of atherosclerosis:
Arteries (brachiocephalic, aortic arch, thoracic aorta) were isolated from ApoE-/- mice. Atherosclerosis was quantified, as previously published (Miller et al., 2013). Briefly, brachiocephalic arteries were fixed in formalin and histological sections were taken in triplicate at 100 µm intervals, beginning at the first section of the artery with a fully intact media. Sections were stained with Masson’s Trichrome. The cross-sectional area of the plaque was measured and standardised to the medial area. A single mean value for atherosclerotic burden for each animal was calculated from the plaque size from each complete serial section throughout the brachiocephalic artery. A single section from each artery (the section exhibiting the largest plaque in cross-section) was chosen for mac-2 immunohistochemistry for macrophage-derived cells. A rat anti-mouse primary antibody was used (1/12000; CL8942AP, VH Bio, Gateshead, UK) with rat IgG (1/12000; I-400, Vector Labs, Peterborough, UK) as a negative control, followed by a goat anti-rat IgG biotinylated secondary antibody (BA-9400, Vector Labs). The area of positive staining was expressed as a proportion of the total plaque area.

- Other organs:
The spleen, liver, heart and kidneys were removed, weighed and stored in 4% formaldehyde for pathological analysis if required based on macroscopic findings.

Statistics:

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related effects on organ weights were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings related to treatment (data not shown) were observed and thus no histopathological analysis was performed on the organs other than lungs.

Neuropathological findings:

not examined

Description (incidence and severity):

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- LUNG: While modest, an increased incidence in minimal chronic bronchoalveolar or alveolar inflammation was observed in the exposed animals compared to the control mice of the 3 strains. The increase did not appear to be related to the percentage Zr-doping.
Particle loaded alveolar macrophages were observed in seven of the eight ApoE-/- mice exposed to nano CeO2 NP. This effect was not observed in ApoE-/- mice exposed to 27% or 78% Zr-doped CeO2 NPs or any other exposure or the control group of the other mouse models (see table 1 in "Any information on results incl. tables"), indicating that Zr-doping may influence particle loading of alveolar macrophages in ApoE-/- mice.
- BALF: In the bronchoalveolar lavage fluid particle loaded macrophages were seen in all the nanoparticle-exposed animals but not in the control groups.

Other effects:

no effects observed

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE (BALF)
- Total and differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytesin) from BALF measured 4 weeks after the last exposure day (mean of 5 to 16 animals) in the 3 mice strains:
No statistically significant differences were observed in the total cell counts or differential cell counts in BALF of the exposed groups compared to the controls for any strain of mouse, except for the total cell count in 5xFAD mice exposed to 27% Zr-doped CeO2 nanoparticles, which was lower than that of the control 5xFAD mice. There was a decrease (p<0.1; post-hoc test for linear trend following one-way ANOVA) in total cell counts (p=0.075), % macrophages (p=0.014), and % neutrophils (p=0.05) in ApoE-/- mice with increasing amounts of Zr-doping. However, no such trends were observed in C57 BL/6J or 5xFAD mice.

- Total protein, Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP) and Gamma-Glutamyl Transpeptidase (GGT) levels in bronchoalveolar lavage fluid (BALF) 4 weeks after exposure (mean of 5 to 16 mice from the each tested strains):
Similarly, no statistically significant differences were observed for LDH, ALP or GGT protein levels between the exposed and control groups.

No constitutive differences in differential cell counts or protein levels were observed between the different mice strains.

EFFECTS ON ATHEROSCLEROSIS IN THE BRACHIOCEPHALIC ARTERY OFApoE-/- MICE AFTER INHALATION
ApoE-/- mice exhibited regions of dense plaques in the aortic arch and branch points of large arteries. Plaques were composed of fibroblastic matrix, smooth muscle cells, lipid cavities and cholesterol crystals. Atherosclerotic burden was quantified in the brachiocephalic artery, with control (air-exposed) mice having a mean plaque size of 94 ± 9% (standardised to the area of the vascular media). Exposure to Zr-doped nano CeO2 did not have a significant effect on the atherosclerotic burden (mean plaque size) of these arteries (p=0.62; One-way ANOVA). However, there was an increase (post-hoc test for linear trend following one-way ANOVA with p=0.09) in the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells) with increasing Zr content of the CeO2 nanoparticles.

Dose descriptor:

NOAEL

Effect level:

> 4 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

Critical effects observed:

Estimated deposition of inhaled Zr-doped nano CeO2 in different regions of the lungs:

The total inhaled dose, estimated as tidal volume (0.203 mL) x breathing frequency (353 min-1) x exposure concentration (≈ 4 x 10-3 μg/mL) x exposure duration (3600 min), was calculated to be 1043 µg and 1056 µg for 27% and 78% Zr-doped nano CeO2, respectively. The total deposited doses (estimated using the MPPD model) were found to be 625 µg for the 27% Zr-doped nano CeO2, and 639 µg for the 78% Zr-doped nano CeO2. The deposited fractions per region, estimated using the MPPD model, were found to be 48.8% and 49.8% (27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively) for the head, 3.4% and 3.3% (27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively) for the tracheobronchial region and 7.9% and 7.5% (27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively) for the alveolar region of the inhaled mass dose. The retained doses in lung, corresponding to the retained dose in the tracheobronchial and alveolar region 4 weeks post-exposure, estimated using the MPPD model, were found to be 16.2 µg and 15.6 µg, for the 27% Zr-doped nano CeO2 and 78% Zr-doped nano CeO2, respectively.

Quantification of cerium and zirconium in tissues (in µg/g of tissues, mean of 4 -6 animals) 4 weeks after the last exposure day:

Table 1: Histopathological findings in lung of C57BL/6J, ApoE-/- and 5xFAD mice.

Histopathological finding →		Chronic broncho-alveolar or alveolar inflammation		Alveolar macrophages
Strain¯	Treatment¯	Minimal	Slight	Minimal
C57BL/6J	Control	60% (3/5)a	-	-
	27% Zr-doped nano CeO₂	40% (2/5)	-	20% (1/5)
	78% Zr-doped nano CeO₂	80% (4/5) -	-	-
ApoE-/-	Control	25% (2/8)	-	-
	27% Zr-doped nano CeO₂	38% (3/8)	13% (1/8)	-
	78% Zr-doped nano CeO₂	50% (4/8)	-	-
5xFAD	Control	69% (11/16)	-	6% (1/16)
	27% Zr-doped nano CeO₂	67% (4/6)	-	-
	78% Zr-doped nano CeO₂	67% (4/6)	-	33% (2/6)

A: Percentage of animals affected. Within brackets the number of animals with histopathological findings versus number of animals evaluated.

Conclusions:

Executive summary:

Four weeks after the last exposure day (i.e day 56 of the study), the animals of each strain were sacrificed, organs weights were measured and the effects of the treatment on the blood (total white blood cell count and lymphocytes, neutrophils, eosinophils and monocytes cell counts) and on the lungs (analyses of the differential cell counts (macrophages, lymphocytes, neutrophils, eosinophils and monocytes) and protein levels (total proteins, ALP, LDH and GGT) in BALF) were evaluated. Furthermore, histopathological analysis of the lungs was done and the tissue distribution of cerium and zirconium was measured using ICP-MS in the lung, heart, kidney, spleen and liver of all animals. The effects of Zr-doped nano-CeO2 on the artherosclerosis burden in the brachiocephalic artery of ApoE-/- mice was also determined in this study to evaluate cardiovascular effects of treatment with nanoparticles by measuring the mean plaque size and of the proportion of plaque staining positive for mac-2 (i.e. macrophage-derived foam cells).

The evaluation of potential neurological effects of treatment on the 5xFAD mice were not reported in this article.

The authors concuded that in this subacute inhalation study, Zr-doped nano CeO2 has a low biological activity in healthy mice and mouse models of atherosclerosis and Alzheimer's disease.

Reference 8

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Justification for type of information:: Read across from studies performed with cerium dioxide and zirconium dioxide. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Reason / purpose for cross-reference:: read-across source
Dose descriptor:: other: read across conclusion
Remarks on result:: other: The reaction mass of cerium dioxide and zirconium dioxide is concluded not to present any systemic toxicity after repeated inhalation exposure.
Remarks:: Conclusion based on the results of the various read across studies performed with bulk and nano zirconium dioxide and bulk and nano cerium dioxide, in addition to the results from a study performed with various Zr-doped nano cerium dioxide forms. In none of these studies, systemic adverse effects have been observed. Further, the observation of loco-regional effects in the lungs seems to be related to cerium dioxide rather than zirconium dioxide, but also appeared to be species-dependent, as such effects have been observed in rats but not in mouse. Moreover, the observed loco-regional effects of cerium dioxide in rats are considered to represent rather a non-specific adaptive response to particle overload of the lung and indicate the sensitivity of rats to this type of condition. Taking all evidence together, it is not considered necessary to perform further repeated dose inhalation toxicity studies with the reaction mass of cerium dioxide and zirconium dioxide, as this is not expected to bring further insights, and it is therefore not considered justifiable taking into account the obligation to avoid animal testing where necessary.
Critical effects observed:: no

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Repeated dose toxicity: oral

No test data are available on repeated dose toxicity of the reaction mass of cerium dioxide and zirconium dioxide for the oral route. Therefore, reliable data for the constituents of the reaction mass were included in a weight-of-evidence approach. The studies included are OECD 422 studies performed with (bulk) cerium dioxide and, for zirconium dioxide, the representative zirconium compound zirconium acetate (which is a 'water soluble' zirconium compound).

The toxicity of (bulk) cerium dioxide was tested in rats via the oral route in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) and in accordance with GLP (Davies, 2010a; Klimisch 1). Following daily oral administration for ~37 days (males) to 40-45 days (females), no toxicologically meaningful effects were observed up to and including at the highest dose of 1000 mg/kg bw/day. Therefore, the unbounded NOEL was 1000 mg/kg bw/day.

In conclusion, the oral toxicity of (bulk) cerium dioxide has been shown to be extremely low based on the absence of adverse effects in rats up to the limit-dose level of 1000 mg/kg bw/day.

The toxicity of (bulk) zirconium acetate was tested in a similar OECD 422 study (Rossiello, 2013; Klimisch 1).

Three groups of 10 males and 10 females each received the test item, by oral gavage, at 100, 300 and 1000 mg/kg bw/day, expressed as bulk zirconium acetate (anhydrous form).

The overall dosing period was 32 days for males, which included 2 weeks before pairing and continuously thereafter up to the

day before necropsy and up to 50 days for females, including 2 weeks before pairing, and thereafter during pairing, gestation and lactation periods until day 3 post partum.

Microscopically, a treatment-related finding was observed in males receiving 300 and 1000 mg/kg bw/day consisting of minimal focal vacuolation of squamous epithelium (limiting ridge) of the non-glandular region of the stomach. This change may be attributed to a local irritant effect of the compound administered by oral gavage and since humans do not have a forestomach or a structure analogous to the forestomach, it is not considered of toxicological relevance. In addition, no abnormalities were found

at the evaluation of the spermatogenic cycle. No effects were noted on reproduction and development at any dose. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for systemic toxicity and reproduction/developmental toxicity could be considered >= 1000 mg/kg bw/day for both males and females.

Based on these results it can be concluded that similar results could be expected with the reaction mass of cerium dioxide and zirconium dioxide in a similar OECD 422 study. No studies are available for nanoforms, however, such studies are available for the inhalation route of exposure, which is considered the most important route to be fully covered for repeated dose toxicity in order to be able to draw conclusions on the nanoforms of the reaction mass.

Repeated dose toxicity: inhalation

In an OECD TG 413 compliant study (Viau, 1994; Klimisch 1), the potential toxicity of bulk cerium dioxide was tested following repeated nose-only inhalation of a dry powder aerosol of cerium dioxide (mass median aerodynamic diameter = 1.8-2.2 µm) to rats (15/sex) for 6 hours a day, 5 days a week, for 13 weeks, at the gravimetric concentrations of 0 (air), 0.005, 0.0505 or 0.5075 mg/L (0, 5, 50.5 or 507.5 mg/m³, respectively).

No treatment-related deaths or clinical signs occurred during the study. There were no effects on ophthalmology, clinical chemistry or urinalysis at any dose level. No behavioural changes following either acute or sub-chronic exposure and no significant differences in motor activity were observed in treated groups in the Functional Observational Battery assessment.

Treatment-related changes were observed in segmented neutrophil counts, lung and spleen weights, lung and lymph node gross appearance at necropsy and lung, respiratory tract and lymphoreticular system histopathology. Microscopically, pigmented material accumulation in the lungs, bronchial, mandibular and mediastinal or pancreatic lymph nodes, trachea, bronchi, larynx, nasal cavity, liver and spleen, as well as alveolar epithelial hyperplasia in the lungs, metaplasia in larynx, and lymphoid hyperplasia in lymph nodes and lungs, correlating with the presence of pigment in these tissues, were seen in treated groups with a clear dose-response relationship. No NOAEC was determined in the study report based on effects at the lowest concentration. This study identified a LOAEC of 5 mg/m3 in rats, based on the increased incidence of lymphoid hyperplasia in the bronchial lymph nodes of male and female rats. However, considering that lymphoid hyperplasia in the bronchial lymph nodes may not represent a specific toxic effect, but rather a non-specific adaptive response to the overloading of the pulmonary alveolar macrophages by inorganic poorly soluble particles in rats, and considering that alveolar epithelial hyperplasia in the lungs represents a more sensitive indication of adverse effects, the concentration of 0.0505 mg/l (50.5 mg/m³) can be regarded as a LOAEC in rats based on the incidence and severity of alveolar epithelial hyperplasia in the lungs following exposure for 13 weeks.

In the case of an inorganic particulate material such as cerium dioxide, a distinction has to be made between chemical-specific and inert particle-induced toxicity. Lung overload-related inflammatory responses are commonly observed in rats following inhalation exposure to poorly soluble particles. The concept of overload applies specifically to poorly soluble particles with low cytotoxicity. The hallmark of particle overload is impaired alveolar clearance which, in rats exposed to poorly soluble particles, is associated with altered macrophage function, pulmonary inflammation, centri-acinar interstitial and alveolar accumulation of particles, and inflammation-induced epithelial cell proliferation (Ref. ILSI Risk Science Institute. The relevance of the rat lung response to particle overload for human risk assessment: A workshop consensus report. Inhalation Toxicology, 12: 1-17, 2000). In terms of risk assessment, lung overload conditions are not relevant to human health because of interspecies differences. The distribution of the retained particles within the lung compartments is different between species. It has been shown that during chronic inhalation exposure, particles are retained to a greater degree in interstitial locations in lungs of non-human primates and dogs than in lungs of rats, and that the interspecies differences in particle location might contribute to corresponding differences in tissue response (Ref. Snipes MB. Current information on lung overload in non-rodent mammals: contrast with rats. Inhalation Toxicology, 8(suppl): 91-109, 1996). These differences combined with the fact that human macrophages have five times the volume of rat macrophages are considered to account for the tendency of rats to respond to poorly soluble particles with more chronic inflammation and epithelial responses compared to humans (Ref. Oberdörster G. Toxicokinetics and effects of fibrous and non-fibrous particles. Inhalation Toxicology, 14: 29-56, 2002).

In the case of the present 13-week inhalation study with cerium dioxide in rats, initially no NOAEC was determined in the study report because of the occurrence of lymphoid hyperplasia in the bronchial lymph nodes at all test concentrations. The lymphoid hyperplasia likely reflects an inflammatory response due to the cerium dioxide particles delivered by dendritic cells from the tracheobronchial region to the bronchial lymph nodes. However from a chemical-specific toxicity standpoint, lymphoid hyperplasia is not known to be on the pathogenetic pathway for adverse effects resulting from continued inhalatory exposure to poorly soluble particles, or to represent an adverse effect in itself. Studies in rats exposed under overloading conditions have shown that enlarged lymph nodes can occur without functional impairment of the antibody-forming function of the lymphocytes (Ref. Bice DE et al. Effects of inhaled diesel exhaust on immune responses after lung immunization. Fundam Appl Toxicol, 5:1075-86, 1985). The negative results obtained with cerium dioxide in the Popliteal Lymph Node Assay conducted on Brown-Norway rats, a rat strain well known for its high sensitivity to immunotoxicants, on popliteal lymph node weights and tritiated thymidine incorporation, as well as on blood Ig E concentrations, further illustrate the absence of immunotoxicity of cerium dioxide. Clinically, a comprehensive review of human lung pathology indicates that bronchial lymph node hyperplasia is not regarded as a diagnostic marker of specific lung diseases (Ref. Travis WD et al. Non-neoplastic Disorders of the Lower Respiratory Tract. American Registry of Pathology & Armed Forces Institute of Pathology,Washington DC. pp. 277-281, 2002). Rather, lymph node hyperplasia appears to be a secondary response to other conditions. Therefore, lymph node hyperplasia should be considered as an adaptive response and alveolar epithelial hyperplasia in the lungs appears more clearly as an indication of adverse effects, and should therefore be considered for the NOAEC/LOAEC determination.

In addition to the study of Viau (1994), literature data on repeated inhalation of nano cerium dioxide, zirconium dioxide or zirconium-doped nano cerium dioxide are available and included in the dossier.

Landsiedel et al. (2014; Klimisch 2) investigated the effects of short-term inhalation of nano cerium dioxide and nano zirconium dioxide in rats. Following administration of test material aerosols (ranging from 0.5 to 10 mg/m3) for five consecutive days with 21-day post-exposure observation, inhaled nano-CeO2 was found in the lung, in alveolar macrophages, and more rarely in the draining lymph nodes, but none was found in extrapulmonary organs according to the authors. Nano-CeO2 induced a transient pulmonary inflammation which was concentration-dependent. Most effects were at least partially reversible during the post-exposure period. The histopathological analysis of lungs revealed no signs of beginning granulomatous changes or fibrosis. Based on cell counts, protein/enzyme concentrations and mediator levels in BALF, the authors derived a NOAEC < 0.5 mg/m3 based on changes of all cytological and biochemical parameters in BALF, particles in macrophages, partial regression of BALF effects and mild diffuse or multifocal alveolar histiocytosis remaining. Therefore, Landsiedel et al. considered nano-CeO2 in a group of materials having a higher toxic potency to lung although a recovery was visible during the post-exposure period. However, only few of these changes (PMN, lymphocyte counts and M-CSL level slightly increased in BALF) were seen at the concentration 0.5 mg/m3 at the end of exposure and recovery occurred for all these parameters, but some mild multifocal alveolar histiocytosis and particles in macrophages were observed also in animals in the recovery group at this concentration. Nevertheless, according to Keller et al. (2014), this reflected an expected physiological response. And thus it could be considered that the NOAEC is lower than or equal to 0.5 mg/m3 in this study. In conclusion, inhaled nano-CeO2 induced only loco-regional effects manifested by a transient pulmonary inflammation. However, no systemic toxicity occurred.

In the same study, inhaled nano-ZrO2 was found in the lungs but was not detectable in the lung-draining lymph nodes at any time in any of the examined animals and not found in extrapulmonary organs according to the authors. Nano-ZrO2 did not elicit local effects in the rat lung. Based on the absence of effects noted, the authors derived a NOAEC >= 10 mg/m³. Therefore, Landsiedel et al. considered nano-ZrO2 in a group of materials causing no lung effects up to the highest tested concentration. No systemic toxicity was observed either.

Dekkers et al. (2017; Klimisch 2) assessed the effect of zirconium doping of cerium dioxide nanoparticles on the potential toxicity in mouse after repeated inhalation exposure. Three different mouse models were used: Atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−), Alzheimer’s disease (5xFAD), and background (non-genetically modified) strain C57BL/6J mice. Female mice were exposed nose-only to clean air, pure nano cerium dioxide, or one of two nano cerium dioxide test materials with various amounts of Zr-doping (27% or 78% Zr), over a 4-week period (4 mg/m3 for 3h/day, 5 days/week). No major toxicological effects have been observed in any of the different mouse models, apart from some modest inflammatory histopathology in the lung, unrelated to the amount of Zr-doping. The authors concluded that nano cerium dioxide had minimal pulmonary and cardiovasuclar effects following subacute inhalation at 4 weeks post exposure in healthy mice and mouse models of atherosclerosis and Alzheimer's disease. Zr-doping of cerium dioxide nanoparticles had limited effects on these responses, although indications that Zr-doping could potentially alter particle loading of alveolar macrophages and increase the inflammatory cell content in atherosclerosis plaques merit further investigation. No systemic toxicity was observed in the study.

Finally, the study of Spiegl et al. (1956; Klimisch 2) performed 30-day repeated dose inhalation toxicity studies with bulk zirconium dioxide in dog, rabbit and rat, as well as 60-day repeated dose inhalation toxicity studies in cat, dog, guinea pig, rabbit and rat. No effects were reported in any of the species tested after inhalation of zirconium dioxide dust (NOAEC >= 100.8 mg ZrO2/m3 air in the 30-day study and NOAEC >= 15.4 mg ZrO2/m3 air in the 60-day study).

Based on the studies discussed above, it is clear that the materials tested (i.e. the individual constituents zirconium dioxide and cerium dioxide, as well as the reaction mass itself), regardless of their nano-status, are not capable of causing systemic toxicity after repeated inhalation exposure. Zirconium dioxide, whether bulk or nano, is consistently found to be toxicologically inert, not resulting in loco-regional adverse effects in the lungs either. For cerium dioxide, whether bulk or nano, loco-regional adverse effects have been consistently observed in rats, but not in mouse. The observed loco-regional effects of cerium dioxide in rats are considered to represent rather a non-specific adaptive response to particle overload of the lung and indicate the sensitivity of rats to this type of condition.

Further, it was demonstrated that the increased presence of zirconium in nano cerium dioxide did not result in the increased observation of obvious adverse effects in mouse.

Repeated dose toxicity: dermal

No reliable studies are available on the repeated dose toxicity via the dermal route for the reaction mass or its constituents cerium dioxide or zirconium dioxide. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2 adaptation, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure on the reaction mass of cerium dioxide and zirconium dioxide.

Repeated dose toxicity	Reaction mass (nano)	Cerium dioxide (bulk)	Cerium dioxide (nano)	Zirconium dioxide (bulk)	Zirconium dioxide (nano)	Zirconium acetate (bulk)
Oral	-	NOAEL >= 1000 mg/kg bw/d (rat, OECD 422)	-	-		NOAEL >= 1000 mg/kg bw/d (rat, OECD 422)
Inhalation	NOAEL > 4 mg/m3 (mouse, cfr. OECD 412, no systemic effects, no/limited loco-regional effects)	LOAEC = 50.5 mg/m3 (rat, OECD 413, no systemic effects, only loco-regional effects)	NOAEL > 4 mg/m3 (mouse, cfr. OECD 412, no systemic effects, no/limited loco-regional effects) NOAEC <= 0.5 mg/m3 (rat, 5-d exposure, no systemic effects, only loco-regional effects)	NOAEC >= 100.8 mg/m3 (dog, rabbit, rat, 30-d, no systemic effects, no loco-regional effects) NOAEC >= 15.4 mg/m3 (cat, dog, guinea pig, rabbit, rat, 60-day, no systemic effects, no loco-regional effects)	NOAEC >= 10 mg/m3 (rat, 5-d exposure, no systemic effects, no loco-regional effects)
Dermal	-	-	-	-	-	-

Justification for classification or non-classification

As no significant effects which could be relevant for human health were observed in the studies performed on the constituents of the reaction mass (cerium dioxide and zirconium dioxide (or, for the oral route of exposure, zirconium acetate as a representative zirconium compound)) or on test material similar to the reaction mass (differently Zr-doped cerium dioxide), regardless the nano-state of the tested materials, it can be concluded that no classification is warranted for the reaction mass of cerium dioxide and zirconium dioxide based on the CLP classification criteria.

Species	Lung (1)	Kidney (2)	Liver	Other
Cat	3/4	0/4	0/4	0/4
Dog	*	*	0/4	*
Guinea pig	9/18	0/18	0/18	0/18
Rabbit	2/10	1/10	0/10	0/10
Rat	8/10	0/10	0/10	0/10

Species	Number of animals	Lung (µg/g)	Pulmonary Lymph Node (µg/g)
Rat	10	158	17
Dog	4	74	731
Rabbit	10	16
Cat	4	20
Guinea pig	18	71

Registration Dossier

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Diss Factsheets

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

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

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Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

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Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Additional information

Justification for classification or non-classification

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