Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1993 - December 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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:
15
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

Examinations

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

Results and discussion

Results of examinations

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

Effect levels

open allclose all
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:
clinical signs
mortality
ophthalmological examination
clinical biochemistry
urinalysis
behaviour (functional findings)
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.