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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1998-05-04 to 1998-07-31 (exposure period only)
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
all concentrations in this study exceed the maximum tolerated dose (MTD) by overwhelming physiological clearnace mechanisms; such conditions are to be avoided (see OECD guidance document 116 and OECD TG 451)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1998-05-04 to 1998-07-31 (exposure period only)
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
all concentrations in this study exceed the maximum tolerated dose (MTD) by overwhelming physiological clearnace mechanisms; such conditions are to be avoided (see OECD guidance document 116 and OECD TG 451)
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of female F344/CrlBR rats were exposed to 10, 50 or 250 mg/m³ pigmentary titanium dioxide via whole body inhalation for 6 hours/day, 5 days/week for 13 week with recovery groups held for an additional 4, 13, 26 or 52 weeks postexposure. At each time point, selected lung responses were examined. The responses studied were chosen to assess a variety of pulmonary parameters including: inflammation, cytotoxicity, and epithelial- and fibroproliferative changes.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
This stock and strain was chosen due to the available historical database on pulmonary disease and particulate exposure available at the performing laboratory and elsewhere.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Wilmington, MA
- Age on receipt: approx. 6 weeks old
- Weight a week after arrival: approx. 87 - 128 g
- Housing: housed 1/cage; housed in 1 m³ stainless steel and glass inhalation chambers in suspended stainless steel caging with wire mesh floors during the acclimatisation, exposure phases and postexposure phase.
- Diet (ad libitum, except during exposure when food was withheld): NIH-07 certified diet
- Water (ad libitum): deionized water
- Acclimation period: approx. 9 days

DETAILS OF FOOD AND WATER QUALITY: drinking water contaminant levels were measured at quarterly intervals.

ENVIRONMENTAL CONDITIONS
- Temperature: 17.8 - 26.1 °C
- Relative humidity: approx. 30 - 70 %
- Air changes: 12 - 15/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
1.44 µm
Geometric standard deviation (GSD):
1.71
Remarks on MMAD:
Standard deviation (MMAD): 0.09
Standard deviation (GSD): 0.23
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m³ H-1000 stainless steel chambers

- System of generating particulates/aerosols: aerosol generation was accomplished using a dust feeder, jet streams of air, a one-stage impactor, and a series of mixing chambers. The dust feeder introduced the particles into the jet streams of air where the particles were broken up and converted to fine particles suspended in air. The aerosol stream exiting the deagglomeration stage passed through a one-stage impactor to remove large particles. The aerosolized powder was then injected into a "pre-chamber" and subsequently distributed and diluted appropriately to the exposure chambers. Prior to the exposures, each chamber was checked for uniformity of distribution of the test substance by measuring the concentration at a minimum of nine positions in the H-1000 chamber without animals but with racks and pans in place.

- Air flow rate: chambers were provided with air at a flow rate of approx. 12 - 15 air changes each hour to ensure an adequate oxygen content of at least 19 %. The airflow rate was monitored and recorde at least once an hour. Total airflow through the H-1000 chamber was monitored by using the pressure drop across an orifice at the inlet of the chamber and adjustments were made with a previously calibrated exhaust fan control through the Andover Infinity system. All chambers were maintained at a slightly negative pressure.

- Temperature, humidity, pressure in air chamber: temperature and relative humidity of the exposure chambers was monitored continuously during exposure and nonexposure periods. Environmental parameters were recorded approx. 6 times during each exposure period. The temperature was maintained at 22.8 - 23.2 °C and relative humidity between 44 to 57 %.

- Method of particle size determination: particle size distribution measurements were carried out at least two times per exposure chamber (excluding the control chamber) during the course of the study, using an aerodynamic sizing device (Micro-Orifice Uniform Deposition Impactor, Model 100).

Preliminary studies were performed prior to the study to confirm the stability of the aerosol.

TEST ATMOSPHERE
- Brief description of analytical method used: Real Aerosol Monitor (Model RAM-S) per H-1000 chamber was used as the primary instrument to continuously measure particle concentration. RAM-S readings were validated by mass weight filters samples. The time-averaged concentration as monitored by the RAM-S was recorded at least six times over the 6 hour exposure system operation period.
Where excursions from the nominal concentrations occurred, manual adjustments were made.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on inhalation exposure" above.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
9.6 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 1.1 mg/m³
Dose / conc.:
47.7 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 5.1 mg/m³
Dose / conc.:
239.1 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 19.3 mg/m³
No. of animals per sex per dose:
59 female rats (6 additional rats/group were included as extra animals to allow for losses in the course of the study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: 4, 13, 26 or 52 weeks of recovery
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the exposure phase and once per day in the post-exposure phase.
- Cage side observations checked: overt clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for the first 17 weeks of the study and biweekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for the first 17 weeks, and biweekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
Five sacrifices were scheduled to occur at the end of the exposure and following 4, 13, 26, or 52 weeks of recovery. Animals remaining after the final scheduled sacrifice were killed and discarded. Subgroups, based on endpoint, were defined as follows:

A) Subgroup 1 (histopathology, cell proliferation, TiO2 body burden; 5 animals/time point):
Animals were implanted, 5 days before euthanasia, with osmotic pumps containing bromodeoxyuridine (BrdU). Pump model 2ML (10 µL/h) was used. BrdU concentration was 5 mg/mL. The right lung lobes and lung-associated lymph nodes were separated from the thoracic viscera, blotted, weighed, and then frozen for the determination of retained TiO2. The left lung was intratracheally infused, under pressure, with 10 % neutral buffered formalin. Following formalin infusion the left lung was fixed for no more than 72 hours (ideally 48 hours in formalin and then changed to 70 % ethanol.
- lung cell proliferation: the fixed left lung lobe was trimmed and embedded in paraffin, along with a piece of duodenum (BrdU incorporation in the duodenum on the same tissue sections serves as a positive control for immunohistochemical staining and delivery of BrdU to the animal). Multiple standarized sections were cut and mounted on coated glass slides and stained for the presence of incorporated BrdU. Immunostaining was performed with an automated immunostainer (Biotek) using a commercially available monoclonal antibody to BrdU. Terminal bronchiolar and alveolar duct cell labelling indices were determiend as well as alveolar cell proliferative labelling indices.
- histopathology: paraffin embedded tissues, from the left lung, were prepared from each animal at the same anatomical site with respected to the mainstem bronchus. Tissues were subjected to routine microtomy at 5 µm and were Trichrome stained using a Masson's method. The Trichrome-stained left lung sections were evaluated for particle-induced histopathologic changes.
- TiO2 lung burden: right lung and lung associated lymph nodes were removed, weighed, and frozen on-site. The subsequent processing and analysis was performed. The tissues were dried and digested using a combination of nitric and hydrofluoric acids. Subsequent analysis for titanium content was by inductively coupled plasma optical emission spectrometry (IPS-OES).

B) Subgroup 2 (BALF, cytology; 5 animals/time point):
The lungs of the animals were lavaged five times with equal volumes of phosphate buffered saline (5 mL). Fluid from the first two lavages was recovered, pooled and placed on ice and the subsequent three lavage fluids were pooled and also placed on ice. The left lobe of the lavaged lung was instilled with freezing medium, tied off and removed, and portions mounted for frozen sectioning. The right lung caudal and accessory lobes were removed and flash-frozen in liquid nitrogen for nucleic acid isolation. The right lung apical and medial lobes were intratracheally infused, under pressure, with 4 % paraformaldehyde fixative. Following infusion the right apical and medial lobes were fixed for no more than 72 hours (ideally 48 hours) in 4 % paraformaldehyde and then changed to 70 % RNAse-free ethanol. These tissues were collected for possible use in in situ hybridization procedures.
- pulmonary toxicity endpoints: cell-free fluid from the pooled first and second bronchoalveolar lavage washes were utilized for the determination of lactate dehydrogenase (LDH), total protein, and phospholipid content. LDH, and total protein were quantitated spectrophotometrically using an automated analyzer. Choline-containing phospholipids were assayed by an enzymatic method using phospholipase D, choline oxidase and peroxidase (Takayama et al., 1977)* using a commercially available kit (Wako).
- cytology: bronchoalveolar lavage fluids were centrifuged to separate cells and supernatant. Cell pellets from all the lavages were pooled and an aliquot used for phospholipid analyses. The remaining cells were resuspended in cell culture medium supplemented with serum, and counted using an automated cell counter. The cell suspensions were diluted with cold medium cytocentrifuged onto glass slides and subsequently air dried, fixed, and stained. Differential counts (200 cells/animal) were obtained using the stained cell preparations.

C) Subgroup 3 (electron microscopy; 3 animals each at the time points 0, 13, and 52 weeks postexposure (0, 10, and 250 mg/m³ concentrations) or 3 animals each at the time point 52 weeks postexposure (50 mg/m³ concentration):
Tracheas were cannulated and the lungs intratracheally infused with glutaraldehyde fixative under pressure. Tthe heart and lungs were removed en bloc and placed in fresh fixative. Once the lungs had fixed for at least 12 hours the fixative was replaced with an appropriate buffer to prevent overfixation.
- electron microscopy: lungs were collected, fixed, and placed in buffer. Sections of plastic-embedded tissue may be examined for localization and interstitialization of the test particles.
- lung volumes: lungs were collected, fixed, and placed in buffer for a minimum of 4 days. The heart and other extraneous tissues were removed and the volume of the lungs was determined using a fluid displacement method (Scherle, 1970)*.

Unscheduled deaths: animals found dead or moribund were necropsied and where possible the target organ, lung, was saved along with the animal's transponder. No effort was made to establish the cause of death or moribund condition.

*References:
- Takayama, M., S. Itoh, T. Nagasaki, and I. Tanimizu. "A new enzymatic method for determination of serum choline-containing phospholipids." Clin Chim Acta 79 (1977): 93-8.
- Scherle, W. "A simple method for volumetry of organs in quantitative stereology." Mikroskopie 26 (1970): 57 - 60.
Statistics:
The analysis included a combination of regression and analysis of variance (ANOVA) methods. All data were tested for normality and homogeneity of variance. If the hypotheses for these assumptions were rejected, common transformations (log, square root etc.) were applied and the data were retested. If the hypotheses were still rejected, nonparametric methods were used.
Incidence data were compared using the appropriate statistical test, generally Fisher's Exact Test. A probability value of 0.05 was used as the critical level of significance within each statistical test, except for the tests of homogeneity and normality, where the significance level was 0.01. However, the critical level of overall significance (alpha level) for each test domain was adjusted to correct for multiple evaluations.
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
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:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
- wet weight of the right lung was increased following 13 weeks of exposure in rats in the 239.1 mg/m³ air dose group and remained elevated at 52 weeks postexposure.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- lung-associated lymph nodes were enlarged (macroscopic evidence). The increase in lymph node size in the TiO2 exposed groups was associated with white aggregations of pigmentary TiO2 (concentration- and time dependent finding).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- alveolar epithelial metaplastic lesions and alveolar septal fibrosis were evident.
- epithelial metaplastic change (bronchiolization of alveoli) was found only in rats exposed to the 47.7 mg/m³ air and 239.1 mg/m³ air TiO2 concentrations.
- fibrotic alveolar septal changes were also limited to the 47.7 mg/m³ air and 239.1 mg/m³ air concentration exposed rats that developed impariment ofalveolar macrophage-mediated lung clearance.
- hypertropic and hyperplastic epithelial changes were in immediate proximity to aggregates of inflammatory cells and particle-laden macrophages. With the exception of the 239.1 mg/m³ concentration exposure rats, the epithelial changes diminished in incidence and severity with the reduction in pulmonary inflammation that occurred over time during the postexposure recovery period.
- in rats of the 239.1 mg/m³ air concentration group, epithelial proliferative changes were associated with metaplastic alteration and septal fibrosis and became more severe during the long-term postexposure periods (26 and 52 weeks postexposure). One of the most significant findings was the finding that the 239.1 mg/m³ exposure concentration group rats developed a preogressively severe epithelial- and fibro-proliferative lesion.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
CYTOLOGY
- number of cells recovered from the lungs by BAL from control animals generally correlated with body weight in rats. Significant increase, over controls, in cell recovery were observed following exposure in rats from all exposure groups . This increase declined with time after exposure.
- exposures to TiO2 resulted in an increase of neutrophils, macrophages, and lymphocytes in rats.
- greatest neutrophil response was seen in animals of the 239.1 mg/m³ air (83 %).
- greatest macrophage response was found in the 239.1 mg/m³ air dose animals.

CELL PROLIFERATION
- increased labeling index was noted in the alveolar cell population in the rats in the 239.1 mg/m³ air dose group through 52 weeks postexposure (p<0.05).

PULMONARY TOXICITY ENDPOINTS
- rats in the 239.1 mg/m³ air and 47.7 mg/m³ air dose groups had persistent elevation of lactate dehydrogenase in BALF, however, those in the 47.7 mg/m³ air dose group had returned to control levels by 26 weeks postexposure.
- rats had significantly elevated persistent levels (to 52 weeks postexposure) of total protein in BALF but this was limited to the 239.1 mg/m³ dose group.
- rat BALF phospholipid concentrations were elevated following exposure but were limited to the 239.1 mg/m³ and 47.7 mg/m³ dose animals. The increased phospholipid levels in BALF from the 239.1 mg/m³ air rats declined with time postexposure but remained significantly greater than the concurrent controls at 52-week postexposure.
- phospholipids associated with cells recovered by lavage, in general, showed a dose dependent elevation in concentration after 13 weeks exposure.
- in the postexpousre period, rats from the 239.1 mg/m³ dose group had significantly elevated phospholipid levels through 52 weeks.
Details on results:
MORTALITY
- no significant exposure-related increase in mortality
- fifteen rats died or were euthanized during the course of the study.
- the majority (7 deaths) of these deaths were in the 47.7 mg/m³ dose group and occurred approx. one year into the study.

BODY WEIGHT AND WEIGHT CHANGES
- a depression in body weight (2 - 3 %) was noted following the end of the exposure period with a subsequent recovery occurring over the next 3 - 4 weeks.
- rats exposed to the 239.1 mg/m³ air concentration of TiO2 demonstrated a consistent pattern of elevated weights (less than 10 %) compared to controls during the latter half of the recovery period (no obvious cause identify).

ORGAN WEIGHTS
- lung-associated lymph node wet weights were elevated in rats following exposure in the 239.1 mg/m³ air dose group and remained elevated until 52 weeks postexposure (probably due to inflammatory response and small parts to the TiO2 burdens).

HISTOPATHOLOGY
- rats had a predominantly centriacinar pattern of particle accumulation.
- rats had focal accumulations of heavily particle-laden macrophages aggregated into "dust macules" in the 47.7 mg/m³ air and 239.1 mg/m3 air dose groups.
- TiO2-induced lung lesions increased in severity with increasing exposure concentration.
- particle-laden macrophage aggregation was noted.
- histopathological appearance of lung lesions correlated very well with the BALF inflammatory indices.
- TiO2-induced inflammation was concentration dependent and limited to the 47.7 mg/m³ and 239.1 mg/m³ cocnentration exposure groups.
- neutrophilic infiltration was observed in rats.
- in general, lesions correlated well with the histopathological occurrence of retained TiO2 particles. This was especially true for inflammatory and epithelial proliferative lesions, both of which were often found around foci of particle-laden macrophages.
- particle retention patterns in the lung were concentration and time dependent. The pattern of lesion change during the postexposure recovery period followed closely the pattern of particle clearance from the lung. Despite the reduction of lung inflammation, certain findings increased in severity over time postexposure, including the aggregation of particle-laden intraluminal macrophages in 47.7 mg/m³ air and 239.1 mg/m³ air dose groups.
- epithelial change in rats was concentrated in areas of heavy dust accumulation.
- rats had a pattern of particle retention that would best be characterized as alveolar intraluminal sequestration of particles.
- rats frequently had particle-laden macrophage accumulations in subpleural regions, with associated epithelial-proliferative changes.

LUNG VOLUMES
- lung volumes did not significantly differ from controls at any time or dose in rats.
- although fibrotic changes of the lung were observed in the rat the magnitude of these changes was not severe enough to translate to the gross architectural changes likely to be detected.

TiO2 BURDENS
- the method quantitation limit (MQL) for the assay was 0.05 µg Ti/mL of sample which translates to values for lung of 0.03 mg TiO2/gram dry weight and for lymph nodes of 2.09 µg TiO2/sample.
- lung burden increased with exposure concentration.
- following 13 weeks of exposure rats exhibited a TiO2 lung burden of 120 mg/gram dry lung.
- burdens decreased in the lung and increased in the lung-associated lymph nodes with time postexposure in rats.
- the lymph node TiO2 burdens of the animals exposed 239.1 mg/m³ air had the greatest increases between 4 and 13 weeks after the exposure ended.
- decreases in lung burdens with time after exposure were sharply different between exposure concentration groups in rats. High and mid dose burdens decreased slowly to approx. 75 % of the initial burden whereas low dose burdens decreased to approx. 15 %.
- pulmonary clearance kinetics of TiO2: rats retained approx. 75 % of the initial burden after 52 weeks recovery.

CELL PROLIFERATION
- rats in the 239.1 mg/m³ dose group had increased terminal bronchiolar cell replication following the 13 week exposure interval, however the labeling index of these cells had returned to control values by 4 weeks postexposure.

CYTOLOGY
- macrophages comprised greater than 90 % of the cells recovered by lavage from control animals in rats.
- recovered neutrophils from control animals were generally a small fraction of the recovered cell population (less than 1 % in rats).
- eosinophils were rare in control rats.
- lymphocytes were minimal in control rats (0.2 %).
- inflammation was most marked immediately following cessation of the 13-week exposure, with a diminution of the inflammatory response during the postexpsoure recovery phase.
Dose descriptor:
NOAEC
Effect level:
9.6 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Dose descriptor:
LOAEC
Effect level:
47.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
other:
Critical effects observed:
not specified
Conclusions:
NOAEC (female rats): 9.6 mg/m³ air (analytical)
LOAEC (female rats): 47.7 mg/m³ air (analytical)

Lung and lung-associated lymph node burdens of TiO2 increased in a concentration-dependent manner. Pulmonary overload was achieved in rats at 47.7 and 239.1 mg/m3. Inflammation was seen at 47.7 and 239.1 mg/m³ (increased numbers of macrophages and neutrophiles and incidences of soluble inflammation markers). Inflammatory responses remained elevated throughout the entire post-exposure recovery period in the most highly exposed animals (239.1 mg/m³). Pulmonary lesions were most severe in rats with progressive epithelial and fibroproliferative changes at 239.1 mg/m³. These epithelial changes were also manifested in rats as an increase in alveolar cell labelling in cell proliferation studies.
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1998-09-14 to 1998-12-11 (exposure period only)
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
all concentrations in this study exceed the maximum tolerated dose (MTD) by overwhelming physiological clearnace mechanisms; such conditions are to be avoided (see OECD guidance document 116 and OECD TG 451)
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of female Lak:LVG(SYR)BR hamsters were exposed to 10, 50 or 250 mg/m³ pigmentary titanium dioxide via whole body inhalation for 6 hours/day, 5 days/week for 13 week with recovery groups held for an additional 4, 13, 26 or 46 weeks postexposure. At each time point, selected lung responses were examined. The responses studied were chosen to assess a variety of pulmonary parameters including: inflammation, cytotoxicity, and epithelial- and fibroproliferative changes.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
hamster, Syrian
Strain:
other: Lak:LVG
Details on species / strain selection:
This stock and strain was chosen due to the available historical database on pulmonary disease and particulate exposure available at the performing laboratory and elsewhere.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Wilmington, MA
- Age on receipt: : approx. 6 weeks old
- Weight a week after arrival: approx. 95 - 137 g
- Housing: housed 1/cage; housed in 1 m³ stainless steel and glass inhalation chambers in suspended stainless steel caging with wire mesh floors during the acclimatisation, and exposure phases. Solid stainless inserts were added to the bottoms of the caging during the nonexposure time periods each day. Animals were housed in filtered microisolated, polycarbonate caging on direct contact celluloase bedding during the postexposure recovery time.
- Diet (ad libitum, except during expsoure when food was withheld): NIH-07 certified diet
- Water (ad libitum, except during expsoure when food was withheld): deionized water
- Acclimation period: approx. 9 days

DETAILS OF FOOD AND WATER QUALITY: drinking water contaminant levels were measured at quarterly intervals.

ENVIRONMENTAL CONDITIONS
- Temperature: 17.8 - 26.1 °C
- Relative humidity: approx. 30 - 70 %
- Air changes: 12 - 15/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
1.36 µm
Geometric standard deviation (GSD):
1.5
Remarks on MMAD:
Standard deviation (MMAD): 0.07
Standard deviation (GSD): 0.11
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m³ H-1000 stainless steel chambers

- System of generating particulates/aerosols: aerosol generation was accomplished using a dust feeder, jet streams of air, a one-stage impactor, and a series of mixing chambers. The dust feeder introduced the particles into the jet streams of air where the particles were broken up and converted to fine particles suspended in air. The aerosol stream exiting the deagglomeration stage passed through a one-stage impactor to remove large particles. The aerosolized powder was then injected into a "pre-chamber" and subsequently distributed and diluted appropriately to the exposure chambers. Prior to the exposures, each chamber was checked for uniformity of distribution of the test substance by measuring the concentration at a minimum of nine positions in the H-1000 chamber without animals but with racks and pans in place.

- Air flow rate: chambers were provided with air at a flow rate of approx. 12 - 15 air changes each hour to ensure an adequate oxygen content of at least 19 %. The airflow rate was monitored and recorde at least once an hour. Total airflow through the H-1000 chamber was monitored by using the pressure drop across an orifice at the inlet of the chamber and adjustments were made with a previously calibrated exhaust fan control through the Andover Infinity system. All chambers were maintained at a slightly negative pressure.

- Temperature, humidity, pressure in air chamber: temperature and relative humidity of the exposure chambers was monitored continuously during exposure and nonexposure periods. Environmental parameters were recorded approx. 6 times during each exposure period. The temperature was maintained at 20.8 - 22.4 °C and relative humidity between 42 to 55 %.

- Method of particle size determination: particle size distribution measurements were carried out at least two times per exposure chamber (excluding the control chamber) during the course of the study, using an aerodynamic sizing device (Micro-Orifice Uniform Deposition Impactor, Model 100).

Preliminary studies were performed prior to the study to confirm the stability of the aerosol.

TEST ATMOSPHERE
- Brief description of analytical method used: Real Aerosol Monitor (Model RAM-S) per H-1000 chamber was used as the primary instrument to continuously measure particle concentration. RAM-S readings were validated by mass weight filters samples. The time-averaged concentration as monitored by the RAM-S was recorded at least six times over the 6 hour exposure system operation period.
Where excursions from the nominal concentrations occurred, manual adjustments were made.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on inhalation exposure" above.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
9.9 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 1.0 mg/m³
Dose / conc.:
49.7 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 4.0 mg/m³
Dose / conc.:
251.1 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 17.3 mg/m³
No. of animals per sex per dose:
59 female hamsters (14 additional hamsters/group were included as extra animals to allow for losses in the course of the study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: 4, 13, 26 or 46 weeks of recovery
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the exposure phase and once per day in the post-exposure phase.
- Cage side observations checked: overt clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for the first 17 weeks of the study and biweekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for the first 17 weeks, and biweekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
Five sacrifices were scheduled to occur at the end of the exposure and following 4, 13, 26, or 46 weeks of recovery. Animals remaining after the final scheduled sacrifice were killed and discarded. Subgroups, based on endpoint, were defined as follows:

A) Subgroup 1 (histopathology, cell proliferation, TiO2 body burden; 5 animals/time point):
Animals were implanted, 5 days before euthanasia, with osmotic pumps containing bromodeoxyuridine (BrdU). Pump model 2ML1(10 µL/h) was used. BrdU concentration was 5 mg/mL. The right lung lobes and lung-associated lymph nodes were separated from the thoracic viscera, blotted, weighed, and then frozen for the determination of retained TiO2. The left lung was intratracheally infused, under pressure, with 10 % neutral buffered formalin. Following formalin infusion the left lung was fixed for no more than 72 hours (ideally 48 hours in formalin and then changed to 70 % ethanol.
- lung cell proliferation: the fixed left lung lobe was trimmed and embedded in paraffin, along with a piece of duodenum (BrdU incorporation in the duodenum on the same tissue sections serves as a positive control for immunohistochemical staining and delivery of BrdU to the animal). Multiple standarized sections were cut and mounted on coated glass slides and stained for the presence of incorporated BrdU. Immunostaining was performed with an automated immunostainer (Biotek) using a commercially available monoclonal antibody to BrdU. Terminal bronchiolar and alveolar duct cell labelling indices were determiend as well as alveolar cell proliferative labelling indices.
- histopathology: paraffin embedded tissues, from the left lung, were prepared from each animal at the same anatomical site with respected to the mainstem bronchus. Tissues were subjected to routine microtomy at 5 µm and were Trichrome stained using a Masson's method. The Trichrome-stained left lung sections were evaluated for particle-induced histopathologic changes.
- TiO2 lung burden: right lung and lung associated lymph nodes were removed, weighed, and frozen on-site. The subsequent processing and analysis was performed. The tissues were dried and digested using a combination of nitric and hydrofluoric acids. Subsequent analysis for titanium content was by inductively coupled plasma optical emission spectrometry (IPS-OES).

B) Subgroup 2 (BALF, cytology; 5 animals/time point):
The lungs of the animals were lavaged five times with equal volumes of phosphate buffered saline (4 mL). Fluid from the first two lavages was recovered, pooled and placed on ice and the subsequent three lavage fluids were pooled and also placed on ice. The left lobe of the lavaged lung was instilled with freezing medium, tied off and removed, and portions mounted for frozen sectioning. The right lung caudal and accessory lobes were removed and flash-frozen in liquid nitrogen for nucleic acid isolation. The right lung apical and medial lobes were intratracheally infused, under pressure, with 4 % paraformaldehyde fixative. Following infusion the right apical and medial lobes were fixed for no more than 72 hours (ideally 48 hours) in 4 % paraformaldehyde and then changed to 70 % RNAse-free ethanol. These tissues were collected for possible use in in situ hybridization procedures.
- pulmonary toxicity endpoints: cell-free fluid from the pooled first and second bronchoalveolar lavage washes were utilized for the determination of lactate dehydrogenase (LDH), total protein, and phospholipid content. LDH and total protein were quantitated spectrophotometrically using an automated analyzer. Choline-containing phospholipids were assayed by an enzymatic method using phospholipase D, choline oxidase and peroxidase (Takayama et al., 1977)* using a commercially available kit (Wako).
- cytology: bronchoalveolar lavage fluids were centrifuged to separate cells and supernatant. Cell pellets from all the lavages were pooled and an aliquot used for phospholipid analyses. The remaining cells were resuspended in cell culture medium supplemented with serum, and counted using an automated cell counter. The cell suspensions were diluted with cold medium cytocentrifuged onto glass slides and subsequently air dried, fixed, and stained. Differential counts (200 cells/animal) were obtained using the stained cell preparations.

C) Subgroup 3 (electron microscopy; 3 animals each at the time points 0, 13, and 52 weeks postexposure (0, 10, and 250 mg/m³ concentrations) or 3 animals each at the time point 52 weeks postexposure (50 mg/m³ concentration):
Tracheas were cannulated and the lungs intratracheally infused with glutaraldehyde fixative under pressure. Tthe heart and lungs were removed en bloc and placed in fresh fixative. Once the lungs had fixed for at least 12 hours the fixative was replaced with an appropriate buffer to prevent overfixation.
- electron microscopy: lungs were collected, fixed, and placed in buffer. Sections of plastic-embedded tissue may be examined for localization and interstitialization of the test particles.
- lung volumes: lungs were collected, fixed, and placed in buffer for a minimum of 4 days. The heart and other extraneous tissues were removed and the volume of the lungs was determined using a fluid displacement method (Scherle, 1970)*.

Unscheduled deaths: animals found dead or moribund were necropsied and where possible the target organ, lung, was saved along with the animal's transponder. No effort was made to establish the cause of death or moribund condition.

*References:
- Takayama, M., S. Itoh, T. Nagasaki, and I. Tanimizu. "A new enzymatic method for determination of serum choline-containing phospholipids." Clin Chim Acta 79 (1977): 93-8.
- Scherle, W. "A simple method for volumetry of organs in quantitative stereology." Mikroskopie 26 (1970): 57 - 60.
Statistics:
The analysis included a combination of regression and analysis of variance (ANOVA) methods. All data were tested for normality and homogeneity of variance. If the hypotheses for these assumptions were rejected, common transformations (log, square root etc.) were applied and the data were retested. If the hypotheses were still rejected, nonparametric methods were used.
Incidence data were compared using the appropriate statistical test, generally Fisher's Exact Test. A probability value of 0.05 was used as the critical level of significance within each statistical test, except for the tests of homogeneity and normality, where the significance level was 0.01. However, the critical level of overall significance (alpha level) for each test domain was adjusted to correct for multiple evaluations.
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
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:
not examined
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
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- lung-associated lymph nodes were enlarged (macroscopic evidence). The increase in lymph node size in the TiO2 exposed groups was associated with white aggregations of pigmentary TiO2 (concentration- and time dependent finding).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- hypertropic and hyperplastic epithelial changes were in immediate proximity to aggregates of inflammatory cells and particle-laden macrophages. The epithelial changes diminished in incidence and severity with the reduction in pulmonary inflammation that occurred over time during the postexposure recovery period.
- in general, lesions correlated well with the histopathological occurrence of retained TiO2 particles. This was especially true for inflammatory and epithelial proliferative lesions, both of which were often found around foci of particle-laden macrophages.
- particle retention patterns in the lung were concentration and time dependent. The pattern of lesion change during the postexposure recovery period followed closely the pattern of particle clearance from the lung. Despite the reduction of lung inflammation, certain findings increased in severity over time postexposure, including the aggregation of particle-laden intraluminal macrophages in 49.7 mg/m³ air and 251.1 mg/m³ air dose groups.
- epithelial change in hamsters was concentrated in areas of heavy dust accumulation.
- hamsters had a pattern of particle retention that would best be characterized as alveolar intraluminal sequestration of particles.
- concentration of particles and associated epithelial changes in central lobar zones, but with particle clearance, there was intense aggregation of heavily particle-laden macrophages in focal lesions.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
CYTOLOGY
- exposures to TiO2 resulted in an increase of neutrophils, macrophages, and lymphocytes in hamsters.
- greatest neutrophil response was seen in animals of the 251.1 mg/m³ air (58 %).
- greatest macrophage response was found in the 251.1 mg/m³ air dose animals.

Details on results:
MORTALITY
- no significant exposure-related increase in mortality
- losses during the exposure period were minimal with the loss of two hamsters.
- hamsters had a great morbidity and mortality presumably due to the occurrence of age-related spontaneous conditions such as chronic renal disease.

BODY WEIGHT AND WEIGHT CHANGES
- a depression in body weight (5 - 11 %) was noted following the end of the exposure period with a subsequent recovery occurring within approx. 6 weeks

ORGAN WEIGHTS
- hamsters had significant lung wet weight changes 4 and 13 weeks postexposure.
- hamsters had no consistent, significant, changes in the weight of the lung-associated lymph nodes.

GROSS PATHOLOGY
- a number of hamsters that died or were removed unscheduled from the study had bilateral, pale, small, firm kidneys with irregular pitted cortical surfaces. A few of these animals had evidence of ascitic fluid and subcutaneous oedema suggestive of nephrotic syndrome. The clinical findings of red discoloured urine support the finding of renal disease in the study hamsters. In addition to unscheduled animals, many of the hamsters scheduled for sacrifice at the 46-week time point had evidence of severe chronic renal disease at necropsy (bilateral, pale, firm, shrunken kidneys).

HISTOPATHOLOGY
- hamsters had a retention pattern more difficult to discern due to the rapid clearance of particles.
- particles in exposed hamsters appeared to be concentrated in central portions of the lung lobes. Although particles were concentrated in central lobar regions in the lungs, they were often aggregated focally within heavily particle-laden macrophages forming "dust macules".
- TiO2-induced lung lesions increased in severity with increasing exposure concentration.
- lesions were generally minimal to mild in severity
- particle-laden macrophage aggregation was noted.
- histopathological appearance of lung lesions correlated very well with the BALF inflammatory indices.
- TiO2-induced inflammation was concentration dependent and limited to the 49.7 mg/m³ and 251.1 mg/m³ concentration exposure groups.
- neutrophils infiltration was observed.

TiO2 BURDENS
- the method quantitation limit (MQL) for the assay was 0.05 µg Ti/mL of sample which translates to values for lung of 0.03 mg TiO2/gram dry weight and for lymph nodes of 2.09 µg TiO2/sample.
- burden values for control hamsters at the end of the exposures (lung and lymph nodes) and 4 weeks postexposure (lung) were above nackground levels. These samples were processed concurrently and the values refelct an increase in the background for that run of the assay rather than the presence of TiO2 in control tissue.
- lung burden increased with exposure concentration.
- following 13 weeks of exposure hamsters had a TiO2 lung burden of 114 mg/gram dry lung.
- burden decreased in the lung and increased in the lung-associated lymph nodes with time postexposure in hamsters.
- the lymph node TiO2 burdens of the animals exposed 251.1 mg/m³ air had the greatest increase between 4 and 13 weeks after the exposure ended.
- pulmonary clearance kinetics of TiO2: hamsters retained approx. 10 % of the initial burden after 46 weeks recovery.

CYTOLOGY
- number of cells recovered from the lungs by BAL from control animals did not correlated with body weight in hamsters. Significant increase, over controls, in cell recovery were observed following exposure in the animals of the 251.1 mg/m³ air dose group. The values return to control levels before the termination of the study.
- macrophages comprised greater than 90 % of the cells recovered by lavage from control animals in hamsters.
- recovered neutrophils from control animals were generally a small fraction of the recovered cell population (1 - 5 % in hamsters).
- eosinophils comprised as much as 13 % of the control cell population in hamsters (possibley due to compromised health in some of the animals).
- lymphocytes ranged from 0.2 to 2 % of the population in hamsters.
- inflammation was most marked immediately following cessation of the 13-week exposure, with a diminution of the inflammatory response during the postexpsoure recovery phase. The TiO2-induced inflammation subsided and was absent in all exposure groups at the time of the final sacrifice (46 weeks postexposure).

PULMONARY TOXICITY ENDPOINTS
- hamsters exposed to the 251.1 mg/m³ air dose of TiO2 had elevated lactate dehydrogenase levels, but the elevations were only significantly different from control through 4 weeks postexposure.
- elevations of total protein were observed in hamsters but were limited to the 251.1 mg/m³ air dose group and had returned to control values by 26 weeks postexposure.
- phospholipid concentration in hamsters was elevated in the 251.1 mg/m³ dose group but had returned to control levels 4 weeks after exposure.
- phospholipids associated with cells recovered by lavage, in general, showed a dose dependent elevation in concentration after 13 weeks exposure.
- persistence of elevated phospholipids associated with cells was observed in hamsters of the 251.1 mg/m³ air dose group but these elevations had returned to control levels by 26 weeks postexposure.

CELL PROLIFERATION
- no significant increases in the labeling index of bronchiolar cells of the hamsters.
- alveolar cells of the hamster lung had no significant increases in cell replication.
- baseline values for alveolar cells of the hamsters increased at the latter time points and may be indicative of the age or health status of these animals. Similar increases in bronchiolar labeling indices were observed in some hamsters of the control and 251.1 mg/m³ groups at 46 weeks postexposure resulting in elevated mean values and statistically, but not biologically, significant decreases in the 9.9 mg/m³ air and 49.7 mg/m³ dose groups.

LUNG VOLUMES
- lung volumes did not significantly differ from controls at any time or dose in hamsters.
Dose descriptor:
NOAEC
Effect level:
9.9 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Dose descriptor:
LOAEC
Effect level:
49.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
other:
Critical effects observed:
not specified
Conclusions:
NOAEC (female hamsters): 9.9 mg/m³ air (analytical)
LOAEC (female hamsters): 49.7 mg/m³ air (analytical)

The following treatment-related findings were recorded for hamsters during the study: significant increase (p<0.05) of the number of neutrophiles recovered by lavage (BAL) of the lungs at the end of exposure were observed. Furthermore, particle-laden macrophages at all post exposure time points (intraluminal and occasionally subpleural) were recorded. Lastly, histopathology revealed minimal type II epithelial cell hypertrophy and hyperplasia, which reduced in incidence and severity with increasing post exposure time.


Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Comparison of selected pulmonary responses of rats, mice, and Syrian golden hamsters to inhaled pigmentary titanium dioxide
Author:
Everitt, J.I. et al.
Year:
2000
Bibliographic source:
Inhalation Toxicology 12 (supplement 3): 275 - 282.
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001
Reference Type:
publication
Title:
Long-term pulmonary responses of three laboratory rodent species to subchronic inhalation of pigmentary titanium dioxide particles
Author:
Bermudez, E.; et al.
Year:
2002
Bibliographic source:
Toxicol. Sci. 70, 86-97
Reference Type:
publication
Title:
Titanium dioxide: Inhalation toxicology and epidemiology
Author:
Hext, P.M. et al.
Year:
2005
Bibliographic source:
Ann. occup. Hyg. 49 (6): 461 - 472.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of female B3C3F1/CrlBR mice were exposed to 10, 50 or 250 mg/m³ pigmentary titanium dioxide via whole body inhalation for 6 hours/day, 5 days/week for 13 week with recovery groups held for an additional 4, 13, 26 or 52 weeks postexposure. At each time point, selected lung responses were examined. The responses studied were chosen to assess a variety of pulmonary parameters including: inflammation, cytotoxicity, and epithelial- and fibroproliferative changes.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): pigmentary TiO2 (supplied by DuPont Haskell Laboratory (Newark, DE))
Specific details on test material used for the study:
not applicable

Test animals

Species:
mouse
Strain:
other: B3C3F1/CrlBR
Details on species / strain selection:
This stock and strain was chosen due to the available historical database on pulmonary disease and particulate exposure available at the performing laboratory and elsewhere.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Wilmington, MA
- Age on receipt: approx. 6 weeks old
- Weight a week after arrival: approx. 18 - 23 g
- Housing: housed 1/cage; housed in 1 m³ stainless steel and glass inhalation chambers in suspended stainless steel caging with wire mesh floors during the acclimatisation, exposure phases and postexposure phase.
- Diet (ad libitum, except during expsoure when food was withheld): NIH-07 certified diet
- Water (ad libitum, except during expsoure when food was withheld): deionized water
- Acclimation period: approx. 9 days

DETAILS OF FOOD AND WATER QUALITY: drinking water contaminant levels were measured at quarterly intervals.

ENVIRONMENTAL CONDITIONS
- Temperature: 17.8 - 26.1 °C
- Relative humidity: approx. 30 - 70 %
- Air changes: 12 - 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
1.39 µm
Geometric standard deviation (GSD):
1.72
Remarks on MMAD:
Standard deviation (MMAD): 0.04
Standard deviation (GSD): 0.29
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m³ H-1000 stainless steel chambers

- System of generating particulates/aerosols: aerosol generation was accomplished using a dust feeder, jet streams of air, a one-stage impactor, and a series of mixing chambers. The dust feeder introduced the particles into the jet streams of air where the particles were broken up and converted to fine particles suspended in air. The aerosol stream exiting the deagglomeration stage passed through a one-stage impactor to remove large particles. The aerosolized powder was then injected into a "pre-chamber" and subsequently distributed and diluted appropriately to the exposure chambers. Prior to the exposures, each chamber was checked for uniformity of distribution of the test substance by measuring the concentration at a minimum of nine positions in the H-1000 chamber without animals but with racks and pans in place.

- Air flow rate: chambers were provided with air at a flow rate of approx. 12 - 15 air changes each hour to ensure an adequate oxygen content of at least 19 %. The airflow rate was monitored and recorde at least once an hour. Total airflow through the H-1000 chamber was monitored by using the pressure drop across an orifice at the inlet of the chamber and adjustments were made with a previously calibrated exhaust fan control through the Andover Infinity system. All chambers were maintained at a slightly negative pressure.

- Temperature, humidity, pressure in air chamber: temperature and relative humidity of the exposure chambers was monitored continuously during exposure and nonexposure periods. Environmental parameters were recorded approx. 6 times during each exposure period. The temperature was maintained at 20.5 - 22.9 °C and relative humidity between 39 to 60 %.

- Method of particle size determination: particle size distribution measurements were carried out at least two times per exposure chamber (excluding the control chamber) during the course of the study, using an aerodynamic sizing device (Micro-Orifice Uniform Deposition Impactor, Model 100).

Preliminary studies were performed prior to the study to confirm the stability of the aerosol.

TEST ATMOSPHERE
- Brief description of analytical method used: Real Aerosol Monitor (Model RAM-S) per H-1000 chamber was used as the primary instrument to continuously measure particle concentration. RAM-S readings were validated by mass weight filters samples. The time-averaged concentration as monitored by the RAM-S was recorded at least six times over the 6 hour exposure system operation period.
Where excursions from the nominal concentrations occurred, manual adjustments were made.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on inhalation exposure" above.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
9.5 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 1.2 mg/m³
Dose / conc.:
47 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 4.6 mg/m³
Dose / conc.:
240.3 mg/m³ air (analytical)
Remarks:
Standard deviation: ± 20.0 mg/m³
No. of animals per sex per dose:
59 female mice (14 additional mice/group were included as extra animals to allow for losses in the course of the study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: 4, 13, 26 or 52 weeks of recovery
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the exposure phase and once per day in the post-exposure phase.
- Cage side observations checked: overt clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for the first 17 weeks of the study and biweekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for the first 17 weeks, and biweekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
Five sacrifices were scheduled to occur at the end of the exposure and following 4, 13, 26, or 52 weeks of recovery. Animals remaining after the final scheduled sacrifice were killed and discarded. Subgroups, based on endpoint, were defined as follows:

A) Subgroup 1 (histopathology, cell proliferation, TiO2 body burden; 5 animals/time point):
Animals were implanted, 5 days before euthanasia, with osmotic pumps containing bromodeoxyuridine (BrdU). Pump model 2001 (1 µL/h) was used. BrdU concentration was 16 mg/mL. The right lung lobes and lung-associated lymph nodes were separated from the thoracic viscera, blotted, weighed, and then frozen for the determination of retained TiO2. The left lung was intratracheally infused, under pressure, with 10 % neutral buffered formalin. Following formalin infusion the left lung was fixed for no more than 72 hours (ideally 48 hours in formalin and then changed to 70 % ethanol.
- lung cell proliferation: the fixed left lung lobe was trimmed and embedded in paraffin, along with a piece of duodenum (BrdU incorporation in the duodenum on the same tissue sections serves as a positive control for immunohistochemical staining and delivery of BrdU to the animal). Multiple standarized sections were cut and mounted on coated glass slides and stained for the presence of incorporated BrdU. Immunostaining was performed with an automated immunostainer (Biotek) using a commercially available monoclonal antibody to BrdU. Terminal bronchiolar and alveolar duct cell labelling indices were determiend as well as alveolar cell proliferative labelling indices.
- histopathology: paraffin embedded tissues, from the left lung, were prepared from each animal at the same anatomical site with respected to the mainstem bronchus. Tissues were subjected to routine microtomy at 5 µm and were Trichrome stained using a Masson's method. The Trichrome-stained left lung sections were evaluated for particle-induced histopathologic changes.
- TiO2 lung burden: right lung and lung associated lymph nodes were removed, weighed, and frozen on-site. The subsequent processing and analysis was performed. The tissues were dried and digested using a combination of nitric and hydrofluoric acids. Subsequent analysis for titanium content was by inductively coupled plasma optical emission spectrometry (IPS-OES).

B) Subgroup 2 (BALF, cytology; 5 animals/time point):
The lungs of the animals were lavaged five times with equal volumes of phosphate buffered saline (1 mL). Fluid from the first two lavages was recovered, pooled and placed on ice and the subsequent three lavage fluids were pooled and also placed on ice. The left lobe of the lavaged lung was instilled with freezing medium, tied off and removed, and portions mounted for frozen sectioning. The right lung caudal and accessory lobes were removed and flash-frozen in liquid nitrogen for nucleic acid isolation. The right lung apical and medial lobes were intratracheally infused, under pressure, with 4 % paraformaldehyde fixative. Following infusion the right apical and medial lobes were fixed for no more than 72 hours (ideally 48 hours) in 4 % paraformaldehyde and then changed to 70 % RNAse-free ethanol. These tissues were collected for possible use in in situ hybridization procedures.
- pulmonary toxicity endpoints: cell-free fluid from the pooled first and second bronchoalveolar lavage washes were utilized for the determination of lactate dehydrogenase (LDH), total protein, and phospholipid content. LDH, and total protein were quantitated spectrophotometrically using an automated analyzer. Choline-containing phospholipids were assayed by an enzymatic method using phospholipase D, choline oxidase and peroxidase (Takayama et al., 1977)* using a commercially available kit (Wako).
- cytology: bronchoalveolar lavage fluids were centrifuged to separate cells and supernatant. Cell pellets from all the lavages were pooled and an aliquot used for phospholipid analyses. The remaining cells were resuspended in cell culture medium supplemented with serum, and counted using an automated cell counter. The cell suspensions were diluted with cold medium cytocentrifuged onto glass slides and subsequently air dried, fixed, and stained. Differential counts (200 cells/animal) were obtained using the stained cell preparations.

C) Subgroup 3 (electron microscopy; 3 animals each at the time points 0, 13, and 52 weeks postexposure (0, 10, and 250 mg/m³ concentrations) or 3 animals each at the time point 52 weeks postexposure (50 mg/m³ concentration):
Tracheas were cannulated and the lungs intratracheally infused with glutaraldehyde fixative under pressure. Tthe heart and lungs were removed en bloc and placed in fresh fixative. Once the lungs had fixed for at least 12 hours the fixative was replaced with an appropriate buffer to prevent overfixation.
- electron microscopy: lungs were collected, fixed, and placed in buffer. Sections of plastic-embedded tissue may be examined for localization and interstitialization of the test particles.
- lung volumes: lungs were collected, fixed, and placed in buffer for a minimum of 4 days. The heart and other extraneous tissues were removed and the volume of the lungs was determined using a fluid displacement method (Scherle, 1970)*.

Unscheduled deaths: animals found dead or moribund were necropsied and where possible the target organ, lung, was saved along with the animal's transponder. No effort was made to establish the cause of death or moribund condition.

*References:
- Takayama, M., S. Itoh, T. Nagasaki, and I. Tanimizu. "A new enzymatic method for determination of serum choline-containing phospholipids." Clin Chim Acta 79 (1977): 93-8.
- Scherle, W. "A simple method for volumetry of organs in quantitative stereology." Mikroskopie 26 (1970): 57 - 60.
Statistics:
The analysis included a combination of regression and analysis of variance (ANOVA) methods. All data were tested for normality and homogeneity of variance. If the hypotheses for these assumptions were rejected, common transformations (log, square root etc.) were applied and the data were retested. If the hypotheses were still rejected, nonparametric methods were used.
Incidence data were compared using the appropriate statistical test, generally Fisher's Exact Test.A probability value of 0.05 was used as the critical level of significance within each statistical test, except for the tests of homogeneity and normality, where the significance level was 0.01. However, the critical level of overall significance (alpha level) for each test domain was adjusted to correct for multiple evaluations.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
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:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
- wet weight of the right lung was increased following 13 weeks of exposure in mice in the 240.3 mg/m³ air dose group and remained elevated at 52 weeks postexposure.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- lung-associated lymph nodes were enlarged (macroscopic evidence). The increase in lymph node size in the TiO2 exposed groups was associated with white aggregations of pigmentary TiO2 (concentration- and time dependent finding).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Particles and associated lesions, characterized by alveolar type II cell hypertrophy, were most prevalent in central lobar regions and virtually absent in the periphery of lung lobes.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
PULMONARY TOXICITY ENDPOINTS (BRONCHOALVEOLAR LAVAGE, BAL)
- lactate dehydrogenase levels were elevated in mouse BALF in the 47.0 mg/m³ air and the 240.3 mg/m³ air dose animals and remained so through 26 weeks postexposure for the 47.0 mg/m³ dose animals and 52 weeks postexposure for the 240.3 mg/m³ dose animals.
- total protein concentrations in mouse BALF, collected from animals of the 47.0 mg/m³ air and 240.3 mg/m³ dose groups, were significantly greater than concurrent controls after exposure and remained elevated through 52 weeks postexposure.
- phospholipid concentration was increased in BALF from mice of all treatment groups following 13 weeks exposure. 9.5 mg/m³ air and 47.0 mg/m³ dose values returned to control levels by 4 and 13 weeks, respectively, however the phospholipid concentration in the 240.3 mg/m³ air dose group remained greater than controls at 52 weeks postexposure.
- phospholipids associated with cells recovered by lavage, in general, showed a dose dependent elevation in concentration after 13 weeks exposure.
- in the postexposure period, mice from the 47.0 mg/m³ air and 240.3 mg/m³ air dose groups had significantly elevated phospholipid levels through 52 weeks.

CYTOLOGY
- number of cells recovered from the lungs by BAL from control animals generally correlated with body weight in mice. Significant increase, over controls, in cell recovery were observed following exposure in the animals of the 240.3 mg/m³ air dose group . This increase declined with time after exposure.
- exposures to TiO2 resulted in an increase of neutrophils, macrophages, and lymphocytes in mice.
- significant increase (p<0.05) of the number of neutrophiles recovered by lavage (BAL) of the lungs at the end of exposure; remained significantly elevated (p<0.05) until the end of the post exposure period (week 52).
- greatest neutrophil response was seen in animals of the 240.3 mg/m³ air (32 %).
- greatest macrophage response was found in the 240.3 mg/m³ air dose animals.
Details on results:
MORTALITY
- no significant exposure-related increase in mortality
- losses during the exposure period were minimal with the loss of two mice.
- a total of 8 mice were removed unscheduled from the study of which 3 died of accidental, cage-related, trauma.
- remaining 5 animals were distributed across the exposed groups.

BODY WEIGHT AND WEIGHT CHANGES
- a depression in body weight (4 - 5 %) was noted following the end of the exposure period with a subsequent recovery occurring over the next 3 - 4 weeks.
- mice exposed to the 240.3 mg/m³ air concentration of TiO2 had a consistent depression, not exceeding 10 % of controls, in body weight during the recovery period (unclear cause).

ORGAN WEIGHTS
- lung-associated lymph node wet weights were elevated in mice following exposure in the 240.3 mg/m³ air dose group (probably due to inflammatory response and small parts to the TiO2 burdens).
- unexpected increases in the control mouse lung and lymph node wet weights 13 weeks postexpousre which resulted in statistically significant decrease in tissue wet weights from TiO2-exposed animals (probably due to error in the weighing of the control tissues).

HISTOPATHOLOGY
- mice appeared to have a panacinar distribution.
- particle-laden macrophages were primarily intraluminal and distributed over a large area of pulmonary parenchyma.
- TiO2-induced lung lesions increased in severity with increasing exposure concentration.
- lesions were generally minimal to mild in severity
- pulmonary overload conditions were evident without histopathological changes
- particle-laden macrophage aggregation was noted.
- in the lungs, there was increased peribronchiolar aggregation of lymphoid cells (age-related finding;)
- histopathological appearance of lung lesions correlated very well with the BALF inflammatory indices.
- TiO2-induced inflammation was concentration dependent and limited to the 47.0 mg/m³ and 240.3 mg/m³ concentration exposure groups.
- neutrophilic infiltration was observed in mice.
- hypertropic and hyperplastic epithelial changes were in immediate proximity to aggregates of inflammatory cells and particle-laden macrophages. The epithelial changes diminished in incidence and severity with the reduction in pulmonary inflammation that occurred over time during the postexposure recovery period.
- in general, lesions correlated well with the histopathological occurrence of retained TiO2 particles. This was especially true for inflammatory and epithelial proliferative lesions, both of which were often found around foci of particle-laden macrophages.
- particle retention patterns in the lung were concentration and time dependent. The pattern of lesion change during the postexposure recovery period followed closely the pattern of particle clearance from the lung. Despite the reduction of lung inflammation, certain findings increased in severity over time postexposure, including the aggregation of particle-laden intraluminal macrophages in 47.0 mg/m³ air and 240.3 mg/m³ air dose groups.
- epithelial change in mice was concentrated in areas of heavy dust accumulation.
- mice had a pattern of particle retention that would best be characterized as alveolar intraluminal sequestration of particles.

- numerous particles and particle-laden macrophages were present in pleural and subpleural regions of 47.0 mg/m³ air dose and especially 240.3 mg/³ air dose animals immediately postexposure, but over time there was a marked preferential loss of particulate material from the periphery of the lung lobes during the postexposure recovery period.

LUNG VOLUMES
- lung volumes did not significantly differ from controls at any time or dose in mice.

TiO2 BURDENS
- the method quantitation limit (MQL) for the assay was 0.05 µg Ti/mL of sample which translates to values for lung of 0.22 mg TiO2/gram dry weight and for lymph nodes of 2.09 µg TiO2/sample.
- lung burden increased with exposure concentration.
- following 13 weeks of exposure mice exhibited a TiO2 lung burden of 170 mg/gram dry lung.
- burdens decreased in the lung and increased in the lung-associated lymph nodes with time postexposure in mice
- the lymph node TiO2 burdens of the animals exposed to 240.3 mg/m³ air had the greatest increase between 4 and 13 weeks after the exposure ended.
- decreases in the lung burdens with time after exposure were sharply different between exposure concentration groups in mice; high and mid dose burdens decreased slowly to approx. 75 % of the initial burden whereas low dose burdens decreased to approx. 15 %.
- pulmonary clearance kinetics: mice retained approx. 75 % of the initial burden after 52 weeks recovery.

CYTOLOGY
- macrophages comprised greater than 90 % of the cells recovered by lavage from control animals in mice.
- recovered neutrophils from control animals were generally a small fraction of the recovered cell population (less than 1 % in mice).
- eosinophils were rare in control mice.
- lymphocytes were minimal in control mice (0.3 %).
- inflammation was most marked immediately following cessation of the 13-week exposure, with a diminution of the inflammatory response during the postexpsoure recovery phase.

CELL PROLIFERATION
- mice in the 240.3 mg/m³ dose group had increased terminal bronchiolar cell replication following the 13 week exposure interval, however the labeling index of these cells had returned to control values by 4 weeks postexposure.
- alveolar cells of the mouse lung had no significant increases in cell replication.

Effect levels

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Dose descriptor:
NOAEC
Effect level:
9.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Dose descriptor:
LOAEC
Effect level:
47 mg/m³ air (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
other:

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEC (female mice): 9.5 mg/m³ air (analytical)
LOAEC (female mice): 47.0 mg/m³ air (analytical)

The following treatment-related findings were recorded for mice during the study: significant increase (p<0.05) of the number of neutrophiles recovered by lavage (BAL) of the lungs at the end of exposure, which remained significantly elevated (p<0.05) until the end of the post exposure period (week 52). Furthermore, lactate dehydrogenase in BAL was significantly (p<0.05) increased at the end of the exposure period and remained significantly elevated (p<0.05) until week 26 post exposure. Protein in BAL was significantly (p<0.05) increased at the end of the exposure period, which remained significantly elevated (p<0.05) until week 52 post exposure. Lastly, histopathology revealed alveolar type II cell hypertrophy (central lobular regions), which is also treatment-related.