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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Inhalation of high concentrations of low toxicity dusts in rats results in impaired pulmonary clearance mechanisms and persistent inflammation
Author:
Warheit, D.B. et al.
Year:
1997
Bibliographic source:
Toxicology and Applied Pharmacology 145: 10 - 22.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male rats were exposed to titanium dioxide particles 6 hours/day, 5 days/week for 4 weeks at concentrations of 5, 50, and 250 mg/m³ and evaluated at selected intervals through 6 months postexposure (0 hour, 1 week, and 1, 3, and 6 months). Indices of pulmonary inflammation as well as alveolar macrophage clearance functions (morphology, in vivo and in vitro phagocytosis, and chemotaxis), cell proliferation, and histopathology endpoints were measured at several post-exposure time periods through 6 months. In addition, amounts of TiO2 in lungs and tracheobronchial lymph nodes were measured to allow an evaluation of particle clearance and translocation patterns.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium dioxide
EC Number:
236-675-5
EC Name:
Titanium dioxide
Cas Number:
13463-67-7
Molecular formula:
O2Ti
IUPAC Name:
dioxotitanium
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): pigment-graded titanium dioxide particles (obtained from the DuPont Co. (Wilmington, DE))
- Particle size (mean diameters of individual particles): 0.25 µm (generally forms 1.0 µm agglomerates)
- Crystal structure: rutile
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: particles were heated to 200 °C for 4 hours to eliminate the possibility of endotoxin contamination.

Test animals

Species:
rat
Strain:
other: Crl:CDBR
Details on species / strain selection:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 7 - 8 weeks

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.4 - <= 1.9 µm
Details on inhalation exposure:
The methods utilized for aerosol generation of titanium dioxide particles have previously been reported (Warheit et al., 1991)*.


*Reference:
- Warheit, D.B., Carakostas, M.C., Hartsky, M.A., and Hansen, J.F. (1991). Development of a short-term inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107, 350 - 368.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Overall mean atmospheric concentrations were very close to the design concentrations.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
5.4 mg/m³ air (analytical)
Remarks:
Standard deviation: 6 mg/m³
Dose / conc.:
51.9 mg/m³ air (analytical)
Remarks:
Standard deviation: 16 mg/m³
Dose / conc.:
252.2 mg/m³ air (analytical)
Remarks:
Standard deviation: 86 mg/m³
No. of animals per sex per dose:
not specified
Control animals:
yes, sham-exposed
Details on study design:
not specified
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: not specified
DETAILED CLINICAL OBSERVATIONS: not specified
BODY WEIGHT: not specified
FOOD CONSUMPTION AND COMPOUND INTAKE: not specified
FOOD EFFICIENCY: not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

LUNG EVALUATIONS:
Following exposures, the lungs of TiO2 exposed animals and aged-matched sham controls were subsequently evaluated by bronchoalveolar lavage fluid analysis, BrdU cell labeling, lung clearance analysis, and in vitro macrophage function at 0 hour, 1 week, and 1, 3 and 6 months postexposure.

Bronchoalveolar lavage procedures and biochemical assays on lavaged fluids were conducted according to methods previously described (Warheit et al ., 1991)*.

- Phagocytosis:
Alveolar macrophage cell culture and phagocytosis assay methods have previously been reported (Warheit et al., 1984a,b)*. For the phagocytic assay for TiO2-exposed macrophages, a suspension of carbonyl iron particles was incubated with normal rat serum for 1 hour at 40°C and sonicatecl to reduce aggregations of particles. A final concentration of 1.75 mg/mL was added to monolayers.

- Chemotaxis:
Alveolar macrophages were collected from TiO2- or sham-expmed rats by lavage as described above. The chemotaxis assay was carried out as described previously using three concentrations (1, 5, and 10%) of zymosan-activated sera as the chemotactic stimulus (Warheit er al., 1984b, 1992)*

- Pulmonary cell proliferation studies:
Pulmonary cell proliferation experiments were conducted according to methods previously described (Warheit et ed., 1992)*.

*References:
- Warheit, D. B., Carakostas, M. C., Hartsky, M.A., and Hansen, J. F. (1991). Development of a short-term inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107, 350-368.
- Warheit, D. B., Hill, L. H ., and Brody, A. R. (1984a). Surface morphology and correlated phagocytic capacity of pulmonary macrophages lavaged from the lungs of rats. Exp. Lung Res. 6, 71-82.
- Warheit, D.B., and Chang, L.Y., Hill, L.H., Hook, G.E.R., Crapo, J.D., and Brody, A.R. (1984b). Pulmonary macrophage accumulation and asbestos-induced lesions at sites of fiber deposition. Am. Rev. Respir. Dis. 129, 301-310.
- Warheit, D.B., Kellar, K.A., and Hartsky, M.A. (1992). Pulmonary cellular effects in rats following aerosol exposures to ultrafine Kevlar aramid fibrils: Evidence for biodegradability of inhaled fibrils. Toxicol. Appl. Pharmacol. 116, 225-239.
Sacrifice and pathology:
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes

Histopathology was performed at 0 hour, 1 week and 1, 3, and 6 months postexposure.

The lungs of rats exposed to TiO2 for 4 weeks were prepared for light microscopy by airway infusion using methods previously reported (Warheit et al., 1984b, 1991)*. Analyses of lung and lymph node burdens were conducted by digesting tissue specimens in hydrofluoric acid and analyzing for titanium using the method of inductively coupled plasma (ICP-AES) spectroscopy.

*References:
- Warheit, D. B., Carakostas, M. C., Hartsky, M.A., and Hansen, J. F. (1991). Development of a short-term inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107, 350-368.
- Warheit, D.B., and Chang, L.Y., Hill, L.H., Hook, G.E.R., Crapo, J.D., and Brody, A.R. (1984b). Pulmonary macrophage accumulation and asbestos-induced lesions at sites of fiber deposition. Am. Rev. Respir. Dis. 129, 301-310.
Other examinations:
not specified
Statistics:
Statistics were carried out using two-tailed Student t-test on a Microsoft Exel software program (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- lesions in the respiratory system varied with exposure concentration and duration of post-exposure recovery.
- 5 mg/m³ concentration: concentration produced only minimal effects. Particle-laden macrophages and a minimal diffuse increase in alveolar macrophages (histiocytosis) were evident at 0 days of recovery. The histiocytosis was no longer evident at 1 week postexposure. Individual particle-laden macrophages could be found in very low numbers within air spaces and lymphoid tissue throughout the entire 6-month postexposure period.
- 50 and 250 mg/m³: concentrations produced a wide spectrum of effects within the lung. Free granular pigment of TiO2 was present on the mucosal surfaces of bronchioles and bronchi at 0 days of recovery. Particle-laden macrophages, found individually, were numerous throughout the air spaces at this same time period. Beginning at 1 week post-exposure and persisting thereafter, many dense aggregates of particle-laden macrophages were within alveoli and alveolar ducts. Cellular hypertrophy and hyperplasia were evident at alveolar wall and duct bifurcations that were adjacent to macrophage aggregates. Mucosal hypertrophy and hyperplasia were also observed within the bronchi and bronchioles.
- number of pigment-laden macrophages found individually and in aggregates was much greater in animals exposed to the highest concentration, and occupied a greater portion of the lung.
- severity of cellular hypertrophy and hyperplasia at alveoli and alveolar duct bifurcations was significantly greater in animals exposed to the highest concentration of TiO2 particles.
- severity and character of the lesions changed with time. Free granular pigment was no longer apparent at 1 week post-exposure in any concentration group. Particle-laden macrophages, found individually, decreased in number with time, but were evident in small numbers within the pulmonary air spaces throughout the entire 6-month recovery period. The numbers and size of the dense aggregates of macrophages within alveoli and alveolar ducts, increased during the first month post-exposure but did not expand throughout the remaining 5-month post-exposure period. Minimal mucosal hypertrophy and hyperplasia in bronchi and bronchioles were evident at 1 month postexposure in the two highest concentration groups. Focal cellular hypertrophy and hyperplasia were associated with aggregates of pigmented macrophages, and were evident at alveoli and alveolar duct bifurcations for the entire 6-month postexposure period in the two higher concentration groups. Pigmented macrophages could also be observed within pulmonary lymphoid tissue throughout this time period.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
LUNG BURDEN ANALYSIS
- four-week exposure to inhaled TiO2 particles produced significant exposure-related accumulations of particles in the lungs of exposed rats.
- animals exposed to 250 mg/m³ TiO2 had lung burdens of 1600 μg/g of fixed lung tissue or 12 mg/lung.
- ratio of mean lung burden/exposure concentration: 51, 54, and 46 µg/mg/m³ (5, 50, 250 mg/m³ dose level, respectively).

BRONCHOALVEOLAR LAVAGE DATA
- four-week exposure to inhaled TiO2 particles produced sustained pulmonary inflammatory responses in animals exposed to 250 mg/m³.
- nearly 100% of alveolar macrophages recovered by lavage from rats exposed to 250 mg/m³ TiO2 contained particles 6 months after the termination of exposures.
- substantial numbers of neutrophils were recovered in rats exposed to TiO2 at 250 mg/m³ dose level and this response was not significantly diminished until 6 months postexposure.
- rats exposed to 50 mg/m³ TiO2 demonstrated small but sustained inflammatory responses which reached control levels by 3 months postexposure.
- LDH and protein values in BAL fluids recovered from TiO2-exposed rats were elevated only in the 250 mg/m³ dose group.

PULMONARY CELL PROLIFERATION
- rats exposed to 250 mg/m³ TiO2 demonstrated substantial increases compared to controls in pulmonary cell proliferation indices measured on lung parenchymal and terminal bronchiolar surfaces and many of these effects were sustained through 3-6 months postexposure.
Details on results:
BRONCHOALVEOLAR LAVAGE DATA
- no significant increases relative to controls in BAL alkaline phosphatase and N-acetylglucosaminidase values were measured for any exposure groups.

LUNG CLEARANCE
- following exposures to 250 mg/m³, clearance of TiO2 particles was evident during the time interval of 1 week through 1 month postexposure but appeared to be substantially diminished thereafter. A plot of the clearance data shows a typically rapid initial clearance followed by a slower phase. The amount measured in the lungs of exposed rats at the end of the 4-week exposure period was proportional to the exposure concentration for the three exposure concentrations. When fitted to a monoexponential clearance model (Morrow, 1977)*, the deposited TiO2 was found to clear with half-times of approximately 68, 110, and 330 days for the 5, 50, and 250 mg/m³ groups, respectively.

PARTICLE TRANSLOCATION TO TRACHEOBRONCHIAL LYMPH NODES
- evaluation of lymph node burdens of rats exposed to 250 mg/m³ TiO2 demonstrated that substantial amounts of TiO2 particles had translocated to tracheobronchial lymph nodes, particularly during the 1- to 3- and 3- to 6-month intervals.
- no appreciable translocation of TiO2 particles to lymph nodes at the other two concentrations.

ALVEOLAR MACROPHAGE FUNCTIONAL RESPONSES
- in vitro phagocytosis study demonstrated that alveolar macrophages exposed to 250 mg/m³ TiO2 were impaired in their phagocytic responses relative to controls and this effect was sustained through 1 month postexposure.
- Chemotaxis study: alveolar macrophages exposed to 250 mg/m³ TiO2 particles were impaired in their chemotactic responses to zymosan-activated sera when compared to controls. This effect was measured immediately after exposure, as well as 1 week and 3 months postexposure in TiO2-exposed animals.

*Reference:
- Morrow, P. E. ( 1977). Clearance kinetics of inhaled particles. In Respiratory Defense Mechanisms. Il. Lung Biology in Health and Disease (J. D.
Brain, Ed.), p. 493. Dekker, New York.

Effect levels

Remarks on result:
other:
Remarks:
4-week inhalation exposures to high dust concentrations produced persistent pulmonary effects, many lasting throughout a 6-month post-exposure period. These included pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance mechanisms, deficits in macrophage function, and morphological evidence of macrophage aggregation.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
4-week inhalation exposures to high dust concentrations produced persistent pulmonary effects, many lasting throughout a 6-month post-exposure period. These included pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance mechanisms, deficits in macrophage function, and morphological evidence of macrophage aggregation.

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