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Administrative data

carcinogenicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented. Data is suitable for read-across.

Data source

Referenceopen allclose all

Reference Type:
Inhalation Toxicity Study on Rats Exposed to Titanium Tetrachloride Atmospheric Hydrolysis Products for Two Years
Lee KP, Kelly DP, Schneider PW, Trochimowicz HJ
Bibliographic source:
Toxicology and Applied Pharmacology 83: 30-45
Reference Type:
other company data
Reference Type:
secondary source
Toxicological profile for titanium tetrachloride.
Bibliographic source:
Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.

Materials and methods

Principles of method if other than guideline:
Rats were exposed to the test substance hydrolysis products by inhalation exposure at aerosol concentrations of 0, 0.1, 1.0 and 10 mg/m3 for 6 h/day, 5 days/week for 2 years.
GLP compliance:
not specified

Test material

Details on test material:
- Name of test material (as cited in study report): Titanium tetrachloride, TiCl4
- Physical state: liquid
- Analytical purity: No data
- Stability under test conditions: The test substance is known to hydrolyze rapidly in air. No unhydrolyzed TiCl4 was found in the exposure chamber.

Test animals

other: Crl:CD
Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratories, Wilmington, Mass
- Weight at study initiation: 240 +/- 10 g
- Housing: individually in stainless-steel, wire-bottomed cages
- Diet: ad libitum, Purina rodent Chow
- Water: ad libitum

- Temperature (°C): 23+-2 °C
- Humidity (%): 50+-10 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
other: air
Details on exposure:
Inhalation Chambers
Special chambers were constructed of Hastelloy, a high-nickel stainless-steel which was found to withstand the corrosive effects of TiCl4 vapor. The chambers were of conventional shape and had a volume of 4.0 m³, with a special vertical-opening, hydraulically operated door.

Atmosphere Generation and Analysis
TiCI4 vapors were generated by passing nitrogen over liquid TiCl4 in a glass vessel placed in a 20°C constant-temperature bath. TiCI4 vapors were mixed with the 1000 liters/min chamber air supply at the chamber top. TiCl4 hydrolysis occurs so rapidly in air that a second nitrogen stream was required to shield the vapor delivery tube to avoid build up of solid hydrolysis products on the delivery tube tip. Chamber atmospheres were monitored by trapping the solid TiCl4 hydrolysis products on cellulose acetate filters which were then analyzed for titanium content by a calorimetric method. Chamber concentrations were reported as mg/m3 of TiCI4 as calculated from the titanium concentration. The aerodynamic diameter (AD) of the chamber particulate was determined twice at the 10 mg/m³ concentration with a Brink cascade impactor. Virtually all the particles were less than 1.6 µm AD, and the mass median diameter was about 0.5 µm with geometric standard deviation of about 2. There was essentially no unhydrolyzed TiCI4 in the chamber atmosphere. This was determined by drawing chamber air through a filter in tandem with a water-filled impinger. If unhydrolyzed TiCI4 vapors were present, they would have passed through the filter and titanium would have been found in the impinger water.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Chamber atmospheres were monitored by trapping the solid TiCl4 hydrolysis products on cellulose acetate filters which were then analyzed for titanium content by a calorimetric method. Chamber concentrations were reported as mg/m³ of TiCI4 as calculated from the titanium concentration.
The aerodynamic diameter (AD) of the chamber particulate was determined twice at the 10 mg/m³ concentration with a Brink cascade impactor. Virtually all the particles were less than 1.6 µm AD, and the mass median diameter was about 0.5 µm with geometric standard deviation of about 2.
Duration of treatment / exposure:
24 months (5 males and 5 females from each group were killed after 3 and 6 months of exposure and, subsequently, 10 males and 10 females were killed after 1 year of exposure. All surviving rats were killed at the end of the 2-year exposure.)
Frequency of treatment:
6 hours/day, 5 days/week
Post exposure period:
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.1, 1.0 and 10 mg/m³
nominal conc.
Doses / Concentrations:
0.1, 1, 10 µg/l

No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle


Observations and examinations performed and frequency:
Sacrifice and pathology:
All rats killed by design, found dead, or killed in extremis were subjected to gross and microscopic evaluation. All surviving rats were killed at the end of the 2-year exposure and their tissues were examined grossly and microscopically. Particular attention was given to lung.
Lung tissue was fixed with Bouin’s solution by intratracheal instillation under low pressure. The trachea, thyroid, adrenal glands, testes, and kidneys were fixed in Bouin’s solution; all other organs and tissues were fixed in a 10% formalin solution for light microscopic examination. Paraffin sections were prepared according to routine histologic techniques. Sections were stained by hematoxylin and eosin, modified trichrome, silver impregnation, and the periodic acid-Schiff (PAS) method. For transmission electron microscopy, the excised lung samples were fixed in 3% glutaraldehyde for approximately 1 hr. Then they were rinsed in Millonig’s phosphate buffer, postfixed for 2 hr at 4°C in 1% osmium tetroxide, dehydrated in alcohol, and embedded in Epon. One-micrometer sections were stained with toluidine blue and used to locate areas for electron microscopy. Elementary analysis of the particulates in the thin sections was performed with a Hitachi H-600 microscope attached to an energy-dispersive X-ray microanalyzer. For scanning electron microscopy (SEM), lung samples were fixed overnight in 3% glutaraldehyde, rinsed in Millonig’s phosphate buffer, and then postfixed in 1% osmic acid for 2 hr. The tissue samples were then dehydrated in graded acetone solutions and dried in a critical point dryer. Finally, the samples were mounted on aluminum stubs, placed on a rotary device in a vacuum evaporator, and coated evenly with approximately 20 pm of gold-palladium. The EDAX was conducted to identify the elemental composition of dust particles by SEM fitted with a Princeton Gamma Tech PGT-1000 EDS system.

Results and discussion

Results of examinations

Details on results:
None of the exposed rats showed any evidence of exposure-related abnormal clinical signs, body weight changes, morbidity, or mortality throughout 2-year exposure when compared to the control groups. The mortality rate was approximately 60 % for females and 40 % for males.

The lung weights of rats at 10 mg/m³ were increased significantly in comparison to those of the control groups, but the lung weights of the other exposed groups were within normal limits.

Compound-related lesions were found only in the respiratory organs and the thoracic lymph nodes.
No abnormality was detected in the lungs of rats exposed to 0.1 mg/m³. At 1.0 mg/m³, tiny yellow foci (ca. 5 mm) were so sparsely scattered in the pleural surface that they were difficult to detect. At 10 mg/m³, yellow foci were increased markedly in number and size (< 1 mm), and were distributed throughout all lobes. Tracheobronchial lymph nodes were enlarged and mottled with tiny yellow foci in rats exposed to 1.0 and 10 mg/m³, but not at 0.1 mg/m³ exposure.

A wide variety of spontaneous neoplastic and non-neoplastic lesions, which were mostly age related, occurred with similar incidence and severity in both control and exposed rats.
The incidence of rhinitis was increased in all groups of exposed rats, but not in a dose-related fashion. Acute or chronic rhinitis occurred in both the anterior and posterior nasal cavity. Some nasoturbinates, the ventral nasal septum, and the ethmoturbinates showed glandular hyperplasia with chronic inflammation. An increased incidence of tracheitis was also observed at 1.0 and 10 mg/m³, but not at 0.1 mg/m³ exposure. The tracheitis was mostly chronic inflammation with lymphoid tissue hyperplasia.
At 0.1 mg/m³, the lungs maintained their normal general architecture. Some alveoli adjacent to the alveolar ducts contained a single or a few aggregated dust-laden macrophages (dust cells). Dust particles were less than 1 µm in diameter and aggregated particles in the dust cells appeared as dark brown granules. The alveoli containing dust cells showed a normal structure. At 1.0 mg/m³, alveoli adjoining the alveolar ducts were filled with aggregated dust cells. The alveolar walls enclosing dust cell aggregates showed only a slight hyperplasia of Type II pneumocytes.
In rats exposed to 10 mg/m³, there was a marked increase in dust cell aggregates in the alveoli adjacent to the alveolar ducts, when compared to those seen at 1.0 mg/m³. The alveoli containing dust cell aggregates showed hyperplasia of Type II pneumocytes but no collagenized fibrosis. Some alveoli adjacent to the terminal bronchioles were lined with ciliated columnar cells (bronchiolarization) contiguous with bronchiolar epithelium. Some alveolar ducts showing bronchiolarization were dilated and contained a mucus secretion. Alveoli were filled with two different types of alveolar macrophages. One was a dust-laden macrophage filled with densely aggregated particles. The other was a foamy alveolar macrophage that contained only a small amount of dust particles (foamy dust cells). Many foamy dust cells showed degenerative changes and were disintegrated in the alveolar air spaces releasing dust particles and granular or fibrinous cellular debris.
Cholesterol crystal clefts were formed within the proteinaceous material or foamy alveolar macrophages. Subsequently, cholesterol granulomas were developed with accumulation of proteinaceous material, foamy dust cells, and cellular debris from disintegrated foamy dust cells. At 10 mg/m³, the incidence of cholesterol granulomas was increased significantly (13 of 69 males, 19 of 74 females) in comparison to those seen at 1.0 mg/m³ (4 of 77 males, 2 of 78 females) or control rats (7 of 79 males, 2 of 77 females). Some alveoli were filled with PAS-positive granular or amorphous proteinaceous material, and microscopic findings were compatible with alveolar proteinosis. The incidence of alveolar proteinosis was only 1 of 69 males and 1 of 74 female rats at 10 mg/m³. The lesion was minute and sharply confined to the alveolar duct region where dust cells were densely aggregated with proteinaceous material. Minute focal pleurisy was developed by an extension of the inflammatory reaction from subpleural cholesterol granulomas and occurred in 9 of 69 males and 8 of 74 females at 10 mg/m³ compared to 4 of 79 males and 2 of 77 females in the control groups. The incidence of bronchiolalveolar adenomas in exposed groups was comparable to that of control groups. However, well-differentiated squamous cell carcinomas were found in 2 of 69 males and 3 of 74 females at 10 mg/m³, but not in the control or the other exposed groups. The carcinomas were developed from the area of the alveoli showing bronchiolarization and squamous metaplasia or dysplasia. Of the five cases of squamous cell carcinoma, two cases were keratinized cystic squamous cell carcinomas. The other three tumors were microscopic-sized, well-differentiated squamous cell carcinomas. There was no tumor metastasis to the regional tracheobronchial lymph nodes or other vital organs in any of the five rats with lung tumors. The keratinized cystic squamous cell carcinomas were characterized by a dermoid cyst-like appearance and the cystic spaces were densely packed with laminated keratin. The cystic walls consisted of well-differentiated squamous cells with various-sized keratin pearls. The squamous cells in the cystic walls showed only a minute microscopic invasion to adjoining alveoli.
The tracheobronchial or mediastinal lymph nodes showed dose-related particle deposition at 1.0 and 10 mg/m³, but not at 0.1 mg/m³. Aggregated dust cells were found in the paracortical and medullary sinusoids, but there was no tissue reaction to accumulated dust particles. The peribronchial or perivascular lymphoid tissue was loaded with dust particles and the peribronchial lymphatics occasionally showed a few migrating dust-laden macrophages. Extrapulmonary particle deposition was also found in the liver in 1 of 74 males and in the spleen in 2 of 74 females at 10 mg/m³. The dust in the liver was located in Kupffer’s cells and in dust-laden macrophages in the portal triads. The spleen revealed particle deposition in the one lymphoid tissue of the white pulp, while the red pulp exhibited minute dust particle deposition. There was no tissue reaction to the accumulated dust particles in the liver or spleen.

Under scanning electron microscopy, the mucosa of the trachea revealed mucus droplets and macrophages containing particles, but no structural alterations were observed in the goblet cells, or in ciliated or microvillous cells in any exposed groups. At 0.1 mg/m³, a few alveoli contained macrophages, but alveolar walls showed normal structure. At 1.0 and 10 mg/m³, there was a dose-related macrophage accumulation in the alveoli associated with minute Type II pneumocyte hyperplasia. In the 1.0 or 10 mg/m³, under transmission electron microscopy, the alveolar walls enclosing dust cells had Type II pneumocytes hyperplasia with increased myelin figures. Type I pneumocytes showed dose-related, fine particulate accumulation in the cytoplasm, but no alterations were found in the cellular organelles. Mature macrophages actively phagocytizing particles (dust cells) were easily identified by long curled cytoplasmic processes, numerous lysosomes, lamellar inclusions, lipid droplets, and numerous phagosomes containing particulates. The particulates had an extremely electron-dense, round configuration measuring approximately 0.2 to 0.7 µm in diameter. Strikingly, there were numerous sickle-shaped phagosomes (approximately 0.3 X 3.0 µm in size) densely ftlled with electron-dense granules in the macrophages.
The foamy dust cells under light microscopy were easily identified by a cytoplasm packed with numerous myelin inclusions, scanty lysosomes, and phagosomes containing a few dust particles. Densely aggregated dust cells in the alveolar air spaces were overloaded with numerous phagosomes, myelin inclusions, and dust particles. The dust cells were fused together by tightly interdigitated cytoplasmic processes. Immature macrophages were increased in number and they were characterized by scanty lysosomes, phagosomes, and short or blunt cytoplasmic processes. Alveoli filled with granular or fibrinous material and disintegrated foamy cells under light microscopy showed an accumulation of myelin or lamellar inclusions in addition to free particles, lysosomes, sickle-shaped phagosomes, and other cellular organelles from disintegrated foamy dust cells. A few dustladen macrophages infiltrated the thickened alveolar septa which also showed proliferating fibrocytes and collagen fiber deposition.

Particles and electron-dense granules in the phagosomes had similar X-ray dispersion patterns when analyzed with EDAX, which were similar to those of particles in the exposure chamber. There were neither degenerative nor necrotic changes in the lymphocytes and reticuloendothelial cells adjacent to dust cell aggregates in the tracheobronchial lymph nodes. The morphology of foamy dust cells in the lymph nodes was similar to that of intraalveolar foamy dust cells.

In a written communication, DuPont (1994) stated that the lung lesions observed in rats by Lee et al. (1986) were reexamined and the conclusion was that three of the lesions should be diagnosed as squamous metaplasia and the other two as proliferative keratin cysts. This reevaluation was consistent with the opinion of an international panel of 13 pathologists who examined similar lung lesions in rats caused by exposure to titanium dioxide. The panel agreed that the lesions were not malignant neoplasms; a majority of the members agreed that the lesions were not neoplastic.

Applicant's summary and conclusion