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Toxicological information

Basic toxicokinetics

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Administrative data

basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Literature published 1989
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride
P. Kopf-Maier and R. Martin
Bibliographic source:
Virchows Archiv B Cell Pathol (1989) 57:213-222

Materials and methods

Objective of study:
Test guideline
no guideline followed
Principles of method if other than guideline:
In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED=effective dose) and a toxic (LD25 ; LD=lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. Further details can be found in the methods below,
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
dicyclopenta-1,3-dien-1-yltitanium(2+) dichloride
Test material form:
solid: crystalline
Specific details on test material used for the study:
Substance: Bis(cyclopentadienyl)titanium(IV)dichloride (C5H5)2TiC12, so-called titanocene dichloride.

Test animals


Administration / exposure

Route of administration:
Details on exposure:
Doses of 60 and 80 mg/kg, dissolved in 0.5 ml of a DMSO/saline mixture (1/9, v/v) which corresponding to effective (ED100) and toxic (LD25) doses were applied intraperitoneally.
Duration and frequency of treatment / exposure:
single dose
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw (total dose)
0.5 ml volume
Dose / conc.:
80 mg/kg bw (total dose)
0.5 ml volume
No. of animals per sex per dose / concentration:
six females per dose group and three females in the solvent control group
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
Not applicable.
Details on study design:
Three animals from each group were sacrificed at 24 and 48 h, respectively. Another three animals, which received only 0.5 ml of the DMSO/saline mixture without drug addition, represented a control group and were killed 48 h after injection.

Preparation for electron microscopy.
The animals were anesthetized with Ketanest (Parke Davis Company, Munich) and perfused for 30 min with 3% glutaraldehyde and 3% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) through the left ventricle of the heart and the aorta. The livers were removed, cut into small pieces, immersed in the fixative solution for 12-24 h, dehydrated and embedded in Epon. Very thin sections (about 30 nm) were prepared and mounted on uncoated 700-mesh grids. All specimens remained totally unstained by osmium or other heavy metals.

Electron microscopy.
The microanalytical investigations were performed using a transmission electron microscope (EM 902; Zeiss, Oberkochen), fitted with an imaging electron energy loss analyzer. Details of the technique of electron spectroscopic imaging using such a filter have been described previously (Ottensmeyer and Andrew 1980; Ottensmeyer 1982, 1984, 1986).
In the present study, the spatial distribution of certain elements, especially titanium (Ti), phosphorus (P) and oxygen (O), was analyzed in the cells constituting the liver tissue. For this purpose, micrographs were taken with an energy loss just greater than the particular absorption edge. These images carry information of the two-dimensional distribution of the particular element as welI as the background i.e. the general nonspecific elemental composition of the specimen. Thus, a reference image was taken at an energy loss just below the particular absorption edge, the difference between the two images representing the distribution of the element under investigation. As the L2.3 edges of P and Ti amount to 132 and 455 eV (electron volt), respectively, the micrographs were taken at 110 ± 10 and 160 ±1 eV for analyzing phosphorus and at 410 ± 10 and 465 ± 10 eV for titanium analysis. The image just above and the image below the particular absorption edge were then aligned to a computer, normalized to equal background densities and subtracted. By this process, elemental net distributions were calculated. The original images as well as the net elemental distributions were then displayed in black and white on a video display system and photographed by a Contax camera, whereby both original images were always taken with equal exposure time.
In order to gain electron energy loss spectra, areas of 5 nm in diameter were analyzed at a constant magnification of 30,000 in the energy loss range of 100-600 eV and spectra were recorded by use of a plotter. By this method qualitative information about the chemical composition of the object within the area investigated was obtained. Based on the determination of the areas under the diverse discontinuities (edges) of the curve at characteristic, element-specific energies, a semiquantitative graduation of the elemental concentration could be drawn. The edge energies of the elements found in most spectra, amount to the following values: P, 132 eV; C, 283 eV; Ca, 346 eC; N, 402 eV; Ti, 455 eV; O, 532 eV.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
One day after application of titanocene dichloride, titanium was enriched in large and numerous cytoplasmic inclusion bodies in cells lining hepatic sinusoids,
i.e. Kupffer cells and, to a minor extent, endothelial cells. Moreover, small spots, containing sparse amounts of titanium in association with phosphorus and oxygen, were detected in the nuclear euchromatin and the nucleolus of endothelial cells. On analyzing the titanium distribution within liver cells at 24 h after application, titanium was found in low concentration within nuclear particles. These particles were either located within the nucleolus or the euchromatin. They were characterized by different size and included, in addition to titanium, phosphorus, carbon, nitrogen, and oxygen in high concentrations. In only a few liver cells, small cytoplasmic inclusions were observed where titanium was loosely scattered.
Analyzing the specimens 48 h after application of titanocene dichloride, titanium was again incorporated into large cytoplasmic inclusion bodies in endothelial and Kupffer cells, in which titanium was always associated with accumulations of phosphorus, carbon, nitrogen and oxygen. Moreover, titanium was also found in numerous cytoplasmic inclusions in macrophages, which were scattered among the hepatocytes of the liver parenchyma.
At this time titanium was only rarely detectable within hepatocyte nucleoli. However, in many cells, titanium-containing particles of different size were either distributed within the euchromatin or associated in a perinucleolar position with cellular nucleoli. Compared with titanium containing cytoplasmic inclusion bodies in endothelial cells or macrophages, these particles contained only small quantities of titanium, but high concentrations of phosphorus, nitrogen and oxygen. More often, titanium was found enriched in cytoplasmic inclusions in liver cells where it was found highly concentrated and associated with phosphorus, nitrogen and oxygen, the quantitative distribution of the diverse elements being similar to the distribution found in the inclusion bodies in endothelial cells and macrophages. Titanium-containing cytoplasmic inclusions assembled in peripheral zones of the cytoplasm of liver cells near bile canaliculi, in a similar fashion to lysomes termed peribiliary dense bodies. Occasionally, titanium-containing material was found in the lumen of bile canaliculi, again associated with carbon and oxygen, but with only small amounts of phosphorus and nitrogen.In the livers of control animals, no titanium signal could be found either in the cytoplasm or the nuclei of Kupffer cells, endothelial cells or liver cells. The lysosomes found in hepatocytes and in Kupffer cells contained phosphorus, nitrogen and oxygen, but no titanium was detectable.
Transfer into organs
Transfer type:
other: transfer into the liver
distinct transfer
Details on excretion:
48 hours after treatment cytoplasmic organelles were the main sites where titanium was found within liver cells. The frequent occurrence of these inclusion bodies near bile canaliculi and the observation of titanium-containing material within the lumen of bile capillaries strongly suggest that titanium-containing metabolites to be eliminated via the bile.

Metabolite characterisation studies

Metabolites identified:
not measured

Applicant's summary and conclusion

Executive summary:

Summary. In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED=effective dose) and a toxic (LD25 ; LD=lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes.

Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an

appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.