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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Literature published 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
distribution
Qualifier:
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:
no
Specific details on test material used for the study:
Substance: Bis(cyclopentadienyl)titanium(IV)dichloride (C5H5)2TiC12, so-called titanocene dichloride.
Radiolabelling:
no
Species:
mouse
Strain:
CF-1
Sex:
female
Route of administration:
intraperitoneal
Vehicle:
DMSO
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
Dose / conc.:
60 mg/kg bw (total dose)
Remarks:
0.5 ml volume
Dose / conc.:
80 mg/kg bw (total dose)
Remarks:
0.5 ml volume
No. of animals per sex per dose:
six females per dose group and three females in the solvent control group
Control animals:
yes, concurrent vehicle
Positive control:
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.
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 type:
other: transfer into the liver
Observation:
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.
Metabolites identified:
not measured
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.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Literature published 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
other: transplacental passage
Qualifier:
no guideline followed
Principles of method if other than guideline:
The passage of titanium-containing metabolites across the placenta into the embryonal compartment was investigated by analyzing the titanium (Ti) content in embryos/fetuses at various intervals between 1 h and 24 h after treatment of pregnant mice with single doses of the antitumor agent titanocene dichloride (CsHs)2TiC12 (60 mg/kg) on days 10, 12, 14 or 16 of gestation.
See methods below for further information.
GLP compliance:
no
Specific details on test material used for the study:
Bis(n5-cyclopentadienyl)dichlorotitanium(IV) (titanocene dichloride, (CsH6)2TiCI z) was prepared and purified. Elemental analyses (C, H, CI) gave deviations ~0.5% of the calculated values. The 1H-NMR, IR and mass spectra showed no evidence of impurities. For application, (CsHs)2TiCI 2 was dissolved in a DMSO-saline (1:9 = v:v) mixture in such a manner that each animal received a total volume of 0.3-0.6 ml (0.01 ml/g body wt.).
Radiolabelling:
no
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Mice were used to test the hypothesis that titanocene dichloride might be unable to cross the placental barrier and enter the embryonal compartment. This study was carried out in response to a previously published study in mice which found 10-50% fetuses had cleft palate when the mothers were treated with titanocene dichloride.
Sex:
female
Details on test animals and environmental conditions:
Female NMRI mice were housed under standardized conditions (tap water and Altromin food ad libitum, room temperature 22-23°C, 12-h light/dark rhythm).
Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
The mice received a single intraperitoneal injection of 60 mg titanocene dichloride/kg on days 10, 12, 14 or 16 of pregnancy (day 0 = day of fertilisation). On day 10, the treated group consisted of 72 animals, on day 12 of 24 animals, and on days 14 and 16 of 12 animals. Another 36 mice were used as control animals, 24 of them received 0.3-0.6 ml of the DMSO-saline mixture (1:9 = v/v) on day 12, the remaining 12 control animals obtained the DMSO-saline injection on day 16.
Duration and frequency of treatment / exposure:
single injection
Dose / conc.:
60 mg/kg bw (total dose)
No. of animals per sex per dose:
72 females were treated on day 10 of pregnancy
24 females were treated on day 12 of pregnancy
12 females were treated on days 14 and 16 of pregnancy
36 females were used in the control group

Control animals:
yes, concurrent vehicle
Positive control:
Not appplicable
Details on study design:
At intervals of 1, 4, 8 and 24 h after application of titanocene dichloride or the mere DMSO-saline mixture, the embryos/fetuses of 18 (day 10), 6 (day 12) or 3 dams (days 14 and 16) were removed and washed 5 times with saline.
The embryos of 6 animals treated on day 10 and of 2 animals treated on day 12 were pooled, whereas, on days 14 and 16, the embryos of one mother
animal always represented one sample. Thus, 3 samples were analyzed at
each time point in each group.
For comparison purposes, blood and liver samples of 6 mother animals treated on day 12 and 6 dams treated on day 16 were taken and analyzed for titanium (Ti) content at 8 (3 animals) and 24 h (3 animals) after application of titanocene dichloride.

Atomic absorption spectroscopy
The analyses were performed in the Analytische Laboratorien Malissa-Reuter, Engelskirchen (F.R.G.) by use of a flameless atomic absorption spectrometer (Perkin-Elmer Model 2380) at X = 363.5 nm. The recovery of Ti following the addition of known quantities of a Ti standard to tissue before evaporation to dryness and digestion with nitric and perchloric acids was 95% in the range of 20-200 mg Ti/kg. The detection limit for Ti was found to be 0.5 tag Ti/kg. The applied method determined total Ti concentrations.
No attempts were made to analyze the biological or chemical stage of titanium.
Details on dosing and sampling:
Histology
The embryos of another 16 pregnant mice were analyzed histologically.
For this purpose, on days 10, 12, 14 or 16, respectively, 4 pregnant animals obtained a single intraperitoneal injection of 60 mg titanocene dichloride/kg dissolved in a DMSO-saline mixture. At intervals of 24 h and 48 h after treatment, the embryos/fetuses of every 2 mother animals were removed by caesarian section. One half of them was fixed in Bouin's solution, dehydrated and embedded in paraplast. Sections 3--5 tam thick were cut and stained with hematoxylin and eosin. The others were fixed in a solution containing 3% glutaraldehyde and 3% paraformaldehyde in cacodylate buffer (pH 7.2), dehydrated and embedded in Mikropal (Ferak, Berlin). Then, 1µm thick sections were cut, mounted and stained with azur according to Richardson.
The embryos of 8 control animals having received only 0.3--0.6 ml of the DMSO-saline mixture on days 10, 12, 14 or 16, respectively, were removed 24 h after injection and prepared in a similar manner.
Details on distribution in tissues:
The amounts of Ti which were registered in control embryos always ranged between 0.4 and 0.9 mg/kg. Similar values were detected in those embryos, the mothers of which had been treated on days 10, 12 or 14 of gestation. Only after application of titanocene dichloride on day 16, the Ti concentrations found in the fetuses clearly exceeded control values, whereby the Ti content in the embryonal compartment steeply rose within 1--8 h after substance application reaching
values 4-fold higher than control concentrations at 8 h. Within the following hours, the Ti content in the fetuses declined and fell to double control values at 24 h.

Analyzing the Ti content in the maternal blood and liver at 8 h and 24 h after application of titanocene dichloride on days 12 and 16 of gestation, clearly shows that higher values were detected in the maternal tissues than in fetuses 8 h and 24 h after treatment on day 16. The maternal blood levels exceeded the fetal concentrations by factors of 3--6, the liver content even by 18--50. These results confirm that only on day 16 of murine pregnancy a limited transplacental transfer of Ti-containing metabolites took place, but that the Ti amounts reaching the embryonal compartment were always much smaller than the Ti concentrations found in maternal tissues.
The histologic analysis of the developing neocortex in mouse embryos/fetuses after treatment of mother animals with titanocene dichloride on days 10, 12, 14 or 16 of pregnancy revealed no signs of any cellular damage at 24 h or 48 h after substance application. Neither the light-microscopic appearance of neuroepithelial cells was altered nor the count of mitoses diminished nor the number of necrotic cells increased in comparison to untreated animals at the same stage of pregnancy.
Transfer type:
blood/placenta barrier
Observation:
distinct transfer
Remarks:
Only after application of titanocene dichloride on day 16, the Ti concentrations found in the fetuses clearly exceeded control values.
Metabolites identified:
not measured
Conclusions:
The results of the present study show that neither titanocene dichloride nor titanium-containing metabolites are able to traverse the placental barrier through the sensitive phase of organogenesis until day 16 of murine pregnancy. Only on day 16, when the morphogenesis of most organs is widely terminated, small amounts of titanium enter the fetal compartment.

Executive summary:

The passage of titanium-containing metabolites across the placenta into the embryonal compartment was investigated by analyzing the titanium (Ti) content in embryos/fetuses at various intervals between 1 h and 24 h after treatment of pregnant mice with single doses of the antitumor agent titanocene dichloride (CsHs)2TiC12 (60 mg/kg) on days 10, 12, 14 or 16 of gestation. The Ti concentration was determined using flameless atomic absorption spectroscopy. After treatment on days 10, 12 or 14, the Ti concentrations were not elevated in comparison to untreated embryos. Only on day 16, i.e. beyond the end of organogenesis, small amounts of Ti were detectable in the fetal compartment 4 -24 h after substance application, exceeding the control values by factors ranging between 2 and 3. These results explain the absence of histologic lesions in developing embryonal organs and the lack of multiple teratogenic effects in new-borns after application of therapeutic doses of (CsHs)2TiCI 2 to pregnant mice during the embryonal organogenesis.

The results of the present study show that neither titanocene dichloride nor titanium-containing metabolites are able to traverse the placental barrier through the sensitive phase of organogenesis until day 16 of murine pregnancy. Only on day 16, when the morphogenesis of most organs is widely terminated, small amounts of titanium enter the fetal compartment.

These findings explain: (i) the absence of histologic lesions in the developing embryonal and fetal tissues, especially the neocortex which proliferates beyond the end of organogenesis; and (if) the lack of gross and multiple malformations in new-borns after treatment of pregnant mice with the anti-proliferative agent titanocene dichloride which is known to exhibit pronounced cytostatic properties against experimental and human tumors. Interestingly, an analogous behavior was shown recently at the murine blood-brain barrier. Here, titanium-containing metabolites were again uncapable to traverse the barrier and to enter the brain compartment in mentionable amounts.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Published in 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
distribution
Qualifier:
no guideline followed
Principles of method if other than guideline:
The pharmacokinetics and organ distribution of titanium (Ti) were analyzed at various intervals up to 96 h after a single i.p. injection of a therapeutic dose of the antitumor agent titanocene dichloride (TDC, 60 mg/kg) by use of flameless atomic absorption spectroscopy in dried organ specimens.

The time-dependent organ distribution of Ti was followed after a single application of titanocene dichloride to mice. These investigations were done to clarify if the accumulation of titanocene dichloride and its metabolites in the liver may be a factor leading to the previously observed hepatic toxicity. This study also questions whether titanium containing metabolites are able to pass the blood-brain barrier and to accumulate in tumor tissue.
GLP compliance:
no
Specific details on test material used for the study:
Bis(n6-yclopentadienyl)dichlorotitanium(IV) (titanocene dichloride, TDC) was prepared and purified. No impurities were detectable by 1H-NMR, IR and mass spectroscopy. Elemental analysis (C, H, C1) revealed deviations < 0.5% of the calculated values.
Radiolabelling:
no
Species:
mouse
Strain:
NMRI
Remarks:
An additional experiment was carried out using CF1, BDF1 and NMRI-nu/nu mice
Sex:
female
Details on test animals and environmental conditions:
Female NMRI mice, obtained from the Zentralinstitut fiir Versuchstierzucht, Hannover, F.R.G., were housed under standardized conditions (tap water and Altromin food ad libitum, room temperature 22-23°C, 12-h light/dark rhythm).
Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
intraperitoneal injection of 60 mg TDC/kg in a DMSO/saline (1:9, v/v) mixture
Duration and frequency of treatment / exposure:
single treatment
Dose / conc.:
60 mg/kg bw (total dose)
Remarks:
total volume of 0.3 - 0.6 ml
No. of animals per sex per dose:
Main experiment: 60 females treated at 60 mg/kg test item and 18 females treated with solvent control
Additional experiment: 12 female CF1 mice, 3 groups of 12 female BDF 1 mice and 12 male NMRI-nu/nu mice (not specified how many were treated with test item or control)
Control animals:
yes, concurrent vehicle
Positive control:
not applicable
Details on study design:
Five days before TDC application, 107 sarcoma 180 cells were inoculated into the flanks of 78 mice. Of these, 60 animals received a single intraperitoneal injection of 60 mg TDC/kg. The other 18 mice were used as control animals, they received 0.3-0.6 ml of the DMSO/saline mixture (1:9, v/v) without TDC. At intervals of 1, 4, 8, 24, 48, and 96 h after application of TDC, 9 mice of the treated group were sacrificed. The same number of animals of the control group were killed 1 and 24 h after administration of the MSO/saline mixture alone. The blood (gathered from the axillary artery), liver, small intestine, brain, leg muscles and tumor of 3 animals of the treated and control groups were removed and prepared for analysis. Because of low organ weights, the kidneys, lungs and hearts of 3 mice of the 9 animals killed at a given time point were pooled to 1 sample each. At additional intervals of 0.5 and 2 h after TDC application, the blood of 3 treated mice was analyzed for titanium content. Thus, at each time point, samples of at least 3 separate animals were determined. Samples from each mouse were analyzed in triplicate. Mean values and standard deviations were calculated for each time point.

In another experiment, the concentration of titanium in various solid tumors other than sarcoma 180 was investigated. For this purpose, 12 female CF1 mice with solid Ehrlich ascites tumor growing in the nuchal region, 3 groups of 12 female BDF 1 mice each bearing colon 38 carcinoma, B16 melanoma or Lewis lung carcinoma, respectively, and 12 male NMRI-nu/nu mice with a xenografted human lung adenocarcinoma (L261) growing in the flanks of the animals, were treated either with 60 mg TDC/kg, applied by i.p. injection, or the mere DMSO/saline mixture. At 4 and 24 h after treatment, 3 animals from each of the treated and control groups were sacrificed, the tumors were removed and prepared for analysis. Mean values and standard deviations were calculated.

Atomic absorption spectroscopy
After removing the organs and the tumors as described, they were washed 3 times in sterile saline and freeze-dried. The analyses were performed in the Analytische Laboratorien Malissa-Reuter, Engelskirchen, F.R.G., by use of a flameless atomic absorption spectrometer (Perkin-Elmer Model 2380) at A = 363.5 nm. The control of quality was performed by determining the following parameters:
(a) The recovery of Ti following the addition of known quantities of a Ti standard to tissue before digestion with nitric and perchloric acids was 95% in the range of 20-200 mg Ti/kg.
(b) The detection limit for Ti was found to be 0.5 µg Ti/kg, corresponding to an absolute amount of 5 ng Ti/ml.
(c) Repeated analyses of the same samples revealed maximum relative deviations of ± 4%.
Only total Ti concentrations were determined by the method mentioned above. No attempts were made to elucidate the molecular constitution of Ti in the organs investigated. The Ti concentrations found in biological tissues were expressed as mg Ti/kg dry weight.
Details on distribution in tissues:
The titanium (Ti) content in the tissues and tumors of the untreated control mice was always lower than 1.0 mg Ti/kg dry weight and ranged between 0.3 and 1.0 mg Ti/kg.
In the blood, the Ti level rose within 1 h after application of TDC to nearly 20 mg Ti/kg dry weight, fell thereafter to 13 mg Ti/kg at 4 h and slowly to 6.6 mg Ti/kg at 96 h, the latter value still representing about a third of the 1-h value. The initial organ concentration was highest in the kidneys 1 h after intraperitoneal substance application and amounted to 51.0 mg Ti/kg. During the following hours, the renal Ti content declined to 30-40 mg Ti/kg, while the concentrations in the liver and the intestine increased within 24 h after administration of TDC, exceeding the kidney values at 4 h and later. At 24 and 48 h after treatment, Ti concentrations as high as 80--90 mg Ti/kg were found in the liver and the intestine. Considering the relative mean liver weight of mice, about 10% of total titanium injected was accumulated in the liver at 24 and 48 h. At these time-points, the liver/blood and liver/intestine ratios amounted to 8-9. At 96 h, still 6-fold higher Ti concentrations were found in the liver and the intestine than in the blood.
Distinctly lower Ti concentrations were recorded in lung, heart and muscles of mice treated with TDC. In the brain, no Ti concentrations significantly higher than control values were measurable at any time during the experimental period.
Regarding the Ti concentrations in various solid tumors, the growth of which is inhibited by TDC, rising Ti concentrations were found in the tissues of experimental tumors between 1 and 24 h after TDC application. In sarcoma 180, where the disposition of Ti was analyzed over a longer period, the Ti concentrations increased continuously up to 15 mg Ti/kg dry weight at 96 h. Whereas the Ti concentrations in experimental tumors amounted to 3-11 mg Ti/kg at 4 and 24 h, the Ti content in the human lung adenocarcinoma L261 exceeded these values and ranged between 13 and 15 mg Ti/kg.
Transfer type:
blood/brain barrier
Observation:
no transfer detectable
Details on excretion:
The observed enrichment of Ti in the liver and the intestine after application of TDC points to both organs as main sites of excretion for titanocene complexes and their metabolites, whereas elimination via the kidneys seems to be less important.
Conclusions:
The organ distribution of Ti after treatment with (C6Hs)2TiC12 showed an accumulation of Ti in the liver and the intestine lasting for several days, whereas the kidneys contained smaller concentrations of Ti, amounting to about half of the concentrations found in the liver. The observed enrichment of Ti in the liver and the intestine after application of TDC points to both organs as main sites of excretion for titanocene complexes and their metabolites, whereas elimination via the kidneys seems to be less important. No transfer of Ti containing metabolites across the blood-brain barrier into brain tissue was detectable after application of TDC.
Executive summary:

The pharmacokinetics and organ distribution of titanium (Ti) were analyzed at various intervals up to 96 h after a single i.p. injection of a therapeutic dose of the antitumor agent titanocene dichloride (TDC, 60 mg/kg) by use of flameless atomic absorption spectroscopy in dried organ specimens.

Highest organ concentrations were found in the liver and the intestine where 80-90 mg Ti/kg dry weight were accumulated at 24 and 48 h, corresponding to liver/blood and intestine/blood ratios of 8-9. However, at no time point after the TDC application, the Ti concentrations in brain tissue exceeded those of control animals. In solid tumors growing subcutaneously in mice, increasing amounts of Ti were found during the course of the experiment, reaching concentrations between 10 and 15 mg Ti/kg at 24 and 96 h after single i.p. application of TDC. These results confirm a typical pattern of organ distribution of Ti-containing metabolites of TDC, which clearly differs from that observed for vanadium after application of vanadocene dichloride or for platinum after treatment with cisplatin.

Endpoint:
basic toxicokinetics, other
Remarks:
Pharmacokinetics in a Phase I clinical trial in adults with advanced solid tumors
Type of information:
other: Phase I clinical trial
Adequacy of study:
weight of evidence
Study period:
The study was published in 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Objective of study:
other: pharmacokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
This Phase I dose-escalation clinical trial of a lyophilized formulation of titanocene dichloride (MKT4) was conducted to determine the maximum tolerated dose, the doselimiting
toxicity (DLT), and pharmacokinetics of titanium (Ti) after a single i.v. infusion of MKT4.The study was performed according to German drug regulations.
The protocol was approved by the ethical boards of the universities of Berlin, Essen, and Hannover.
GLP compliance:
no
Remarks:
Study was performed according to good clinical practice guidelines
Specific details on test material used for the study:
Substance referred to as MKT4: a lyophilized formulation of titanocene dichloride, was supplied by medac GmbH in 25-mg vials.
Radiolabelling:
no
Species:
other: human
Strain:
other: not applicable
Details on species / strain selection:
Eligible for the study were patients with histological proof of malignancy for which conventional treatment had failed or was not available. Measurable disease was not required but, when present, was evaluated before dosing and every 22 days. Other eligibility criteria were: age between 18 and 75 years; Karnofsky-performance index _70%; life expectancy >3 months; no chemotherapy or radiotherapy within the 4 weeks preceding the study; written informed consent; adequate bone marrow (leukocyte, >3.500/mm3; platelet count, >100.000/mm3); and renal (serum creatinine, <1.5 mg/l00 ml)
and hepatic (serum bilirubin, <1.5 mg/l00 ml; alanine aminotransferase and aspartate aminotransferase less than two times the upper limit of normal values, unless secondary to metastatic liver disease) function. Exclusion criteria were: acute infectious disease, left ventricular hypertrophy, persistent toxicity of prior therapy (except alopecia or actinic dermatitis), and refusal of informed consent. The patients were hospitalized for treatment.
Sex:
male/female
Route of administration:
intravenous
Vehicle:
other: malic acid
Dose / conc.:
15 other: mg/m^2
Dose / conc.:
30 other: mg/m^2
Dose / conc.:
50 other: mg/m^2
Dose / conc.:
75 other: mg/m^2
Dose / conc.:
105 other: mg/m^2
Dose / conc.:
135 other: mg/m^2
Dose / conc.:
180 other: mg/m^2
Dose / conc.:
240 other: mg/m^2
Dose / conc.:
315 other: mg/m^2
Dose / conc.:
420 other: mg/m^2
Dose / conc.:
560 other: mg/m^2
Details on study design:
Study Design.
Baseline values before treatment comprised complete blood cell count with differential and reticulocytes, sodium, potassium, calcium, creatinine, creatinine clearance, urea, uric acid, cholinesterase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, alkaline phosphatase, -y-GT, lactic dehydrogenase, serum glucose, triglycerides, thyroid hormones, and plasmatic coagulation parameters. Complete physical examination and history, chest X-ray, electrocardiogram, and tumor measurements as appropriate were performed in addition.
After infusion, patients had weekly assessments including all laboratory tests mentioned above. Physical examination, evaluation of performance status and drug-related toxicity according to the WHO criteria were assessed before each treatment course. Response evaluation was performed according to the WHO criteria in patients with measurable disease after each treatment cycle.
Details on dosing and sampling:
Drug Administration.
Immediately before infusion, MKT4 was reconstituted in 25 ml of 50 mM malic acid to a final concentration of 1 mg of titanocene dichloride/ml of malic acid (pH = 3.5). After reaching the 420 mg/m2 dose level, the increased infusion volume necessitated an increase of the titanocene dichloride concentration. Three patients then received 75-mg vials. The
content was reconstituted in 37.5 ml of 25 mM malic acid to a final concentration of 2 mg of titanocene dichloride/ml of malic acid (pH = 3.0). The MKT4 infusion was given light-protected via a central iv. line over 30 mm (15-180 mg/m2) or over 60 mm (180-560 mg/m2). Blood pressure, pulse, and temperature were recorded before therapy and 1, 4, 8, and 24 h after therapy. Serum glucose monitoring was performed every 6 h up to 24 h after MKT4 infusion. Subsequent courses were repeated every 21 days. A delay of 1 week was permitted. Individual therapy was continued until there was objective evidence of disease
progression, nephrotoxicity WHO grade 2, other nonhematological toxicity WHO grade 3, hematological toxicity WHO grade 4, or a >20% treatment-related decline of the Karnofsky performance index. Treatment was also discontinued at the discretion of the treating physician or according to the patient’s decision. Antiemetics were only used if there was nausea/vomiting during the previous treatment course.

Dose-Escalation Procedures.
The starting dose of titanocene dichloride was 15 mg/m2. The dose increase was based
on a modified Fibonacci scheme and proceeded as follows: 30,
50, 75, 105, 135, 180, 240, 315, 420, and 560 mg/m2. A minimum of three patients were entered at each dose level. There was no dose escalation in individual patients. MTD was defined as the titanocene dichloride dose leading to DLT in at least two of six patients. DLT was defined as nephrotoxicity grade 2, other nonhematological toxicity grade 3, or hematological toxicity grade 4. The titanocene dichloride dose was only escalated to the next dose level if there was no toxicity or no
more than one case of nephrotoxicity (elevation of serum creatinine)
grade 1, other nonhematological toxicity grade 2, or hematological toxicity grade 3. If one patient had nephrotoxicity grade 2, nonhematological toxicity grade 3, or hematological toxicity grade 4, further evaluation of toxicity at this level was performed by treating three additional patients.
Details on distribution in tissues:
The pharmacokinetics of total Ti were concomitantly determined in plasma and whole blood in six comparative analyses in the first four patients treated with titanocene dichloride doses between 135 and 240 mg/m2. A constant ratio of 1:2 was obtained for AUC0-x in plasma and whole blood in four of six patients, and analysis was then carried out in plasma alone.
The pharmacokinetics of total Ti were analyzed in plasma and urine for 14 cycles in 10 patients. The biological half-life t1/2β in plasma was thus 22.8 ± 11.2 h (harmonic mean xh ± pseudo-SD), and the peak plasma concentration cmax was ~30 µg/ml at a dose of 420 mg/m2. The distribution volume Vss was 5.34 ± 2.1 L (arithmetic mean xa ± SD), and the total clearance Cltotal was 2.58 ± 1.23 ml/min.
There was a good correlation between the concentration time curves of Ti in plasma and the MKT4 dose administered. This was confirmed when the Ti AUC0-∞ in plasma was plotted against the titanocene dichloride dose and resulted in linear regression analysis with a 0.8856 correlation coefficient.
Comparing the AUC0-∞ of total Ti in plasma and ultrafiltrate reveals a plasma protein binding of Ti in the range of 70-80%.
Details on excretion:
The determination of the AUC0-∞ of total Ti in urine showed that between 3% and 16% of the total Ti administered is renally excreted during the first 36 h after administration.

The mean number of courses/patient was two. Thirteen patients received one treatment course; one of these patients (dose level 420 mg/m2) died of unexpected rapid tumor progression 5 days after treatment and, thus, can not be evaluated. Of the other patients, 20 received two treatment courses, 4 received three courses, 2 received four courses, and 1 received five courses.

Thirty-seven patients were treated with a 1 -mg/ml-concentrated solution of titanocene dichloride in malic acid buffer up to the level of 560 mg/m2. The infusion time was extended, and the concentration increased with further dose and infusion volume increases. Subsequently, the next three patients (38-40) were infused at dose level 315 mg/m2 with a concentrated solution of 2 mg/ml titanocene dichloride. To prevent nephrotoxicity, prehydration with I liter of isotonic saline over 2 h and posthydration with 2 liters of isotonic saline over 4 h was administered to these patients.

Thirty-five patients were withdrawn from the study with progressive disease. In three of those patients, renal toxicity was an additional reason for treatment discontinuing. One patient (dose level 30 mg/m2) decided to discontinue the study after five treatment courses at his own request, despite stable disease. One patient treated with a 2-mg/ml concentration of 315 mg/m2 titanocene dichlonide was taken out of the study because of renal toxicity (WHO grade 3). There were three deaths on study. A patient with hepatocellular carcinoma died 20 days after one course at the 1 5 mg/m2 dose level due to gastrointestinal bleeding in conjunction with tumor infiltration into the bowel, as confirmed by autopsy. Another patient suffering from testicular cancer with multiple lung metastases died of respiratory failure 5 days after one treatment course at the 420 mg/m2 dose level. Pulmonary embolism was suspected, but no further diagnostic evaluations were performed in this patient. The third patient who had biliary cancer with multiple liver metastases developed fatal hepatic and renal failure 10 days after an infusion of 420 mg/m2 titanocene dichloride. The early deaths of the first two patients were clearly related to rapid tumor progression. In the third patient, an additional influence of the study drug could not be excluded. Thus, possibly one death was at least partially drug-related due to grade 4 hepatotoxicity.

Determining the clinical pharmacokinetics of titanocene dichlonide was one of the objectives of the present Phase I study. However, up to the 135 mg/m2 level, titanocene dichloride could not be clearly identified in plasma or urine by high-performance liquid chromatography with spectrophotometric detection at 250 nm used according to in vitro investigations of titanocene dichlonide solutions. Thus, it is necessary to develop effective methods for ex vito stabilization in whole blood as well as further sensitization of titanocene dichloride detection. In consequence, we determined total Ti as the method applied in the early pharmacokinetic investigations of cisplatin to determine total platinum in plasma by atomic absorption spectroscopy before development of intact cisplatin detection methods. Thus, only 14 pharmacokinetic investigations in 10 patients treated with a dose range of 135-560 mg/m2 titanocene dichlonide could be performed.

Executive summary:

This Phase I dose-escalation clinical trial of a lyophilized formulation of titanocene dichloride (MKT4) was conducted to determine the maximum tolerated dose, the dose limiting toxicity (DLT), and pharmacokinetics of titanium (Ti) after a single i.v. infusion of MKT4. Forty patients with refractory solid malignancies were treated with a total of 78 courses. Using a modified Fibonacci scheme, 15 mg/m2initial doses of titanocene dichloride were increased in cohorts of three patients up to level 11(560mg/m2) if DLT was not observed. The maximum tolerated dose was 315 mg/m2, and nephrotoxicity was DLT. Two minor responses (bladder carcinoma and non-small cell lung cancer) were observed.

The pharmacokinetics of plasma Ti were assessed in 14 treatment courses by atomic absorption spectroscopy. The ratio for the area under the curve0-∞in plasma and whole blood was 1.2. The following pharmacokinetic parameters were determined for plasma, as calculated in a two-compartment model: biological half-lifet1/2β,in plasma was 22.8 ± 11.2 h (xh± pseudo-SD), peak plasma concentration cmax~30 µg/ml at a dose of 420 mg/m2, distribution volume Vss= 5.34 ± 2.1 L (xa± SD), and a total clearance Cltotal=2.58 ±1.23 mI/min (xa± SD). There was a linear correlation between the area under the curve0-∞of Ti in plasma and the titanocene dichloride dose administered with a correlation coefficientr2of 0.8856. Plasma protein binding of Ti was in the 70-80% range. Between 3% and 16% of the total amount of Ti administered were renally excreted during the first 36 h. The recommended dose for Phase II evaluation is 240 mg/m2given every 3 weeks with i.v. hydration to reduce renal toxicity.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Executive summary:

Tissue samples of heart, liver, lung, and spleen from five male and five female rats given 0, 31,or 125 mg/kg were analyzed for titanium residues. The highest levels of titanium were found in the spleen and liver.

Titanium levels in the heart, liver, and lung ranged from approximately 15 to 39 ppm for males and 15 to 42 ppm for females. However, the titanium levels in the spleen were much higher (100 to 180 ppm for males; 110 to 230 ppm for females) at both 15 months and 2 years.

 

Titanium, determined by plasma-atomic emission spectroscopy,wasfound to be present in other tissues including the heart, liver, lung, and spleen. This procedure measured total titanium and could not distinguish between atomic titanium and the parent compound or metabolites. Bioaccumulation occurred slowly with the maximum titanium concentrations in the organs tested being reached by 15 months.

No further increase was observed at 2 years, indicating that steady state concentrations had been achieved. The spleen accumulated the highest levels of titanium.

This suggests that the parent compound, titanocene dichloride, was toxic at the site of exposure, whereas the titanium-containing metabolite reaching other organs was relatively nontoxic.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Phase I trail in Humans
Reason / purpose:
reference to same study
Objective of study:
other: pharmacokinetics
Qualifier:
no guideline followed
GLP compliance:
no
Remarks:
The study was approved by the local ethics committee and conducted according to the principles of good clinical practice as laid out in the Committee for Proprietary Medical Products (CPMP) guidelines "Good Clinical Practice in the European Community."
Radiolabelling:
no
Species:
other: huiman
Sex:
male/female
Route of administration:
intraperitoneal
Vehicle:
other: buffer solution. The pH of this solution was 3.2
Details on exposure:
Drug Administration
TD was provided in lyophilized form in amber injection bottles that contained 80 mg (this formulation has the name MKT 5). Reconstitution
of the solution was performed just before administration, with the enclosed malate buffer to a concentration of 2 mg of TD in 1 mL of
buffer solution. The pH of this solution was 3.2. TD was administered no more than 1 hour after reconstitution with prehydration and
posthydration in the following way: (1) 500 mL 0.9% saline plus 5 mmol potassium plus 1 mL 50% magnesium sulfate (MgSO4) over 1
hour; (2) 100 mL 10% mannitol over 15 minutes; (3) TD as a 1-hour infusion at a constant rate; (4) 1,000 mL 0.9% saline plus 10 mmol
potassium plus 2 mL 50% MgSO4 over 2 hours. TD was administered via a central venous catheter protected from the light on a weekly
schedule in the outpatient department. Immediately preceding infusion, pulse, blood pressure, respiratory rate, and body temperature were
measured.

Study Design
The initial dose for this weekly schedule was 70 mg/m 2/wk. The planned doses for subsequent dose levels were 80 mg/m2/wk, 105
mg/m 2/wk, 140 mg/m 2/wk, 185 mg/m 2/wk, and 240 mg/m2/wk. It was anticipated that three patients would be treated at each dose level before
a dose escalation if no DLT was seen. A patient needed to receive one course of TD to be assessable for acute toxicity. If the adverse events did
not exceed CTC grade 2 or nephrotoxicity grade 1, the patient could continue treatment at the same dose level until progression of disease.
Treatment was discontinued if CTC grade 3 toxicity or CTC grade 2 nephrotoxicity occurred. If no toxicity occurred or just one patient
showed grade I nephrotoxicity (creatinine) or grade 2 organ toxicity, the next dosage level was entered. If toxicities occurred in two or all three
patients with at least one case of grade 2 nephrotoxicity, grade 3 organ toxicity, or grade 4 hematotoxicity, we would consider DLT to have
been observed. In all other cases, more patients, up to six, would be treated at the same dosage level. If DLTs also developed in this second
group, up to three additional patients were to be enrolled at the previous dosage level. Provided that no more than 33% of patients at the second
highest dose level experienced DLT, this dose is defined as the maximum-tolerated dose (MTD) and recommended for phase II evaluation.

Pharmacokinetic Studies
Cooperative patients who consented underwent blood sampling for pharmacokinetic analysis. Blood samples were taken at 0, 5, 15, 30, 45,
and 60 minutes during the 1-hour infusion, at 5, 10, 15, 30, 45, and 60 minutes during the first hour after the end of the infusion, and, where
possible, at 2, 4, 8, 12, 24, 48, 96, and 139 hours after the end of the infusion. At each sampling time, 10 mL of blood was placed into
heparin tubes and centrifuged at room temperature. One milliliter of the plasma was stored in tubes that contained 0.1 mL of 2-mol/L hydrochloride
for the detection of titanium (this step was completed in 3 to 4 minutes) for specimens in which we attempted to detect TD; for
detection of titanium, nonacidified specimens were used. A 0.5-mL quantity of the plasma was placed into the upper reservoir of a
disposable Ultrafree filtration unit (molecular weight cut-off, 30,000; Centrifree (no. 4104), Amicon, Inc, Beverly, MA) and centrifuged for
10 minutes at room temperature. Urine was also collected for 24 hours after the commencement of the infusion, where possible. All of the
specimens were stored at -180C until analyzed. Titanium was measured using electrothermal-atomization atomic absorption spectrometry at a
wavelength of 364.3 nm on a Varian Spectra AA-400 equipped with deuterium background correction. Analysis was performed directly
following dilution with 0.1% nitric acid (HNO 3) that contained 0.1% each of nonionic surfactant and antifoam reagent; no other modifier was
used. Dilutions were dried up to 120 0C, ashed at 1,3000 C, and atomized at 2,9000 C. The limit of detection with this method is 0.1 pg/mL for
plasma and urine and 0.03 pg/mL for ultrafiltrate. For calibration, identically treated matrix-matched standards were used. Standards were
prepared using inorganic titanium (BDH, Poole, United Kingdom), the analytical behavior of this form of the element having been found to
match that of titanium present in solutions of TD in 0.1% HNO3. Pharmacokinetic analysis was performed as previously described,
using TOPFIT version TOPAS 2.0.0, to one-, two-, or threecompartment open pharmacokinetic models.24 Although data could be
modeled with a three-compartment model, the area under the concentration- time curve from 0 to 8 hours (AUCo.8) of total titanium gave
physically implausible results because of the long half-life (t1/ 2) and the large extrapolation required. Therefore, a noncompartmental analysis
was used for all data sets, which allowed an AUC from 0 to 139 hours to be estimated. The terminal tj/2 was calculated from 20 hours to the last
data point.
Details on excretion:
Analysis was undertaken for total plasma titanium (TPTi) and ultrafiltrable titanium (UFTi). For TPTi, the pharmacokinetics were assessed in eight patients after the first course of
TD. In three patients treated at - 140 mg/m2, UFTi levels of titanium were high enough to be detected and analyzed.
The pharmacokinetic data for TPTi modeled poorly, in part due to the long terminal t% of elimination requiring extensive extrapolation; thus, a noncompartmental analysis was undertaken. There
was a correlation of 0.80 between dose and AUCO-139. There was also a high correlation of 0.97 between peakTPTi levels and TD dose . The median values for calculated parameters for TPTi were volume of distribution at steady-state (Vss), 10 L; clearance, 0.61 mL/min; and terminal tl/2, 165 hours. The percentage of total dose of titanium excreted in the urine in the first 24 hours ranged between 7% and 24.3%. Only five collections were analyzed- too few to predict accurately whether renal clearance relates to nephrotoxicity.
UFTi data was obtained in three patients. UFTi is only a small fraction of TPTi, being 5.2% + 2.5% for three patients at maximum concentration (Cmax). Correspondingly, the AUC values are much smaller than that of TPTi when fitted to a one-compartment model with a mean t% for UFTi of 0.41 ± 0.21 hours.

TD is a promising metallocene anticancer drug. Some clinical characteristics of TD pharmacology are reminiscent of cisplatin. At the chemical level, TD, like cisplatin, can undergo aquation in a pH-dependent manner.25 In close analogy to total plasma titanium (after TD administration), the pharmacokinetics of total plasma platinum (after cisplatin) are described by a triphasic elimination. The first phase of cisplatin elimination occurs with a t1 12 of approximately 30 minutes and is believed to represent intact drug not protein-bound drug. 26,27 The low levels of UFTi are also reminiscent of results with cisplatin. The longer elimination tl/2 of cisplatin of 24 hours is believed to be due to clearance of cisplatin-protein complexes, and it would seem likely that the same explanation holds true for TD. However, we have not demonstrated which plasma components TD binds to, but this is the subject of continuing investigation.
Conclusions:
Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (tl/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a onecompartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% ± 2.5% those of TPTi.
Executive summary:

Purpose: To determine the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of a weekly schedule of titanocene dichloride (TD) and to define the pharmacokinetics of titanium in plasma and urine. Patients and Methods: Twenty patients with a median age of 58 years received 83 courses of TD. TD was given as 1-hour infusion at escalating doses from 70 to 185 mg/m 2/wk. Pharmacokinetic analysis was performed in eight patients for total plasma titanium (TPTi) and in three patients for ultrafiltrable titanium (UFTi). Results: At the fifth dose level (185 mg/m 2/wk), a variety of DLTs were seen in five patients: fatigue in three, bilirubinemia in one, and hypokalemia in two. A

further six patients were treated at 140 mg/m 2; only one had dose-limiting creatinine elevation and this dose was therefore defined as the MTD. No myelosuppres sion or alopecia were observed. One patient with adenocarcinoma of unknown primary had a minor response. Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (tl/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a onecompartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% ± 2.5% those of TPTi. Conclusion: The MTD of TD given on a weekly schedule is 140 mg/m 2, with cumulative, but reversible creatinine and bilirubin elevation being the DLTs.

Description of key information

1. Estimation of absorption kinetics (see attachments)

 

Metallocenes are organometallic complexes with a central metal atom attached to aromatic ligands. Two aromatic ligands attached to the metal are a basic definitional criterion for metallocenes. Titanocene, with group-IV titanium as the metal, has two cyclopentadiene rings and two chlorine molecules attached to the metal(Patty’s Metallocenes 2012).

 

1.1. Oral absorption:

The molecular weight (< 500 (249)) indicates that the substance may easily be absorbed orally. The substance is a red solid (medium particle size and dustiness. The substance does not have ionisable groups, which makes it easier to be absorbed.

However, no determination of the water solubility of the parent test item was possible or relevant due to rapid hydrolysis in contact with water (supported by Toney et al 1985), as indicated by available literature and supported by the extreme solution pH values observed (that saturated aqueous solutions have a pH of 0.8), attributed to the liberation of hydrochloric acid during hydrolysis. 

Analysis of saturated solutions generated using the standard regulatory method resulted in a mean solution concentration equivalent to 103 g/L of solution at 20.0 ± 0.5 °C, monitoring response attributed to soluble hydrolysis products formed on dissolution.The probably hydrolysis products are shown in the section on metabolism below.

 

Also the octanol-water partition coefficient could not be determined for the parent substance due to the fact that the parent substance is hydrolytically unstable. The measured Log P for the hydrolysis products is -1.35 at 20°C. A log P between -1 and 4 favours absorption by passive diffusion, the hydrolysis products fall in this category.

 

In oral toxicity studies up to 2-years signs of local toxicity were observed at the sight of first contact in the forestomach of rats. Systemic toxicity were also observed in these studies indicating that oral absorption does occur. Titanium-containing metabolite(s) reaches the organs, since titanium accumulation is observed, but was relatively nontoxic.

 

As a consequence, the bioavailability of the substance itself is considered to be low, following hydrolysis in the stomach. The absorption of the hydrolysis products is considered to be high via oral route. A default absorption rate of 100% is set for oral route.

 

 

1.2. Dermal absorption:

The molecular weight of the substance does not favor dermal uptake and the substance is a medium dustiness solid. Since the substance hydrolyses rapidly in water the water solubility and Log P cannot be used for estimating the dermal absorption. The substance will not evaporate from the skin.

The substance is not corrosive or irritating to skin. No effects, systemic or local, were observed in an acute dermal study or in a GPMT. Other toxicological studies via the dermal route are not available.

Since no measured data is available dermal absorption for the substance is set by default at 100 % for risk assessment purposes; however, it is likely that this value presents in fact an overestimation.

 

1.3. Absorption via inhalation:

Data on absorption via inhalation is not available. The vapor pressure of the substance is very low and the substance decomposes before melting from approximately 227 °C without any evidence of melting. At ambient working conditions vapors will not be formed and inhalation exposure may occur via inhalation of dust. The medium particle size of the substance shows that when inhaled it will end up in the nose and throat. Through the action of clearance mechanisms the substance may be transported out of the respiratory tract and swallowed after inhalation exposure. Toxicological studies via the inhalation route are not available.

The substance is classified as a respiratory irritant based on the fact that it hydrolyses in contact with water and HCl may be released. Any degradation products based on the water solubility Log P have the potential to be absorbed directly across the respiratory tract epithelium. This result to no principal difference in absorption compared to oral route i.e. also set to 100%.

 

 

1.4. Distribution and metabolism

In oral toxicity studies up to 2-years signs of local toxicity were observed at the sight of first contact in the forestomach of rats. Systemic toxicity were also observed in these studies indicating that oral absorption does occur. Titanium-containing metabolite(s) reaches the organs, since titanium accumulation is observed, but was relatively nontoxic (NTP 1991).

 

In a study performed by the NTP (1991) tissue samples of heart, liver, lung, and spleen from five male and five female rats given 0, 31,or 125 mg/kg were analyzed for titanium residues. The highest levels of titanium were found in the spleen and liver. Titanium levels in the heart, liver, and lung ranged from approximately 15 to 39 ppm for males and 15 to 42 ppm for females. However, the titanium levels in the spleen were much higher (100 to 180 ppm for males; 110 to 230 ppm for females) at both 15 months and 2 years. Titanium, determined by plasma-atomic emission spectroscopy, was found to be present in other tissues including the heart, liver, lung, and spleen. This procedure measured total titanium and could not distinguish between atomic titanium and the parent compound or metabolites. The results from the 2-year study suggest that the parent compound was toxic at the site of exposure, whereas the titanium-containing metabolite reaching other organs was relatively nontoxic (NTP 1991).

 

Titanium compounds are generally considered to be poorly absorbed upon ingestion and inhalation. However, detectable amounts of titanium can be found in the blood, brain, and parenchymatous organs of individuals in the general population; the highest concentrations are found in the hilar lymph nodes and the lung. Titanium is excreted with urine, and gastrointestinal excretion via the bile is possible (WHO EHC 1982). See below the full text from the EHC on titanium.

 

Figure2a and 2b from Kostova (2009) based on results from Toney at al (1985) shows what happens with the substance in unbuffered solutions of water (pH ~ 3) or in 0.32 M KNO3. This gives an indication of the potential metabolities that are formed, although under physiological conditions other factors such as enzymes are also present. In these studies reversible rapid hydrolysis of the first chloride ligand occurs to give an aquated intermediate, followed by a slower dissociation of the second chloride [50 min = Ti;], From Fig. (2a), it can be seen that both the concentration and solution pH will affect the equilibrium reactions, with the rate of chloride hydrolysis expected to be decreased at low pH and in high concentrations of salt since the equilibrium lies in favour of the metallocene dichloride. The estimated half live for loss of the Cp rings in Cp2TiCl2 water or in 0.32 M KNO3 at 37°C and at low pH (pH ~ 3), is 57 hours Fig. (2b).

 

From Kostova 2009, see attachment.

 

Based on the information, the substance and or its metabolites are expected to be widely distributed throughout the body after uptake.

 

The substance has been under investigation as an anticancer drug and for this purpose several studies have been performed to investigate pharmacokinetics via the IP and IV routes. Summaries of these studies are given below:

 

·        In a clinical trial twenty patients with a median age of 58 years received 83 courses of the substance called TD in the article. TD was given as 1-hour infusion (IV) at escalating doses from 70 to 185 mg/m 2/wk. Pharmacokinetic analysis was performed in eight patients for total plasma titanium (TPTi) and in three patients for ultrafiltrable titanium (UFTi). Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (tl/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a one compartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% ± 2.5% those of TPTi. (Christodoulou 1998).

 

·        It was shown in mice after single IP injections that neither titanocene dichloride nor titanium-containing metabolites are able to traverse the placental barrier through the sensitive phase of organogenesis until day 16 of murine pregnancy. Only on day 16, when the morphogenesis of most organs is widely terminated, small amounts of titanium enter the fetal compartment (Köpf -Maier, 1988).

 

·        The organ distribution of Ti after treatment with (C6Hs)2TiC12 (IP) showed an accumulation of Ti in the liver and the intestine lasting for several days, whereas the kidneys contained smaller concentrations of Ti, amounting to about half of the concentrations found in the liver. No transfer of Ti containing metabolites across the blood-brain barrier into brain tissue was detectable after application of TDC (Köpf –Maier et al., 1988).

 

·        Köpf –Maier et al. (1988) studied the pharmacokinetics and organ distribution of titanium (Ti) at various intervals up to 96 h after a single i.p. injection of a therapeutic dose of the antitumor agent titanocene dichloride (TDC, 60 mg/kg) by use of flameless atomic absorption spectroscopy in dried organ specimens. Highest organ concentrations were found in the liver and the intestine where 80-90 mg Ti/kg dry weight were accumulated at 24 and 48 h, corresponding to liver/blood and intestine/blood ratios of 8-9.

However, at no time point after the TDC application, the Ti concentrations in brain tissue exceeded those of control animals. In solid tumors growing subcutaneously in mice, increasing amounts of Ti were found during the course of the experiment, reaching concentrations between 10 and 15 mg Ti/kg at 24 and 96 h after single i.p. application of TDC.

 

·        Korfel et al (1998) This Phase I dose-escalation clinical trial of a lyophilized formulation of titanocene dichloride (MKT4) was conducted to determine the maximum tolerated dose, the dose limiting toxicity (DLT), and pharmacokinetics of titanium (Ti) after a single i.v. infusion of MKT4. Forty patients with refractory solid malignancies were treated with a total of 78 courses. Using a modified Fibonacci scheme, 15 mg/m2initial doses of titanocene dichloride were increased in cohorts of three patients up to level 11(560mg/m2) if DLT was not observed. The maximum tolerated dose was 315 mg/m2, and nephrotoxicity was DLT. Two minor responses (bladder carcinoma and non-small cell lung cancer) were observed.

The pharmacokinetics of plasma Ti were assessed in 14 treatment courses by atomic absorption spectroscopy. The ratio for the area under the curve0-in plasma and whole blood was 1.2. The following pharmacokinetic parameters were determined for plasma, as calculated in a two-compartment model: biological half-lifet1/2β,in plasma was 22.8 ± 11.2 h (xh± pseudo-SD), peak plasma concentration cmax~30 µg/ml at a dose of 420 mg/m2, distribution volume Vss= 5.34 ± 2.1 L (xa± SD), and a total clearance Cltotal=2.58±1.23 mI/min (xa±SD). There was a linear correlation between the area under the curve0-of Ti in plasma and the titanocene dichloride dose administered with a correlation coefficientr2of 0.8856. Plasma protein binding of Ti was in the 70-80% range. Between 3% and 16% of the total amount of Ti administered were renally excreted during the first 36 h. The recommended dose for Phase II evaluation is 240 mg/m2given every 3 weeks with i.v. hydration to reduce renal toxicity.

 

 

1.5. Excretion and bioaccumulation:

In a study where mice were exposed IP (Kopf-Maier and Martin 1998), 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.

 

In another study (Köpf –Maier et al. 1988), the observed enrichment of Ti in the liver and the intestine after application of TDC points to both organs as main sites of excretion for titanocene complexes and their metabolites, whereas elimination via the kidneys seems to be less important.

 

In a study performed by the NTP (1991), described above showed that bioaccumulation occurred slowly with the maximum titanium concentrations in the organs tested being reached by 15 months. No further increase was observed at 2 years, indicating that steady state concentrations had been achieved.

 

TDC and/or its metabolites have no bioaccumulation potential as such. Titanium is found to accumulate in several organs until a steady state is reached. Toxicity is not observed.

 

5. Conclusion

TDC is a mono-constituent substance, it is a solid at room temperature. Based on information from property profiling as well on information on studies on TDC, the following statements can be made:

-Oral absorption: the bioavailability of TDC, following dissociation of the salt in the Gastro- Intestinal tract, is considered to be high via oral route. A provisional absorption rate of 100% is set for the oral route.

-Dermal absorption: Dermal absorption of TDC is expected to be low. A provisional absorption rate of 100% is set for the dermal route, due to lack of data.

-Respiratory absorption: Likelihood of exposure via inhalation is based on the particle size. Particles will deposit mainly in upper airways, and will be subsequently swallowed after mucociliary transportation to pharynx. No principal difference is therefore expected in absorption between exposures via inhalation route and oral route as high in respiratory tract. Absorption via inhalation therefore also set to 100%.

-It is predicted that TDC and/or its metabolites are rapidly absorbed, distributed over the whole body.

- TDC and/or its metabolites have no bioaccumulation potential as such. Titanium is found to accumulate in several organs until a steady state is reached. Toxicity is not observed.

 

6. References

 

Not elsewhere in IUCLUD; reviews.

 

Irena Kostova, (2009)Titanium and Vanadium Complexes as Anticancer Agents.Anti-Cancer Agents in Medicinal Chemistry, 9, pp827-842

 

Toney et al., (1985) Hydrolysis chemistry of the metallocene dichlorides M(ƞ5-C5H5)2Cl2, M = titanium, vanadium, or zirconium. Aqueous kinetics, equilibria, and mechanistic implications for a new class of antitumor agents.  J. Am. Chem. Soc., 1985, 107 (4), pp 947–953.

 

WHO (1982) EHC24 (http://www.inchem.org/documents/ehc/ehc/ehc24.htm)

 

 

EHC 24 on titanium:

 

Summary:

“Quantitative information on absorption through inhalation is lacking. Absorption of titanium from the gastrointestinal tract takes place, but the extent of this absorption is not known. Based on average titanium concentrations found in human urine of about 10 µg/litre, it can be calculated that the absorption is about 3%, assuming a daily intake of at least 500 µg.

 

The highest concentrations of titanium have usually been found in the lungs, followed by the kidney and liver. In most studies on concentrations of titanium in blood, levels reported have been about 0.02-0.07 mg/litre. Titanium crosses the blood-brain barrier and is also transported through the placenta into the fetus. It seems to accumulate with age in the lungs, but not in other organs.

In the two reports available, the biological half-life for titanium in man has been calculated to be about 320 days and 640 days, respectively.

 

Most ingested titanium is eliminated unabsorbed. In man, titanium is probably excreted with urine at an approximate average rate of 10 µg/litre. Excretion by other routes is unknown.

 

Titanium compounds are poorly absorbed from the gastrointestinal tract.

 

ADME

Data on the absorption of titanium compounds are very limited and very little quantitative information is available with regard to absorption by inhalation. Ingested titanium is apparently absorbed from the gastrointestinal tract (Schroeder et al., 1964) but there is little information regarding the extent of absorption, and comparative studies using different titanium compounds have not been made. Lloyd et al. (1955) tested the suitability of titanium dioxide as a marker for digestibility. The recovery of only 92% of the titanium dioxide fed to rats at a dietary level of 2.5 g/kg remained largely unexplained. The minute absorption of titanium from the gastrointestinal tract was demonstrated in a study in

which mice were given 44Ti intragastrically (without marker). The whole body count after 24 h did not exceed the background level (Thomas & Archuleta, 1980). A comparison of organ contents of 44Ti

after oral and intravenous administration of the isotope (3 µCi), indicated a gastrointestinal absorption of less than 5% in lambs (Miller et al., 1976). When male and female rats were fed a diet containing titanium dioxide (100 g/kg) for a period of about 32 days, a significant retention of titanium of 0.06 and 0.11 mg/kg wet weight was found only in the muscles; no retention was observed in the liver, spleen, kidney, bone, plasma, or erythrocytes (West & Wyzan, 1963). The same authors administered 5 g of titanium dioxide to 5 male adult volunteers on 3 consecutive days. This did not cause any significant increase in the urinary content of titanium.

 

The clearance of titanium dioxide from the lungs was studied in rats after inhalation of 15 or 100 mg/m3. The average median aerodynamic diameter of the titanium dioxide particles was 1.48 µm.

After a single exposure, about 40-45% of the deposited particles were cleared from the lung in 25 days. At 15 mg/m3, 0.7% was found in the hilar lymph nodes indicating penetration of titanium dioxide particles from alveoli into the lymphatic system and partial clearance by the lymphatic route. The clearance rate was similar after intra-tracheal administration of titanium dioxide. At an exposure of 100 mg/m3, the clearance rate decreased drastically (Ferin & Feldstein, 1978). Elo et al. (1972) demonstrated the presence of titanium dioxide in the lymphatic systems of 3 workers employed in processing titanium dioxide pigments.

 

The distribution of titanium in the organs of mice following the administration of a tetravalent, soluble titanium salt (titanium potassium oxalate) in drinking-water at a concentration of 5 mg/litre was reported from a life span study by Schroeder et al. (1964). The results were compared with organ concentrations in control mice fed drinking-water without the addition of titanium, and in wild field mice (Table 5). Organs of treated and wild animals displayed concentrations of roughly the same order of magnitude, whereas untreated mice showed lower levels, the differences being more pronounced in males.

 

Table 5. Titanium concentrations in the organs of mice given titanium in the drinking-water at 5 mg/litre throughout the life span (values in mg/kg wet weight)a

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                   No.  Heart Lung Spleen Liver Kidney

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Treated mice

 

     Male         41   8.80  4.81 6.83   1.81  2.86

     Female       37   4.10  1.66 3.70   2.05  2.89

 

Untreated mice

 

       Male       31   0.34  0.13 0.94   0.38  0.33

       Female     51   1.08  0.66 1.10   0.67  0.55

 

Wild field mice    9    6.93  3.03 -      4.10  1.03

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a From: Schroeder et al. (1964).

 

Following intravenous injection of rats with 50 mg of titanium dioxide (250 mg/kg body weight), there was an exponential disappearance rate with only about 30% remaining after 10 min.

After intravenous injection of 250 mg/kg body weight of titanium dioxide in rats, about 70% of the injected dose was detected in the liver, 5 min after administration, and almost 80%, 15 min after injection. The highest concentration was found in the liver followed by the spleen after 6 h, whereas, after 24 h, the highest concentration was found in the celiac lymph nodes, which filter the lymph from the liver. One year after the injection, the highest concentrations were still found in these lymph nodes (Huggins & Froehlich, 1966).

 

 

Human studies

 

Little information is available on the absorption of titanium compounds by man. With respect to absorption by inhalation, there is evidence showing that titanium containing particles in the air

are in the upper respirable size range (Johansson, 1974). The titanium retained in the peripheral part of the lungs does not seem to account for the observed titanium levels in lung tissue (Schroeder et al., 1963). Experiments on rats suggest that titanium may be taken up by the lungs from the blood. On the basis of rather rough calculations, Schroeder et al. (1963) concluded that a third or less of the inspired titanium may be retained in the lungs.

 

Few studies have been reported on the absorption of titanium from the gastrointestinal tract in man. Perry & Perry (1959) reported a mean concentration of 10 µg/litre in pooled urine indicating absorption; however, the extent of the absorption is not known. Accepting this amount in the urine, and assuming a daily intake of 300 µg of titanium, Schroeder et al. (1963) calculated that about 3% of the dietary dose would be absorbed.

 

Wide variations in titanium levels in different organs in man have been found, the lungs frequently containing the highest amount. Hamilton et al. (1972/1973) using X-ray fluorescence found concentrations of 3.7 mg/kg wet weight in the lung and 0.8 mg/kg in the brain, demonstrating that titanium passes the blood-brain barrier. Earlier, Tipton & Cook (1963) and Schroeder et al. (1963) had also found that concentrations in lung tissue were higher than in other human tissues. In a male worker not occupationally exposed to metals, the highest concentration was found in the hilar lymph nodes (150 mg/kg dry weight) followed by the lung (33 mg/kg dry weight) (Teraoka, 1980). Comparison of tissue levels of titanium between American subjects and people from other geographical areas showed similar high concentrations in pulmonary tissues (Perry et al., 1962).

 

In the study by Schroeder et al. (1963), accumulation of titanium started in the lung after the third decade and did not occur in the kidney, skin, or aorta. Infant kidneys contained several times the adult concentration of titanium (Tipton & Cook, 1963).

 

Metal and mineral concentrations in the lungs of West Virginian bituminous coal miners were studied by Crable et al. (1967, 1968). The mean concentration of titanium, among other metallic constituents, in the lungs of 26 miners (with 23-50 years service) was 119 mg/kg dry weight compared with a normal level of 19 mg/kg. Röthig & Wehran (1972) found concentrations of titanium in the lungs of patients with silicosis ranging from 4 to 24.3 mg/kg. Levels in the lymph nodes ranged from 12.2 to 120 mg/kg. The average titanium concentration rose with increasing severity of silicosis, the concentration of titanium in the hilar lymph nodes being much higher than that found in the lungs.

 

A mean titanium concentration in blood of 0.07 mg/litre was reported by Hamilton et al. (1972/1973), not much different from the 0.02-0.03 mg/litre previously reported by Maillard & Ettori (1936a; 1936b). A somewhat higher mean level of 0.123±0.005 mg/litre was found in 20 healthy subjects, 20-43 years of age, by Mozajceva (1970). Timakin et al. (1967) reported a mean level of 0.054±0.002 mg/litre in the serum of 200 healthy persons from the USSR. Smysljaeva et al. (1971) determined the distribution of titanium in the blood of children in the age range of 1-14 years. They found a ratio of 2:3 between erythrocytes and plasma; this ratio decreased slightly with age. The range of the ratios was

0.5-1.

 

Titanium was qualitatively detected in leukocytes, using electron probe microanalysis (Carroll & Tullis, 1968). There are some indications that titanium levels in the blood may change in a variety of diseased states (Bredihin & Soroka, 1969; Kas'janenko & Kul'skaja, 1969; Mozajceva, 1970; Alhimov et al., 1971).

 

Schroeder et al. (1963) demonstrated the presence of titanium in the tissues of newborn infants, indicating that titanium passes the placenta. The fact that titanium was not detectable in all fetuses may reflect the sensitivity of the analytical methods used; however, Scanlon (1975) interpreted this finding as evidence of titanium not being an essential element for man.

 

Most of ingested titanium is eliminated unabsorbed with the faeces. Under normal circumstances, titanium is excreted with the urine probably at a rate of about 10 µg/litre (Perry & Perry, 1959; schroeder et al., 1963). Higher urinary excretion levels of 0.41 and 0.46 mg/litre have been reported in two adults (Tipton et al., 1966). Other routes of excretion are not known.

 

Biological half-life

 

Few attempts have been made to calculate the biological half- life of titanium in man or experimental animals. The lung is considered to be the primary target organ in man and the residence time of titanium dioxide in the lung has been regarded as long (ICRP, 1959). In one report, the biological half-life of titanium in man was calculated to be 320 days (ICRP, 1959). Following the intraperitoneal and intravenous administration of 44Ti in mice, a mean biological half-life of 640 days was calculated. On the basis of experience with the biological half-life of uranium dioxide in rats, monkeys, and dogs, the authors speculated that the whole-body retention of titanium in man may be even longer than the reported 640 days in mice (Thomas & Archuleta, 1980).”

 

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information