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Carcinogenicity

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Description of key information

The NTP performed a series of studies with the substance. A 2 -year carcinogenicity study was performed where rats were dose by gavage on a daily basis at 25 and 50 mg/kg bw/d.

Local carcinogenicity

Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity' of titanocene dichloride in male F344/N rats based on a marginal increase in the incidence of fore-stomach squamous cell papillomas, squamous cell carcinoma, and basosquamous tumor benign. There was equivocal evidence of carcinogenic activity of titanocene dichloride in female F344/N rats based on a marginal increase in the incidence of fore-stomach squamous cell papillomas.

Nonneoplastic lesions associated with the administration of titanocene dichloride for up to 2 years included erosions and inflammation of the gastric mucosa,hyperplasia and metaplasia of the fundic glands with fibrosis of the lamina propria in the glandular stomach, and acanthosis (hyperplasia) and hyperkeratosis of the forestomach epithelium.

In these 2-year studies of titanocene dichloride, the forestomach tumors in dosed male and female rats given titanocene dichloride by oral gavage were considered equivocal evidence of carcinogenic activity because:

1) the numbers of tumors were small and only slightly above historical controls;

2) there was no dose-related increase in tumor incidence in male rats;

3) the tumors were mostly benign (the only squamous cell carcinoma occurred in a low dose male); and,

4) the biological potential of the relationship of hyperplasia and the papillomas is not clear.

The 2 year study shows that the parent compound, titanocene dichloride, was toxic at the site of exposure, whereas the titanium-containing metabolite reaching other organs was relatively nontoxic. Recent studies by Kopf-Maier and Martin (1989) have shown that following the intraperitoneal injection of titanium dichloride, titanium accumulates in both the cytoplasm and nucleus of cells. Other studies suggest that the cytotoxicity of this compound results from the interaction with nucleic acid constituents (Toney et al.,1986).

The effects may be explained by what occurs when the substance comes into contact with water. According to an assessment of the water solubility of the test item (Envigo Study Number FD03TF), no determination of the water solubility of the parent test item was possible or relevant due to rapid hydrolysis in contact with water. Analysis of saturated solutions resulted in a mean solution concentration equivalent to 103g/L of solution at 20.0+0.5 ◦C, monitoring the response attributed to soluble hydrolysis products formed on dissolution. Extreme low pH values for the solution were noted, pH 0.8, consistent with the liberation of hydrochloric acid during hydrolysis as which is supported by Toney et al (1985).

Therefore, also in contact with the forestomach of a rat such a reaction may occur. The pH in the forestomach of the rat is between 4.5 -6 and there is no protection from mucus secretion as there is in the human stomach (Proctor et al., 2007). Furthermore, the dosing is done in bulk by gavage. The severity of the reaction would probably less in the human stomach.

Furthermore, since humans have no squamous epithelium in the stomach, the potential risk from non-genotoxic forestomach carcinogens involves exposure of the mouth, pharynx and oesophagus at dose levels that exert irritating action. It seems very unlikely that exposure to concentrations far below those having irritating potential is hazardous to man (Kroes and Wester, 1986).

In case of workers oral exposure is not expected. The major routes of exposure for workers are inhalation and dermal. In case of exposure on skin these effects observed in the stomach are not relevant. Skin irritation studies and an acute dermal study do not show any effects on the skin.

The substance is positive in the Ames test and in the MLA. Due to the complex structure QSAR predictions are not relevant for this substance. For in vivo genotoxicity a study is proposed to further elucidate this endpoint.

Other local effects

At 25 mg/kg bw/d are:

End of the study:

·        A high incidence of abnormal respiratory sounds.The respiratory sounds were probably due to exudate in the upper respiratory tract and were related to the pulmonary and nasal cavity lesions present in dosed animals.

·        Acute inflammation of the nasal mucosa and Acute and/or granulomatous inflammation of the lung in male and female rats. The lung and nose lesions were considered to be causally related to the reflux and aspiration of the gavage solution due to degenerative and inflammatory lesions in the stomach.

·        Fat proliferation was seen in the stomach. Nonneoplastic lesions in the forestomach were observed: epithelial hyperplasia and hyperkeratosis and inflammation was seen in the glandular stomach.

 

At 50 mg/kg bw/d are:

15 month evaluations:

·        Nonneoplastic lesions associated with the administration of titanocene dichloride occurred in stomach and were similar to those seen at two years.

End of the study:

·        The same except the effects on the glandular stomach are more severe: inflammation, hyperplasia/dysplasia, erosion, fibrosis and metaplasia.

Systemic effects

The following systemic effects were observed in this study at both dose levels:

·        At the 15 month interim evaluation a statistically significant decrease in body weight for males given 25 or 50 mg/kg and for females given 50 mg/kg. Absoluteandrelative liver weights were significantly decreased in males given 50 mg/kg and significantly increased in females given 50 mg/kg. In males asignificantly decreased organ to body weight ratios at 50mg/kg dose of the brain and liver and at 25mg/kg of the brain. In females a significantly decreased organ to body weight ratios at 50mg/kg dose of the brain, heart, liver and lung and at 25mg/kg of the liver.At the end of the study decreased body weights were observed and final bodyweight were 91-96% for males and females.

·        At the end of the study in both males and females and dose levels granulomatous inflammation/pigmentation of the liver was seen and in males at the high dose lesions with eosinophilic focus and mixed focus.

·        In males significantly lower survival rates were seen at the end of the study.

·        At the 15 month evaluations at both dose levels accumulations of macrophages laden withblue-gray to gray-green pigment were seen in several organs of dosed male and female rats, but occurred predominantly in mesenteric lymph nodes and lungs.In afew selected animals, special stains were employed in an effort to further characterize the pigment. In these animals, the pigment was negative for the periodic acid-Schiff reaction and Perl’siron stainand was not believed to be hemosiderin or lipofuscin. The pigment was thought to containtitanium.

·        Hematological parameters were measured in the 15 month interim evaluation and significant dose-related decreases in hematocrit, haemoglobin, meancellvolume, and meancell hemoglobin values were observed for male andfemale ratsgiven titanocene dichloride at both dose levels. These changes in hematology values are consistent with a mild microcytic anemia of the type commonly accompanying various chronic inflammatory processes.

Based on the results of this study the LOAEL is, for local and systemic effects, 25 mg/kg bw/d.

 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: NTP protocol
Version / remarks:
Details can be found: https://ntp.niehs.nih.gov/testing/types/cartox/index.html
GLP compliance:
yes
Remarks:
FDA Good Laboratory Practice Regulations (21 CFR Part 58)
Specific details on test material used for the study:
Strem Chemicals (Newbury Port, MA) provided lot no. 13574-S,which was used for the 2-year studies. Identity, purity, and stability analyses were conducted at the analytical chemistry laboratory, Midwest Research Institute (MRI), Kansas City, MQ. The study chemical, a dark red, microcrystalline solid, was identified as titanocene dichloride by infrared and nuclear magnetic resonance spectroscopy. The purity of lot no. 13574-S was determined to be greater than 99% by titration, elemental analysis, and Karl Fischer water analysis. Stability studies performed by titration indicated that titanocene dichloride was stable as a bulk chemical for at least 2 weeks at temperatures to 60ºC when protected from light. Based on the stability study results, the bulk chemical was stored at 0º ± 5º C at the testing laboratory throughout the study period. The stability of the bulkchemical was monitored by elemental analysis and by titration periodically during all phases of the studies. No change in the study material was detected.
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
Rats used in the 2-year studies were obtained at 4 weeks of age from the Frederick Cancer Research Facility (Frederick, MD). Males were quarantined for 11 days and females were quarantined for 13 days. During this time, animals were checked daily. To assess the health status of the rats, 5 animals per sex were killed prior to study initiation and were examined for infectious and parasitic diseases. The rats were about 6 weeks of age at the beginning of the study. The health of the animals was monitored during the course of the studies according to the protocols of the NTP Sentinel Animal Program.

Animal Maintenance
Rats were housed five per cage. Feed and water were available ad libitum. Racks were rotated in the room every two weeks, and cages were rotated from top to bottom within each group every two weeks.

Method of Animal Distribution
Animals of each sex randomized into cage groups and then cage randomized to dosed and control groups by a table of random numbers

Method of Animal Identification
Ear punch

Feed
NIH-07 open formula meal diet; (Zeigler Bros., Inc., Gardners, PA), available ad libitum

Maximum Storage Time for Feed
120 days after milling

Feeders
Stainless steel, gang style (Scientific Cages, Inc.,Bryan, TX), filled twice weekly, changed weekly

Water
Automatic watering system, (Edstrom Industries, Inc., Waterford, WI), ad libitum

Cages
Polycarbonate (Lab Products, Inc, Rochelle Park, NJ),changed twice weekly

Bedding
Aspen bed or Beta Chips; changed twice weekly

Cage filters
Non-woven fiber (Snow Filtration,Cincinnati, OH)

Racks
Stainless steel (Lab Products, Inc., Ruchelle Park, NJ), changed once every two weeks

Animal Room Environment
Temperature: 22.4º ± 1.4º C
Humidity: 45.2% ± 6.8%
Light: fluorescent, 12 hour day
Room air changes: 10-12 changes/ hour
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The dose formulations were prepared by mixing appropriate amounts of titanocene dichloride and corn oil. During the studies, the dose formulations were stored at 0º ± 5º C for no longer than 2 weeks.

Vehicle
Each lot of corn oil vehicle used in these studies was analyzed for peroxides at monthly intervals by Official Method Cd 8-53 of the American Oil Chemists' Society (Mehlenbacher et al., 1972). The peroxide content of the vehicle ranged from 1.00 to 3.65 mg/kg, well below the tolerance limit of 10
mg/kg. Results of periodic referee analyses of the dose formulations performed by MRI using an ul traviolet spectroscopy (Cary 219 spectrometer, acetonitrile as diluent, absorbance measured at 251 to 253 nm) were in agreement with the results from the study laboratory.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies showed no decrease in titanocene dichloride concentration after storage of the sus pensions for 2 weeks in the dark at 5º C or 25ºC, or under simulated animal dosing conditions. The study laboratory conducted periodic analyses of the titanocene dichloride dose formulations using high performance liquid chromatography or ultra-violet spectrophotometry. During the 2-year studies, the dose formulations were analyzed at approximately 8-week intervals and were within is 10% of th e target concentrations 96% of the time. The corn oil vehicle was analyzed for peroxides at monthly intervals; the peroxide content of the vehicle was within acceptable limits. Results of periodic referee analyses of the dose formulations performed by MRI were in agreement with the results from the study laboratory.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
5 days per week
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Groups of 70 rats of each sex
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 70 rats of each sex were administered 0, 25, or 50 mg/kg titanocene dichloride in corn oil by gavage at a dose volume of 5 mL/kg for 5 days per week for 104 weeks. Ten rats per dose group were evaluated (necropsy, organ weights, histopathology, tissue residues of titanium, and hematology analyses) after 15 months of chemical administration. In addition to these 70 animals, another ten rats per dose group were used in a separate research project that was not part of the 2-year carcinogenesis studies described in this report. One low-dose female in the separate research project died early and was included in the pathology analyses for the carcinogenesis studies. Thus, 61 low-dose females were used for the pathology and statistical evaluations for the 2-year studies, while 60 animals of each sex in the remaining dose groups were evaluated.
Observations and examinations performed and frequency:
All animals were observed twice daily, and clinical findings were recorded monthly or as necessary.Moribund animals were killed. Individual body weights were recorded prior to study initiation, once per week for the first 13 weeks of the studies, and every 4weeks thereafter. Mean body weights were calculated for each group.
Sacrifice and pathology:
After 15 months, ten rats per dose group were killed for evaluation of organ weights, hematology parameters, tissue residues of titanium, and gross and microscopic pathology. The 104-week treatment period was followed by a one-week observation period, after which surviving animals were killed. A necropsy was performed on all animals including those found dead. Necropsies of all dosed rats were conducted within 7 working days of the end of the observation period; control animals were killed and necropsied within 9 working days of the end of the observation period. During necropsy, all organs and tissues were examined for grossly visible lesions. Portions of the heart, liver, lung, and spleen were collected from 10 randomly selected animals per dose group,frozen in liquid nitrogen, and shipped to MRI for analysis of titanium residues. Remaining tissues
were preserved in 10% neutral buffered formalin and routinely processed for microscopic examination(embedded in paraffin, sectioned at 4 to 6 pm, and stained with hematoxylin and eosin). A complete histopathologic evaluation inclusive of gross lesions was performed on all animals.Upon completion of the microscopic evaluation by the laboratory pathologist, the slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slideblock match, and wet tissue audit. The slides, individual animal data records, and pathology tables were sent to an inde
pendent pathology quality assessment laboratory. The individual animal records and pathology tables were compared for accuracy, slide and tissue counts were verified, and histotechnique was evaluated. All tissueswith a diagnosis of neoplasia; the liver, stomach, lungs, and nose from all male and female rats; the pancreas from all males; and all tissues from a randomly selected 10% of the control and high-dose rats were reevaluated microscopically by a quality assessment pathologist. In addition, sections of the spleen, thymus, duodenum, jejunum, bone marrow, and lymph nodes were reviewed for all rats to confirm the reported incidence of pigmentation.
The quality assessment report and slides were submitted to the NTP Pathology Working Group (PWG) chair, who reviewed the aforementioned tissues and any other tissues for which there was a disagreement in diagnosis between the laboratory and quality assessment pathologists. Representative examples of potential chemical-related non-neoplastic lesions and neoplasms, lesions for which there was a difference diagnosis between the study pathologist and reviewing pathologist, and lesions of general interest were selected by the chair for review by the PWG. The PWG consisted of the quality assessment pathologist and other pathologists experienced in rodent toxicologic pathology. This group examined the tissues without knowledge of dose groups or previously rendered diagnoses. When the consensus opinion of the PWG differed from that of the laboratory pathologist, the diagnosiswas changed. Thus, the final diagnoses represent a consensus of contractor pathologists and the PWG. Details of these review procedures have been described by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analysis of pathology data, the diagnosed lesions for each tissue type are evaluated separately or combined according to the guidelines of McConnell et aL (1986).

Necropsy
Complete necropsy performed on all animals. Organ weights of brain, heart, right kidney, liver, lung, and spleen were recorded.

Histopathology
Complete histopathologic examinations were performed on all control and highdose animals evaluated at 15 months and on all animals in the core study. Tissues examined included:gross lesions and tissue masses (and regional lymph nodes), blood smear (15-month only), esophagus, cecum, colon, rectum, duodenum, ileum, jejunum, liver, pancreas, salivary glands, stomach, heart, adrenal glands, parathyroid glands, pituitary gland, thyroid gland, epididymis, preputiaklitoral gland, prostate gland, seminal vesicles, testes, ovaries, uterus, bone marrow, mandibular and mesenteric lymph nodes , spleen, thymus, skin, mammary gland, bone (sternebrae), brain, lungs and bronchi, nose, trachea, kidney, urinary bladder. Tissues examined in lowdose animals at the 15-month interim evaluations we re gross lesions and bone marrow, brain, kidney, liver, lung, mandibular and mesenteric lymph nodes, spleen, and stomach.

Clinical Pathology
Hematology: 10 rats per dose were killed at 15 months for evaluation of hematology parameters including hematocrit, hemoglobin, erythrocyte count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet, reticulocyte, leukocyte count and differential.

Supplemental Studies
Sections of heart, liver, lung, and spleen were collected from 10 rats per dose at 15 months and at 2years. These tissues were evaluated for titanium residues.
Statistics:
Survival Analyses
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were censored from the survival analyses at the time they were found dead of other than natural causes or were found to be missing; animals dying from natural causes were not censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two-sided.

Calculation of Incidence
The incidence of neoplastic or non-neoplastic lesions is given as the ratio of the number of animal
s bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators include only those animals for which the site was examined histologically.However, when macro-scopic examination was required to detect lesions (e.g. skin or mammary tumors) prior to histologic sampling, or when lesions had multiple potential sites of Occurrence (e.g., mononuclear cell leukemia), the denominators consist of the number of animals on which a necropsy was performed.

Analysis of Continuous Variables
The nonparametric multiple comparison procedures of Dunn (1964) or Shirley (1977) were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of organ weight data, clinical chemistry, and hematology.Jonckheere’s test (Jonckheere, 1954) was used to evaluate the significance of dose-response trends and to determine whether Dunn’s or Shirley’s test was more appropriate for pairwise comparisons.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The administration of titanocene dichloride was associated with a high incidence of abnormal respiratory sounds in male and female rats which were first noticed approximately eight months into the study. These low intensity respiratory sounds were audible only in close proximity to affected animals (auscultation was not performed). The respiratory sounds were probably due to exudate in the upper respiratory tract and were related to the pulmonary and nasal cavity lesions present in dosed animals. These sounds were noted in low-dose males (42/80), low-dosefemales (14/80), high-dose males (58/80), and high-dose females (24/80). In comparison, abnormal respiratory sounds were observed in only 1 of the 80 control males and none of the 80 control females.
Mortality:
mortality observed, treatment-related
Description (incidence):
Low- and high- dose females had similar survival rates; although these rates were lower than the control value, the difference was not statistically significant. Treated males had survival rates significantly less than that of control males.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
15-Month Interim Evaluations
A dose-related decrease in mean necropsy body weight was observed in treated rats of both sexes. This decrease was statistically significant for males given 25 or 50 mg/kg and for females given 50 mg/kg. Absolute and relative liver weights were significantly decreased in males given 50 mg/kg and significantly increased in females given 50 mg/kg. No other notable organ weight differences were observed.

End of study
Mean bodyweights of male and female rats given 25 mg/kg titanocene dichloride were marginally lower (1% to 7%) than the control after week 14. Mean body weights of high-dose male rats were within 10% of controls for the first 33 weeks of dosing; after week 33, the mean body weights of highdose males were 9% to 14% lower than those of the control animals. High- dose female rats had bodyweights within 10% of controls for the first 49 weeks of treatment; during the second year of the study, high-dose females had body weights 11% to 15% lower than controls. The final mean bodyweight for rats given titanocene dichloride were 96% and 91% for low- and high- dose males and 96% and 89% for low- and high- dose females.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
15-Month Interim Evaluations
Significant dose-related decreases in hematocrit, haemoglobin, mean cell volume, and mean cel lhemoglobin values were observed for male and female rats given titanocene dichloride. These changes in hematology values are consistent with a mild microcytic anemia of the type commonly accompanying various chronic inflammatory processes.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
15-Month Interim Evaluations
Males - significantly decreased organ to body weight ratios at 50mg/kg dose of the brain and liver and at 25mg/kg of the brain.
Females - significantly decreased organ to body weight ratios at 50mg/kg dose of the brain, heart, liver and lung and at 25mg/kg of the liver.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
15-Month Interim Evaluations
Nonneoplastic lesions associated with the administration of titanocene dichloride occurred in stomach primarily the and were similar to those seen at two years. Accumulations of macrophages laden with blue-gray to gray-green pigment were seen in several organs of dosed male and female rats, but occurred predominantly in mesenteric lymph nodes and lungs. In a few selected animals, special stains were employed in an effort to further characterize the pigment. In these animals, the pigment was negative for the periodic acid-Schiff reaction and Perl’s iron stain and was not believed to be hemosiderin or lipofuscin. The pigment was thought to contain titanium.

End of study
Stomach
The principal lesions associated with the administration of titanocene dichloride by oral gavage for up to 2 years occurred in the stomach. The lesions were similar to those seen in the 13-week studies and at the 15-month interim evaluations, but were generally more extensive or severe.In the forestomach, hyperplasia of the epithelium (diagnosed as acanthosis in the 2-year studies) and hyperkeratos is usually occurred on or near the limiting ridge (junction of the forestomach and glandular stomach). There was variable thickening and folding of the stratified squamous epithelium due to an increase in cells composing the stratum spinosum, as well as an increase in basal cells, and accumulations of keratin on the surface. In some rats the squamous epithelium extended beyond the forestomach and covered parts of the adjacent glandular mucosa. A spectrum of interrelated lesions occurred in the glandular stomach of dosed rats including erosions, inflammation, hyperplasia and metaplasia of the glandular epithelium, fibrosis of the lamina propria, fat proliferation, and pigmentation. The mucosa glandular immediately adjacent to the limiting ridge of the forestomach was often the most severely affected. The erosions were superficial foci of necrosis with loss of the surface epithelium, gastric pits and fundic glands; the erosions generally did not extend across the muscularismucosa into the submucosa. Infiltrates of neutrophils and mononuclear cells were present in the lamina propria and submucosa, and there was a variable increase in fibrous connective tissue in the lamina propria. Throughout the sections of stomach examined, there was atrophy of some fundic glands with concomitant hyperplasia or metaplasia of others. The atrophic glands exhibited loss of parietal and chief cells and were lined by nondescript cuboidal to columnar cells. In other glands the parietal and chief cells were replaced by less differentiated cuboidal cells which occasionally appeared multilayered. Two low-dose males and one low-dose female had a more severe form of hyperplasia (termed adenomatous hyperplasia) characterized by marked focal thickening of the mucosa and disorganization of the glands. The epithelium in these lesions was relatively uniform with minimal atypia and there was no extension through the muscularis mucosa. There was some uncertainty about the biological nature of these lesions, but it was the consensus of the Pathology Working Group that these lesions were not neo-plasm. Single cells or clusters of cells similar to pancreatic acinar cells were present in scattered fundic glands (metaplasia). Mineralization was characterized by focal clusters of coarse basophilic granular material in areas of erosion and/or inflammation. The fat proliferation seen in dosed rats consisted of focal accumulations of well-differentiated adipocytes in the submucosa of the glandular stomach. This lesion also may represent a metaplasia or transdifferentiation of fibroblasts into adipocytes. The single lipoma found in a low-dose male was a largenodular mass of mature adipocytes. Whether it was related in any way to the fat proliferation is unknown.Macrophages containing blue-gray to gray-green pigment similar to that seen in other organs were also present in the lamina propria and submucosa of the glandular stomach.

Lung and Nose
There was a chemical-related increased incidence of acute inflammation of the nasal mucosa in male and female rats. In males, fungal hyphae were often associated with the inflammatory lesions, whereas only a few females were similarly affected. Acute and/or granulomatous inflammation of the lung also occurred with a dose-related increase in treated rats. The inflammatory lesions were located near the terminal bronchioles and, occasionally, globules of yellow refractile material were present in the lesions. The foreign material may represent the corn oil vehicle. The lung and nose lesions were considered to be causally related to the reflux and aspiration of the gavage solution due to degenerative and inflammatory lesions in the stomach. Infiltration of macrophages (histiocytes) in the lung also occurred more frequently in dosed rats than in controls. The macrophages were often located around small vessels and in alveoli near the pleural su rface. In dosed rats, some macrophages contained blue-gray to gray-green pigment believed to be titanium.

Liver
Granulomatous inflammation occurred at increased incidence in rats receiving titanocene dichloride. The lesions were often located adjacent to or around portal areas and consisted of lymphocytes, and macrophages. Pigment, similar to that found in other organs, was observed in some of the macrophages in these lesions as well as in individual Kupffer cells scattered throughout the lobules. The incidences of mixed cell focus and eosinophilic focus were also increased in male rats, although those of basophilic focus and clear cell focus were not increased. The various types of foci are distinguished on the basis of stain affinity, and the mixed cell foci usually contain a mixed population of basophilic and clear cells or eosinophilic and clear cells. The staining affinity of these cells usually corresponds to increases in cytoplasmic rough endoplasmic reticulum (basophilic cells), smooth endoplasmic reticulum (eosinophilic cells), or glycogen (clear cells). Whether these modest increases in mixed and eosinophilic foci are related to administration of titanocene the dichloride is uncertain.

Mesothelium of Peritoneal Cavity and Tunica Vaginalis
Mesothelioma occurred in 3/60 low-dose and 4/60 high-dose male rats, but not in controls. The incidences of mesothelioma in NTP historical controls is 106/2,762 (3.8%) with as many as 6/50 in one control group. The marginal increase in mesotheliomas in dosed males is due largely to the lower than expected incidence in controls. Therefore, the small number of mesotheliomas in dosed males was not considered related to the administration of titanocene dichloride.

Pigmentation of Various Organs
Small amounts of pigment similar to that seen in the stomach, lung, and liver as described were seen within macrophages in various lymphnodes, spleen, and intestinal tract of dosed rats. Since macrophages in the spleen and lymph nodes commonly contain hemosiderin, it was not always easy to discern those believed to contain titanium from those which contained only hemosiderin.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
15-Month Interim Evaluations
There were no apparent treatment-related neoplasms in male or female rats.

End of study
Stomach
Squamous cell papillomas occurred at low incidence in the forestomach of dosed rats, but not in controls. In males, papillomas of the forestomach occurred in four low-dose and one high-dose animal, whereas in females papillomas were seen in one low-dose and two high-dose animals. A squamous cell carcinoma was seen in one low-dose male,which also had a squamous cell papilloma, and a basosquamous tumor was seen in one high- dose male. Squamous cell papilloma or carcinoma of the forestomach are uncommon neoplasms in control rats. The incidence of squamous cell papilloma or carcinoma combined in NTP historical controls is 912,735 for males and 812,748 for females with no more than two occurring in any single control group. The papillomas were exophytic growths with squamous epithelium overlying a fibrovascular core, whereas the squamous cell carcinoma demonstrated invasion of the submucosa by cords and clusters of anaplastic squamous cells. The basosquamous tumor was a small circumscribed nodular mass projecting into the submucosa and consisting of basal cells and occasional small clusters of squamous cells. The tumor resembled benign basal cell neoplasms of the skin.

Liver
There was a marginally increased incidence of hepatocellular adenomas in high-dose male rats relative to controls (control, 4/60;low-dose, 3/59;high-dose 7/60); an hepatocellular adenoma or carcinoma occurred in one high-dose male. The incidence of hepatocellular adenoma or carcinoma combined in NTP historical control male rats is 82/2,758 (3%) with as many as in one control 7/50 group. Because the increase was slight and the incidence of these neoplasms in the high-dose group was within the range of historical controls, they were not considered related to the administration of titanocene dichloride.

Clitoral Gland
There was a decreased incidence of clitoral gland adenomas or carcinomas in treated female rats (13/56;9/55;2/56). The incidence of clitoral gland adenomas or carcinomas in the control group was higher than the NTP historical control incidence of 13812,763 (5%); previously no more than 9/50 had been seen in a given control group. Therefore, the apparent decreased incidence of these tumors in treated females may be spurious and not related to chemical administration.

Pituitary Gland
There was a decreased incidence of pituitary gland adenomas in treated females (29/59;23/59; 16/60). The incidence of pituitary gland adenomas in NTP historical control females is 1,045/2,710 (39%) with as many as 32/49 in a single control group. In addition, the combined incidence of malignant tumors at all sites was also decreased in treated females(30/60,25/61;18/60). The incidence of malignant tumors at all sites in NTP historical control female rats is 1,01812,763 (37%) with as many as 28/50 in one control group. The decreased incidences of pituitary gland tumors and of malignant tumors at all sites in the high dose females were marginally significant (P=0.041 and P=0.044) and were not considered to be clearly related to chemical administration.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Tissue Analysis for Titanium Residues
Titanium levels in the heart, liver, and lung ranged from approximately 15 to 39 ppm for males and 15 to 42ppm 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.
Key result
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
haematology
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
other: Equivocal evidence of carcinogenic activity based on a marginal increase in the incidence of fore stomach squamous cell papillomas, squamous cell carcinoma, and basosquamous tumor benign.

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Titanocene Dichloride

Variable

Male F344/N Rats

Female F344/N Rats

Doses

0, 25, or 50 mg/kg in corn oil

by gavage

0, 25, or 50 mg/kg in corn oil by gavage

Body weights

Dosed lower than controls

Dosed lower than controls

2-year survival rates

41/60, 30/60, 24/60

37/60, 30/61, 31/60

Non-neoplastic effects

Glandular stomach:

erosions -1/58, 9/59, 13/58;

inflammation -0/58, 9/59, 10/58;

hyperplasia -0/58, 10/59,

24/58;

metaplasia -0/58, 26/59,

36/58;

fibrosis -0/58, 30/59, 37/58; fat proliferation -0/59, 2/59, 14/60

Forestomach acanthosis (epithelial hyperplasia) -8/57, 25/59, 26/59;

hyperkeratosis -5/57, 13/59, 17/59

Liver granulomatous inflammation -0/60, 16/59, 14/60

Various organs: pigmentation

Glandular stomach

erosions -2/60, 11/60, 10/60;

inflammation -0/60, 4/60,2/60,

hyperplasia -0/60, 24/60,23/60;

metaplasia -0/60, 36/60, 51/60;

fibrosis -0/60, 39/60,51/60;

fat proliferation -0/60, 15/60, 41/60

Forestomach acanthosis (epithelial

hyperplasia) -11/60,20/60, 27/60;

hyperkeratosis -10/60, 23/60, 21/60

Liver: granulomatous inflammation - 6/60,24/60, 33/60

Various organs: pigmentation

Neoplastic effects

Forestomach:

squamous cell papilloma - 0/60, 4/60,1/60;

squamous cell carcinoma - 0/60, 1/60,0/60;

basosquamous tumor benign - 0/60, 0/60, 1/60

Forestomach:

squamous cell papilloma -0/60, 1/61,2/60

Level of evidence of carcinogenic activity

Equivocal evidence

Equivocal evidence

Conclusions:
Under the conditions of these 2-year gavage studies, there was equivocal evidence* of carcinogenic activity' of titanocene dichloride in male F344/N rats based on a marginal increase in the incidence of fore-stomach squamous cell papillomas, squamous cell carcinoma, and basosquamous tumor benign. There was equivocal evidence of carcinogenic activity of titanocene dichloride in female F344/N rats based on a marginal increase in the incidence of fore-stomach squamous cell papillomas.
Nonneoplastic lesions associated with the administration of titanocene dichloride for up to 2 years included erosions and inflammation of the gastric mucosa,hyperplasia and metaplasia of the fundic glands with fibrosis of the lamina propria in the glandular stomach, and acanthosis (hyperplasia) and hyperkeratosis of the forestomach epithelium.

*Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of neoplasms that may be chemically related.
Executive summary:

Toxicology and carcinogenesis studies were conducted by administering titanocene dichloride (greater than 98% pure) in corn oil by gavage to groups of F344/N rats for 2 years. Body Weight and Survival in the 2-Year Studies The doses selected for the 2-year studies in rats (0, 25, and 50 mgkg) were based on the potentially life-threatening nature of the glandular stomach lesions and the decreased body weight gain seen in 13-week studies. The final mean body weights of high-dose males and females were 91% and 89% of controls, respectively. The 2-year survival rates for males in the control, low-, and high-dose groups were 41/60, 30/60, and 24/60;survival rates for female rats were 37/60, 30/61, and 31/60. Non-neoplastic and Neoplastic Effects in the 2-Year Studies The principal toxic effects associated with the administration of titanocene dichloride for 2 years occurred in the stomach. The lesions in the stomach were seen at the 15-month interim evaluations and were similar to, but less severe than, those observed at 2 years. The lesions included focal erosions of the glandular mucosa with an associated inflammatory response, hyperplasia and metaplasia of the epithelium of the fundic glands, and fibrosis of the lamina propria and submucosa. Forestomach lesions included focal acanthosis (hyperplasia) and hyperkeratosis of the stratified squamous epithelium. Squamous cell papillomas of the forestomach were seen in four low-dose males, one high-dose male, one low-dose female, and two high-dosefemales; none were observed in controls. A squamous cell carcinoma of the forestomach occurred in one low dose male and a benign basosquamous tumor occurred in one high-dose male. Accumulations of macrophages with blue-gray pigment believed to contain titanium were present in many organs of dosed rats including the gastro-intestinal tract, liver, lung, and lymph nodes. A doserelated

increase in the incidence of inflammation of the nasal mucosa and lung also occurred and was attributed to reflux and/or regurgitation and aspiration of gavage solution due to the severe stomach lesions.

Under the conditions of these 2-year gavage studies:

- There was equivocal evidence of carcinogenic activity of titanocene dichloride in male F344/N rats based on a marginal increase in the incidence of fore-stomach squamous cell papillomas, squamous cell carcinoma, and basosquamous tumor benign.

- There was equivocal evidence of carcinogenic activity of titanocene dichloride in female F344/N rats based on a marginal increase in the incidence of fore-stomach squamous cell papillomas.

Nonneoplastic lesions associated with the administration of titanocene dichloride for up to 2 years included erosions and inflammation of the gastric mucosa, hyperplasia and metaplasia of the fundic glands with fibrosis of the lamina propria in the glandular stomach, and acanthosis (hyperplasia) and hyperkeratosis of the forestomach epithelium.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
25 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
One K1 study is available
System:
gastrointestinal tract
Organ:
stomach

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

References:

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.

 

Proctor et al., (2007) REVIEW Mode-of-Action Framework for Evaluating the Relevance of Rodent Forestomach Tumors in Cancer Risk Assessment. TOXICOLOGICAL SCIENCES 98(2), 313–326

 

Kroes, R. and Wester, P. W. (1986)Forestomach carcinogens: possible mechanisms of action.Fd Chern. Toxic.Vol. 24, No.l0/11, pp. 1083-1089

Justification for classification or non-classification

Based on the available information the substance is not classified for this endpoint.