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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted in 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: NTP protocol
Version / remarks:
Details can be found: https://ntp.niehs.nih.gov/testing/types/cartox/index.html
Principles of method if other than guideline:
Groups of 10 rats of each sex were administered 0, 8, 16, 31, 62, or 125 mgkg titanocene dichloride in corn oil by gavage 5 days per week for 13 weeks. Additional groups of 5 rats per sex received 0, 31, or 125 mgkg titanocene dichloride on the same schedule and were used for the determination of tissue residues of titanium. See methods below for further information
GLP compliance:
yes
Remarks:
FDA Good Laboratory Practice Regulations (21 CFR Part 58)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch number 0708501022
Description Red crystalline solid
See certificate of analysis:
Purity 99.8%
Storage requirements Store under a nitrogen atmosphere in a cool, dry, well-ventilated area away from flammable materials and sources of heat or flame.
Expiration date September 1, 2012
Specific details on test material used for the study:
Lot no. PB013180 from Pfaltz and Bauer, Inc. (Waterbury, CT)
The study chemical, a dark red, microcrystalline solid, was identified as titanocene dichloride by infrared and nuclear magnetic resonance spectroscopy. Lot no.PB013180 was greater than 98% pure, as determined by titration and elemental analysis.
Stability studies performed by titration indicated that titanocene dichloride was stable as a bulkb chemical for at least 2 weeks at temperatures to 60º C 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 bulk chemical was monitored by elemental analysis and by titration periodically during all phases of the studies. No change in the study material was detected.

Test animals

Species:
rat
Strain:
other: F344/N
Details on species / strain selection:
Male and female F344/N rats were obtained from Frederick Cancer Research Facility (Frederick, MD). Animals were observed for 5 to 20 days, distributed to weight classes, and assigned to groups according to tables of random numbers. The rats were 8 to 9 weeks old when the study began.
Sex:
male/female
Details on test animals and environmental conditions:
Rats were housed five per cage. Feed and water were available ad libitum. Animals were observed twice daily for morbidity and mortality. Moribund animals were killed and necropsied. Individual animal weights were recorded at study initiation, weekly throughout the dosingperiod, and at the end of the study.

Method of Animal Identification
Ear punch

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

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

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 (American Excelsior Co., Baltimore, MD), 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: 19.4ºC-25.0º C
Humidity: 11%-50%
Light: fluorescent, 12 hour/day
Room air changes greater than 12 changes/hour

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Groups of 10 rats of each sex were administered 0, 8, 16, 31, 62, or 125 mgkg titanocene dichloride in corn oil by gavage 5 days per week for 13 weeks. Additional groups of 5 rats per sex received 0, 31, or 125 mgkg titanocene dichloride on the same schedule and were used for the determination of tissue residues of titanium.
Vehicle:
corn oil
Details on oral 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 ultraviolet 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:
Studies of the stability and homogeneity of dose formulations were performed at MRI. Samples were diluted in chloroform, filtered through a 0.5 µ Millipore filter into 5 mL septum vials, and analyzed by high performance liquid chromatography using a Varian 5000 liquid chromatograph with an ultraviolet detector (254 nm) and a mobile phase of 20% hexane and 80% chloroform:tetrahydrofuran (80:20). No decrease in titanocene dichloride concentration was found after storage of the solutions for 2 weeks in the dark at 5º C or 25º C, or under simulated animal dosing conditions (open to air and light for 3 hours). During the studies, the dose formulations were stored at 0 ± 5º C, brought to room temperature, and hand agitated prior to administration. Unused formulations were discarded 14 days from the date of preparation.

The study laboratory conducted periodic analyses of the titanocene dichloride dose formulations using high performance liquid chromatography or ultraviolet spectroscopy.
During the 13-week studies, samples were also diluted in chloroform, but the three lowest concentrations (0.8 to 3.1 mg/mL) were analyzed by high-performance liquid chromatography and the three highest concentrations (6.2 to 25.0 mg/mL) were analyzed by ultraviolet spectroscopy. At the end of the subchronic studies, ultraviolet spectroscopy was used to analyze all dose formulatiom, but the diluent used was acetonitrile instead of chloroform and absorbance was measured at 252 nm instead of 256 nm. All measured concentration were within 10 % of the target concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
8 mg/kg bw/day (nominal)
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
31 mg/kg bw/day (nominal)
Dose / conc.:
62 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males 10 females; an and additional 5 per sex in control, mid- and highdose- groups for tissue residue studies
Control animals:
other: negative control, not specified if vehicle or no treatment
Details on study design:
After 13 weeks, all surviving animals were killed. A complete necropsy was performed on all animals except those used for the determination of titanium levels in tissues. A complete histopathologic examination was performed on all core study animals in the control and 125 mgkg dose groups, and selected tissues were examined from animals in the lower dose groups. Prior to processing, organ weights were determined for brain, heart, right kidney, liver, lung,thymus, and right testis (males) of all core study animals. The heart, liver, lungs, and spleen were collected from the animals predesignated for the titianium tissue level studies and were frozen and stored at -70º C. The frozen tissue samples were sent to MRI for analysis of titanium residues by inductively coupled plasma-atomic emission spectroscopy after a wet digestion procedure.

Examinations

Observations and examinations performed and frequency:
Observed twice daily. Clinical observations recorded as necessary. Individual bodyweights recorded at study initiation, weekly during dosing, and at study termination.
Sacrifice and pathology:
Necropsy
Complete necropsy performed on all animals except those used for the determination of titanium residues. Organ weights were recorded for brain, heart, right kidney, liver, lung, thymus, and right testis (males).

Histopathology
Histopathologic examinations were performed on all animals dying early, controls, and high dose animals. Tissues examined included gross lesions and tissue masses, blood smear, mandibular and mesenteric lymph nodes, salivary glands, heart, esophagus, stomach, brain, sternebrae (including marrow), thyroid gland, parathyroid glands, small intestine, cecum, colon and rectum, liver, testes, epididymis, prostate gland, seminal vesicles, ovaries and uterus, lungs and bronchi, nasal cavity and nasal turbinates, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary gland, mammary gland, skin, and preputial or clitoral glands. Tissues were examined in animals in the lower dose groups. These included the spleen and stomach of females given 8 to 62 mg/kg.

Supplemental Studies
Sections of heart, liver, lung, and spleen from 5 animals of each sex in control, 31, and 125 mg/kg groups were evaluated for titanium residues.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat given 125 mg/kg died during week 4 of treatment and one control male died during week 9 due to a cage maintenance accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weights and the mean body weight changes for male and female rats in the 62 and 125 mg/kg dose groups were significantly lower than control. Rats given 62 mg/kg weighed 8% to 10% less than controls at the end of the study,while rats given 125 mg/kg weighed 13% to 20% less than controls. Bodyweights for the other dose groups were similar to controls. All females given 125 mg/kg appeared thin and pale during the studies, and dyspnea was present in 2 of the 15 high-dose females.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significant negative trends were observed for the absolute mean weights of the heart, liver, and thymus in male rats and of the thymus in female rats. Mean heart weights were significantly lower than the control for all groups of dosed males except those given 31 mg/kg titanocene dichloride. Mean liver weight was significantly lower than control for males given 125 mg/kg only, and mean thymus weights were significantly lower than control for male and female rats in the 62 and 125 mg/kg dose groups. Males given 125 mg/kg had significantly lower relative liver and thymus weights, and males given 8 mg/kg had significantly lower relative heart weights than control. The absolute and relative organ weight changes were considered related to the lower body weights of treated animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Lesions associated with the administration of titanocene dichloride were observedonlyin the stomach of rats. Hyperplasia and/or hyperkeratosis of the squamous epithelium of the forestomach involved the limiting ridge and the immediately adjacent area. It consisted of an increased number of basophilic cells in the basal cell layers and slight thickening of the overlying keratin layer. There were also superficial erosions of the glandular stomach mucosa resulting from focal necrosis of the surface epithelium and the subjacent gastric pits and upper portions of the gastric glands. An acute inflammatory response was usually associated with these lesions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Regenerative hyperplasia of the mucosa of the stomach was also observed and was characterized by the replacement of mucous cells in the surface epithelium and gastric pits and parietal and chief cells in the gastric glands by less differentiated cells with enlarged nuclei. The affected glands had dilated lumens and irregular profiles. In a few scattered gastric glands of some rats, the parietal and chief cells were replaced by cells resembling pancreatic acinar cells (metaplasia). They had large nuclei with basophilic cytoplasm in the basal region of the cell and bright eosinophilic granules in the apical region.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
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

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic effects
Effect level:
31 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Remarks:
Local effects
Effect level:
< 8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
8 mg/kg bw/day (nominal)
System:
other: digestive system
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

 Lesions of the Stomach in Rats in the13-Week Gavage Studies of Titanocene Dichloride           

Organs and Diagnosis

Vehicle control

8 mg/kg

16 mg/kg

31 mg/kg

62 mg/kg

125 mg/kg

Males

Forestomach(a)

(10)

-(b)

-

-

-

(10)

Epithelial hyperplasia

0

 

 

 

 

3

Hyperkeratosis

0

 

 

 

 

4*

Glandular stomach

(10)

-

-

-

-

(10)

Hyperplasia (c)

0

 

 

 

 

7**

Erosion

0

 

 

 

 

9**

Fibrosis

0

 

 

 

 

4*

Metaplasia

0

 

 

 

 

5*

Inflammation

0

 

 

 

 

10**

Females

Forestomach(a)

(10)

(10)

(10)

(10)

(10)

(10)

Epithelial hyperplasia

0

6**

6**

7**

6**

3

Hyperkeratosis

0

2

6**

6**

4*

8**

Glandular stomach

(10)

(10)

(10)

(10)

(10)

(10)

Hyperplasia (c)

0

2

1

5*

6**

8**

Erosion

0

2

3

4*

4*

9**

Fibrosis

0

0

0

0

0

6**

Metaplasia

0

0

0

2

3

7**

Inflammation

0

5*

7**

6**

8**

9**

 

* Significantly different (P≤0.05) from the control group by Fisher exact test

** P≤0.01

(a) The number in parentheses is the number of animals with organ examined microscopically. More than one lesion may occur within the same organ.

(b) Tissue not examined for this dose group

(C) Lesion diagnosed as dysplasia by the laboratory pathologist.

Applicant's summary and conclusion

Conclusions:
In the 13-week studies there was no dose-related mortality, but decreased body weight gain was seen in the 62 and 125 mg/kg dose groups. Toxic lesions were observed in the forestomach of 125 mg/kg animals, and erosions, fibrosis, hyperplasia, and metaplasia of the glandular stomach were present in females given 31, 62, or 125 mg/kg titanocene dichloride and in males receiving 125 mg/kg. Although histologic examination of the stomach was not performed for males in the 8, 16, 31, or 62 mg/kg dose groups, the stomach lesions in females from the 31 mg/kg dose groups were minimal in severity.
Executive summary:

The 13-week studies were conducted by administering titanocene dichloride at doses of 0, 8, 16, 31, 62, or 125 mg/kg bw/d. One female rat in the 125 mg/kg bw/d dose group died from chemical toxicity during the fourth week of the studies. Bodyweight gain was lower in rats given 62 or 125 mg/kg bw/d than in control groups. Treatment associated histopathologic lesions were seen in the stomachs of high dose males and all groups of females given titanocene dichloride. These lesions included hyperplasia and metaplasia of the glandular stomach and hyperplasia and hyperkeratosis of the forestomach.