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Repeated dose toxicity: oral

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Administrative data

chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
A bioassay of titanium dioxide for possible carcinogenicity was conducted by administering the test chemical in the diet to Fischer 344 rats and B6C3F1 mice for 103 weeks.
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium dioxide
EC Number:
EC Name:
Titanium dioxide
Cas Number:
Molecular formula:
Titanium dioxide
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Titanium dioxide
- Substance type: inorganic
- Physical state: solid; white pigment possessing great covering or opacifying power
- Analytical purity: > 98 %
- Lot/batch No.: Lot Nos. 402110C46, 402129A29, 402129B20; Three lots of titanium dioxide anatase, designated Unitane® 0-220, were obtained from American Cyanamid Company, Wayne, New Jersey., USA.
- Other: Moisture content < 0.4 %

Test animals

Details on test animals or test system and environmental conditions:
On arrival at the laboratory, the rats were quarantined for 30 days and the mice for 15 days, determined to be free from observable disease or parasites, and assigned to the dosed or control groups based on initial individual body weight, so that the of mean animal body weights per group were approximately equal.
- Source: Frederick Cancer Research Center, Maryland, USA
- Age at study initiation: 36 days
- Weight at study initiation: not reported
- Fasting period before study: not reported
- Housing: in polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets. Mice were housed 5 per cage throughout the study.
- Diet: ad libitum
- Water: ad libitum

- Temperature (°C): 20-24
- Humidity (%): 45-55
- Air changes (per hr): 12
- Photoperiod: 12 hours dark/light cycle

Administration / exposure

Route of administration:
oral: feed
corn oil
Details on oral exposure:
- Rate of preparation of diet (frequency): Diets were prepared once per week and stored at room temperature until used.
- Mixing appropriate amounts with (Type of food): TiO2 was incorporated into the basal diet of Wayne® Lab Blox animal meal (Allied Mills, Inc., Chicago, 111.) by thorough mixing in a Patterson-Kelly twin-shell blender equipped with an intensifier bar.
- Storage temperature of food: RT

VEHICLE: corn oil
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: Corn oil (Duke's, C. F. Sauer Co., Richmond, Va.) was added to the dosed diets and to the diets for the matched controls to give a final concentration of 2%.
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
As a quality control measure, selected samples from freshly prepared mixtures were stored at 4°C and aliquots from these samples, containing approximately 50 micrograms of titanium dioxide were later analyzed for titanium dioxide by the method described by the Association of Official Analytical Chemists (1975). At each dietary concentration, the mean value obtained by the analytical method was within 4% of the theoretical value, although the coefficient of variation was nearly 30%. This variation appears to be due to the difficulty in obtaining a homogeneous mix of a fine powder in feed.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Doses / Concentrations:
0 ppm
nominal in diet
Doses / Concentrations:
25000 ppm
nominal in diet
Doses / Concentrations:
50000 ppm
nominal in diet
No. of animals per sex per dose:
50 males and 50 females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Subchronic feeding studies were conducted to estimate the maximum tolerated doses of titanium dioxide, on the basis of which two concentrations were selected for administration in the chronic studies. On the basis of results from a 14-day (repeated dose) oral range-finding study, doses of 6,250, 12,500, 25,000, 50,000, or 100,000 ppm were administered in the diet in the subchronic studies. Ten males and 10 females of each species were administered the test chemical at each dose, and 10 males and 10 females received basal diets. Dosed animals received the test compound for 13 consecutive weeks. There were no deaths, and dosed animals had mean body weight gains that were comparable to those of the controls. No gross or microscopic pathology was found that could be related to the administration of the test chemical. On the basis of these results, the high dose in the chronic studies was set at 50,000 ppm, the maximum amount allowed for use in chronic bioassays in the Carcinogenesis Testing Program, and the low dose was set at 25,000 ppm.
Positive control:


Observations and examinations performed and frequency:
- Time schedule: All animals were observed twice daily for signs of toxicity. Clinical signs and the presence of palpable masses were recorded every week.

- Time schedule: weekly

- Time schedule for examinations: every 2 weeks for the first 12 weeks and every month thereafter.

- Food consumption was recorded every 2 weeks for the first 12 weeks and every month thereafter.

- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data






Sacrifice and pathology:
The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions from killed animals and from animals found dead. The tissues were preserved in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: brain (frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), pituitary, spinal cord (if neurologic signs were present), eyes (if grossly abnormal), esophagus, trachea, salivary glands, mandibular lymph node, thyroid, parathyroid, heart, thymus, lungs and mainstem bronchi, liver, gallbladder (mice), pancreas, spleen, kidney, adrenal, stomach, small intestine, colon, urinary bladder, prostate or uterus, testes or ovaries, sternebrae, femur, or vertebrae including marrow, mammary gland, tissue masses, and any gross lesion.
Other examinations:
not reported
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) extensions of Cox's methods for testing for a dose-related trend. One-tailed P values have been reported for all tests except the departure from linearity test, which is only reported when its two-tailed P value is less than 0.05.
To determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals the one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor incidence of a control group with that of a group of dosed animals at each dose level. When results for a number of dosed groups (k) are compared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made. The Bonferroni inequality (Miller, 1966) requires that the P value for any comparison be less than or equal to 0.05/k. In cases where this correction was used, it is discussed in the narrative section. It is not, however, presented in the tables, where the Fisher exact P values are shown.
The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used when appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The clinical signs observed in the dosed groups were comparable with those of the control group and included protrusion of the eyes, bloody crust surrounding the eyes, palpable nodules, tissue masses and/or wart-like lesions, localized sores, irritation and swelling of the testes, hunched appearance, and/or thinness. Alopecia (localized or generalized) was noted in all the control and dosed groups; however, more was observed in the control females than in the dosed females. The areas of alopecia were primarily located around the nose and head and progressed to generalized alopecia in some of the animals. The type of feed-hopper used in this study may have caused the alopecia around the nose. Animals in all of the dosed groups had white feces.
mortality observed, treatment-related
Description (incidence):
In male mice, the result of the Tarone test for dose-related trend in mortality is not significant, but in females, the result of the Tarone test shows a significant (P = 0.001) positive dose-related trend. Forty out of fifty (80%) of the high-dose males, 40/50 (80%) of the low-dose males, and 32/50 (64%) of the matched-control males were still alive at week 104. In females, 33/50 (66%) of the high-dose group, 39/50 (78%) of the low-dose group, and 45/50 (90%) of the matched controls were alive at week 104.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Administration of TiO2 had no effect on the mean body weights of either the male or the female mice.
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 examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
A low incidence of neoplasia was observed in both the control mice and dosed mice. These neoplasms were of the usual number and type observed in mice of this age and strain. A slightly increased number of hepatocellular carcinomas was observed in the high-dose males; the incidence of tumors was not increased over that observed in historical-control groups of mice of this age and strain. Degenerative, proliferative, and inflammatory lesions were also of the usual number and kind observed in aged B6C3F1 mice.

Effect levels

Dose descriptor:
Effect level:
50 000 ppm
Based on:
test mat.
Basis for effect level:
other: With the exception of white faeces, there was no other clinical sign that was judged to be related to titanium dioxide exposure.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

In the male and female mice, no tumors occurred in dosed groups at incidences that were significantly higher than those for corresponding control groups.

Applicant's summary and conclusion

Based on the histopathological examination, TiO2 was neither toxic nor carcinogenic to mice under the conditions of this bioassay.
Executive summary:

In a chronic toxicity study titanium dioxide (> 98% purity) was administered to 50 B6C3F1 mice per sex and dose in the diet at dose levels of 0, 25000, and 50000 ppm for 103 weeks.

Administration of titanium dioxide had no appreciable effect on the mean body weights of mice of either sex. With the exception of white feces, there was no other clinical sign that was judged to be related to the administration of titanium dioxide. Survival of the male mice at the end of the bioassay was not affected by the test chemical; mortality in female mice was dose related. Sufficient numbers of dosed and control mice of each sex were at risk for development of late-appearing tumors.

A NOAEL of 50000 ppm was established. Based on the histopathological examination, titanium dioxide was neither toxic nor carcinogenic to mice under the conditions reported.