Titanium tetrachloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study design is not guideline-conform: two dose levels only; satellite group missing; body weight and food consumption measured once a week during the first 13 weeks; haematology, urinalysis, and clinical chemistry missing; findings of gross pathology not presented: histopathology incomplete (large intestine, sin, and peripheral nerve missing)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 50 male and 50 female B6C3F1 mice each were fed a diet containing 2% corn oil and 25000 or 50000 ppm titanium dioxide for 103 weeks (7 days per week). A control group receiving corn oil in the diet was run concurrently. After the administration period the animals were observed for 1 additional week. The following parameters were assessed and presented: clinical signs, mortality, detailed clinical observations, body weight, and histopathology.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Titanium dioxide

EC Number:

236-675-5

EC Name:

Titanium dioxide

Cas Number:

13463-67-7

Molecular formula:

O2Ti

IUPAC Name:

dioxotitanium

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): titanium dioxide anatase (designated Unitane® 0-220; obtained from American Cyanamid Company, Wayne, New Jersey)
- State of aggregation: white pigment
- Analytical purity: min. 98 %
- Moisture content: < 0.4 %
- Lot Nos.: 402129A29 (used from weeks 0 - 51) and 402129B20 (used from weeks 52 - 102)

- Impurities (identity and concentrations):
Lot 402129A29: niobium, chlorine, and phosphorus in the range 0.1 - 1.0 %
Lot 402129B20: niobium, chlorine, phosphorus, silicon, and potassium in the range 0.1 - 1.0 %

- Composition of test material, percentage of components:
Lot 402129A29: 1.6 % titanium
Lot 402129B20: 1.5 % titanium

Specific details on test material used for the study:

not applicable

Test animals

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center, Maryland
- Age at study initiation: 36 days
- Housing: housed in polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets; mice were housed 5/cage; bedding material: heat-treated harwood chip bedding (Sani-Chips®)
- Diet (ad libitum): basal diet of Wayne® Lab Blox animal meal (Allied Mills, Inc., Chicago, Ill.)
- Water (ad libitum): well water
- Quarantine period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 45 - 55 %
- Air changes: 12/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

not specified

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with basal diet of Wayne® Lab Blox animal meal: a quantity of the bulk chemical was sifted to remove any large particles, and the amount required for each dose mixture was weighed out under a hood. This quantity was then incorporated into the diet by thorough mixing in a Patterson-Kelly twin-shell blender equipped with an intensifier bar. Corn oil was added to the dosed diets and to the diets for the matched controls to give a final concentration of 2 %.
- Rate of preparation of diet (frequency): once per week
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

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)*.
Duplicate 100 mg subsamples of feed were ashed, and the residues fused with 2 g of potassium pyrosulfate. The fusion mixture was quantitatively transferred to a 100 mL volumetric flask using a 1:1 mixture of sulfuric acid and water, and diluted to volume with water. With a Tiron indicator, the transmittance of this solution was read at 410 nm. Concentrations of titanium dioxide were determined by comparison with standard solutions.
Recoveries were also determined from duplicate analyses of spiked samples worked up simultaneously with each set of dosed feed samples. The average recovery from the 2.5 % spiked samples was 97.5 %, and from the 5.0 % spiked samples, 100.3 %.

Results:
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.

Theoretical concentrations in diet: 2.5 and 5.0 % in diet
Sample analytical mean: 2.4 and 4.9 % in diet (coefficient of variation: 26.3 and 29.5 %, respectivley)
Range: 2.2 - 2.9* and 4.79 - 6.85*
*Ranges exclude the two samples at each level during weeks 35 and 45 which analysed at only 40 - 50 % of the theoretical; these samples were included in the Number of Samples, Sample Analytical Mean, and Coefficient of Variation.

*Reference:
- Association of Official Analytical Chemists, Official Methods of Analysis of the Association of Official Analytical Chemists, 12th edition, Horwitz, W., ed., Association of Offical Analytical Chemists, Washington, B.C., 1975, p. 7.ebrc09

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

7 days/week (ad libitum)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 000 ppm

Remarks:

equivalent to 3750 mg/kg/day (recalculated from ppm value (factor (mouse): 0.150)

Doses / concentrations 2

Dose / conc.:

50 000 ppm

Remarks:

equivalent to 7500 mg/kg/day (recalculated from ppm value (factor (mouse): 0.150)

No. of animals per sex per dose:

50 males / 50 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: subchronic feeding study was conducted to estimate the maximum tolerated doses of titanium dioxide, on the basis of which two concentrations were selected for administration in the chronic study. On the basis of results from a 14-day (repeated dose) oral range-finding study, doses of 6250, 12500, 25000, 50000, or 100000 ppm were administered in the diet in the subchronic study. Ten male and 10 female mice 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 for mice in the chronic study was set at 50000 ppm and the low dose was set at 25000 ppm.

- Rationale for animal assignment: animals were assigned to the dosed or control groups based on initial individual body weight, so that the mean body weights per group were approx. equal.

- Post-exposure period: 1 week

Positive control:

not examined

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: signs of toxicity and survival

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: every week

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

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

FOOD EFFICIENCY:
- 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: No

WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Animals that were moribund and those that survived to the termination of the study were killed. 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, embedded in paraffin, sectioned, and stained. 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), oesophagus, trachea, salivary glands, mandibular lymph node, thyroid, parathyroid, heart, thymus, lungs and mainstem bronchi, liver, gallbladder, 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.
A few tissues from some animals were not examined, particularly from those animals that died early. Also, some animals may have been missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathologic evaluation.

Statistics:

Product-limit procedure of Kaplan and Meier, method of Cox, Tarone's extensions of Cox's methods, linearity test, one-tailed Fisher exact test, Bonferroni inequality, Cochran-Armitage test for linear trend in proportions with continuity correction, and life-table methods

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

- female mice: the result of the Tarone test for dose-related trend in mortality shows a significant (P = 0.001) positive dose-related trend.
- female mice: 33/50 (66%) of the 50000 ppm dose group, 39/50 (78%) of the 25000 ppm dose group, and 45/50 (90%) of the matched controls were alive at week 104.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- 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. Alopecia was more often 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.
- animals in all of the dosed groups had white faeces.

MORTALITY
- male mice: the result of the Tarone test for dose-related trend in mortality is not significant.
- male mice: forty out of fifty (80%) of the 50000 ppm dose males, 40/50 (80%) of the 25000 ppm dose males, and 32/50 (64%) of the matched-control males were still alive at week 104.

BODY WEIGHT AND WEIGHT GAIN
- administration of titanium dioxide had no appreciable effect on the mean body weights of either the male or the female mice

HISTOPATHOLOGY: NON-NEOPLASTIC (data from the study were put in context with historical control data by the evaluator of this study)
The study describes several non-neoplastic findings observed in mice without however attributing any adversity to these. By putting these into context with historical control data, the effects can be considered to lack toxicological significance. The findings can be summarised briefly as follows:

- spleen haematopoiesis: no effects in mice (6-10% M / 4-8% F)

- kidney chronic inflammation: male mice only; incidence 8% (low) and 10% (high) vs. 6% in control; females not affected (0-6%)
historical controls (HC)* male mice (lymphatic infiltration used as correlate for chronic inflammation): 24.5%
Conclusion: the findings observed in male mice can be considered to be within the historical control data of this strain taken from a publication.

- liver necrosis: male mice only; incidence 16% (high) vs. 0% in control and low dose; females (0-2%) not affected
HC 7% M 5.9%F
Conclusion: the incidence in male mice is higher than historical control data for this strain of mice, but it needs to be noted that the incidence is already much higher than the HC in the concomitant control group, and the incidence in the low and high dose group is not dose related. It is therefore considered implausible that treatment with the test item is responsible for these findings observed in male mice.

- Uterus/endometrium cystic hyperplasia: mice only; incidence 86% (low) and 78% (high) vs. 35% in control (no dose relation)
Conclusion: the incidence observed in the control, low and high dose groups is within the range of historical control data.

HISTOPATHOLOGY: NEOPLASTIC
- sufficient numbers of mice of each sex were at risk for the development of late-appearing tumors.
- 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 50000 ppm dose males. The incidence of tumours 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.

- the results of the Cochran-Armitage test for positive dose-related trend in incidences of tumors and those of the Fisher exact test for higher incidences of tumors in dosed groups than in control groups are not significant for any type of tumor occurring in either sex.
- a significant trend (P = 0.037) in the negative direction is observed in the incidence of follicular-cell adenomas of the thyroid in female mice, in which the incidence in the control group exceeds the incidences in the dosed groups.
- results of the Fisher exact test (P = 0.035 in the negative direction) for the comparison of the incidence of combined lymphomas and leukemias in the female 25000 ppm dose group with that in the corresponding controls are above that of 0.025 required for significance in multiple comparisons. This negative result may be accounted for by the difference in survival, since the dosed animals did not live as long as the control animals.
- in each of the 95% confidence intervals of relative risk the value of one is included; this indicates the absence of significant positive results. It should also be noted that each of the intervals has an upper limit greater than one, indicating the theoretical possibility of the induction of tumors by titanium dioxide, which could not be detected under the conditions of this test.

*Sources for historical control data:
Hirouchi Y., et al. (1994): Historical Data of Neoplastic lesions in B6C3F1 mice, J. Toxicol Pathol 7: 153-177
Goodman D.G., et al. (1978): Neoplastic and Non-neoplastic Lesions in Aging F344 Rats, Tox and Appl Pharmacol 48, 237-248
Coleman G.L. (1977): Pathological Changes During Aging in Barrier-reared Fischer 344 Male Rats, J Gerontol 32 (3):258-278

Effect levels

1

Dose descriptor:

NOEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOEL

Effect level:

7 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: recalculated from the ppm value in food with the factor of 0.150 (mouse)

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOEL (systemic toxicity; mice): 50000 ppm (equivalent to 7500 mg/kg/day)

According to the study authors, there was no clinical sign that was judged to be related to titanium dioxide exposure, with the exception of white faeces. The study describes several non-neoplastic findings observed both in mice, without however attributing any adversity to these. By putting these into context with historical control data, the effects can be considered to lack toxicological significance. The findings can be summarised briefly as follows:

- spleen haematopoiesis: no effects in mice (6-10% M / 4-8% F)

- kidney chronic inflammation: male mice only; incidence 8% (low) and 10% (high) vs. 6% in control; females not affected (0-6%)
historical controls (HC)* male mice (lymphatic infiltration used as correlate for chronic inflammation): 24.5%
Conclusion: the findings observed in male mice can be considered to be within the historical control data of this strain taken from a publication.

- liver necrosis: male mice only; incidence 16% (high) vs. 0% in control and low dose; females (0-2%) not affected
HC 7% M 5.9%F
Conclusion: the incidence in male mice is higher than historical control data for this strain of mice, but it needs to be noted that the incidence is already much higher than the HC in the concomitant control group, and the incidence in the low and high dose group is not dose related. It is therefore considered implausible that treatment with the test item is responsible for these findings observed in male mice.

- Uterus/endometrium cystic hyperplasia: mice only; incidence 86% (low) and 78% (high) vs. 35% in control (no dose relation)
Conclusion: the incidence observed in the control, low and high dose groups is within the range of historical control data.