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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

repeated dose toxicity: inhalation
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1981-07-09 to 1979-06-04
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented study.
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
Pulmonary Responses of Rats Exposed to Titanium Dioxide (TiO2) by Inhalation for Two Years.
Lee, K.P.; et al.
Bibliographic source:
Toxicol Appl Pharmacol 79:179-192
Reference Type:
Characterization and reclassification of TiO2-related pulmonary lesions
Warheit D.B.; Frame S.R.
Bibliographic source:
J. Occup. Env. Med. 48, 1308-1313

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Investigation of potential adverse effects of TiO2 in rats after chronic inhalation exposure.
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:
Constituent 2
Reference substance name:
Rutile (TiO2)
EC Number:
EC Name:
Rutile (TiO2)
Cas Number:
Details on test material:
- Name of test material (as cited in study report): Titanium dioxide
- Physical state: solid
- Analytical purity: 99.0%
- Impurities (identity and concentrations): 1.0% cooxidised Al2O3

Test animals

Crj: CD(SD)
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories, Wilmington, Mass.)
- Age at study initiation: 3 weeks old
- Weight at study initiation: 70.5 g (males) and 70.7 g (females)
- Housing: Animals were housed pair wise (separate sexes) in stainless steel wire-mesh cages.
- Diet: ad libitum, Purina Rodent Chow
- Water: ad libitum
- Acclimation period: approximately 17 days

- Temperature (°C): 23 +/- 2
- Humidity (%): 50 +/- 10
- Photoperiod: 12 hours dark/light cycle
No further details are given.

Administration / exposure

Route of administration:
Type of inhalation exposure:
whole body
other: air
Remarks on MMAD:
MMAD / GSD: 1.5 - 1.7 µm; 84 % < 13 µm MMD
Details on inhalation exposure:
- Exposure apparatus: inhalation chambers made of metal (not reactive with TiO2)
- Chamber volume: 3.84 m^3
- Method of holding animals in test chamber:
- System of generating particulates/aerosols: Atmospheres of TiO2 were generated by metering the dust into an apparatus containing a vertical elutriator connected in series to a settling chamber. An ACCU-RATE, Model 502, variable-speed screw-feeder was used to meter TiO2 dust into a Plexiglas sample-delivery tube attached perpendicularly to the vertical axis of the elutriator. The dust was dispersed by an air jet directed along the sample delivery tube axis and passed into the elutriator. Initial settling of the heavier nonrespirable dust particle took place in the elutriator; the lighter particles passed into the settling chamber from which the respirable particles were diverted into the exposure chamber. Chamber concentrations were maintained by controlling the TiO2 delivery rate into the generation apparatus and by diluting the dust particle stream as it entered the chamber.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity of the exposure chambers were targeted at 23 +/- 2°C and 50 +/- 10%, respectively. These were measured at least once daily.
- Air flow rate: >800 L/min

- Particle size distribution: yes. Aerodynamic particle sizing was performed for at least seven exposures in each chamber over the course of the study. Two types of in-stack cascade impactors were used for these determinations: Monsanto 5-Stage Impactor with Cyclone Preseparator and Sierra, Model 210, 8-Stage Impactor with Cyclone Preseparator. The mass median aerodynamic diameter, geometric standard deviation and the fraction of respirable particles were determined graphically. Those particles with a MMD of 10 µm or less were considered respirable.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.5 - 1.9 µm.
- The TiO2 in the test atmospheres had a mean respirable fraction of 93.7% or greater.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Chamber concentrations were determined gravimetrically. Approximately every half hour and from each exposure chamber, a predetermined volume of chamber atmosphere was drawn through a preweighed Gellman, Type-A/E, glass-fibre filter, 47 mm diameter. Each chamber concentration was calculated from the net weight of TiO2 collected on the filter. The mean daily chamber concentrations were calculated as the time-weighted averages (TWA) over each 6-hour exposure period.
Duration of treatment / exposure:
up to 2 years
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Doses / Concentrations:
10.6 ± 2.1 mg/m³
analytical conc.
Doses / Concentrations:
50.7 ± 6.65 mg/m³
analytical conc.
Doses / Concentrations:
250.1 ± 24.7 mg/m³
analytical conc.
No. of animals per sex per dose:
4 groups of 100 male and 100 female rats
Control animals:
Details on study design:
- Dose selection rationale: Results from previous inhalation studies.
- Rationale for animal selection: Selection of the CD rat was based on extensive experience with the strain and its suitability relative to longevity, hardiness, sensitivity and low incidence of spontaneous disease.
- Rationale for animal assignment: Rats of each sex were divided by computerised, stratified randomisation into groups of 100 males and groups of females such that the mean of body weights of each group of rats within a sex were approximately equal.
Positive control:
no data


Observations and examinations performed and frequency:
- Time schedule: Cage site examinations to detect moribund or dead rats and abnormal behaviour and appearance among rats were conducted at least twice daily throughout the study. At least once weekly during the first 3 months and at least every other week during the remainder of the study, each rat was individually handled and carefully examined for abnormal behaviour and appearance.


- Time schedule for examinations: During the first 3 months of the study all rats were weighed once weekly. They were weighed approximately once every other week for the remainder of the study.



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


- Time schedule for collection of blood: Approx. 3, 6, 12, 15 and 18 months after the study's initiation, haematological analyses were conducted.
- How many animals: 10 males and 10 females
- Parameters checked: basophil count (BASO), eosinophil count (EOSIN), erythrocyte count (RBC), haematocrit (Ht), haemoglobin (Hb), Leukocyte count (WBC), Lymphocyte count (LYMPH), mean cell haemoglobin (MCH), mean cell volume (MCV), mean corpuscular haemoglobin concentration (MCHC), monocyte count (MONO) and neutrophil count (NEUT).

- Time schedule for collection of blood: Approx. 3, 6, 12, 15 and 18 months after the study's initiation, clinical chemical analyses were conducted.
- How many animals: 10 males and 10 females
- Parameters checked: analnine aminotransferase activity (GPT), alkaline phosphatase activity (AP), bilirubin (BILRN), calcium, phosphorus, total protein (TROT) and urea nitrogen (BUN).

- Time schedule for collection of urine: Approx. 3, 6, 12, 15 and 18 months after the study's initiation.
- Parameters checked: quantitative measurements (volume, osmolality, pH) and qualitative measurements (bilirubin, blood, protein, sugar, urobilinogen); in addition the appearance of each urine speciem was noted and the sediment from pooled urine specimens was examined microscopically.

Sacrifice and pathology:
Gross and histopathological examinations were conducted on 5 rats/sex/treatment group after 3 and 6 months exposure, on 10 rats/sex/treatment group after 12 months exposure and on all rats alive after 24 months exposure.
Rats which had been designated for clinical chemical evaluation were not included among those selected for the interim sacrifices.
All rats found dead or sacrificed in extremis (integrity or tissue permitting), were examined grossly and histopathologically.

Lung tissue was fixed for light microscopy. The trachea, thyroid, adrenal glands, testes, and kidneys were fixed in Bouin's solution; all other organs and tissues were fixed in Formalin. Paraffin sections were prepared and stained for examination in the electron microscope.

Representative specimens of the following organs and tissues were taken from all rats: cardiovascular system, respiratory system, digestive system, urinary system, hematopoietic system, reproductive system (males and females), endocrine system, nervous system, musculoskeletal system, special sense organs, integumentary system and other (adipose tissue and all gross lesions).
Other examinations:
no data
Body weight and weight gain data were evaluated with a one-way analysis of variance and the least significant difference test. Organ weight and particle size data were evaluated with a one-way analysis of variance with pair wise comparison being made with the LSD and/or Dunnett's tests, and a test for linear trend. Clinical laboratory data were evaluated by a partially nested and crossed analysis of variance and by the LSD test. Outliners within the clinical laboratory and organ weight data were evaluated and excluded from calculations of the means by using the Dixon Criterion. The Bartlett test was also used to evaluate the organ weight data.
Significance was judged at the 5% level of probability.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
The following observations were associated with the TiO2 exposures:
- Coloured dischanges around the eyes and nose were observed approximately 1.4- to 8-fold less frequently among TiO2-exposed rats than the controls.
- Irregular respiration and abnormal lung noise were observed with a greater incidence and earlier in the study among the TiO2-exposed rats than the controls. The incidence was greater for TiO2-exposed females than males. A clear dose-response relationship was not observed.
Exposure to TiO2 resulted in no abnormal clinical sign or excess mortality in any exposed group.
- Stained and/or wet perineum was observed with a greater incidence among female rats exposed to TiO2. At the higher TiO2 concentrations, the incidence was greater and the observation was made earlier in the study than at the lower concentrations.

The mortality rates for all groups within a sex were similar. The mortality rates for females were 1.2- to 2-fold higher than those for male rats.

Mean body weights of all TiO2-exposed groups were generally lower that those of the controls. Although there was no dose-related trend for these body weigh effects, the mean weights for male and female rats exposed to 250 mg/m^3 was consistently lower than those for rats exposed to 10 or 50 mg/m^3. The differences in the mean body weight gain among all treatment groups parallelled the mean body weight effects.

There were several changes in haematological parameters related to exposure of rats to TiO2. When compared to the controls over course of this study:
- RBCs for rats in the 250 mg/m^3 treatment groups were slightly greater (up to 9%; not statistically significant).
- Hts and Hbs for rats in the 250 mg/m^3 treatment groups were greater (up to 12 and 11%, respectively).
- MCVs and MCHs in male rats from all exposed groups were greater (up to 14%); there were only minimal differences among these groups.
- WBCs in all treatment groups were greater (up to 80% for the high-dose group). The values for rats in the 250 mg/m^3 treatment group were generally higher than those of the other treatment groups.
- NEUTs in all treatment groups were greater (up to 80% in male and 117% in female rats in the 250 mg/m^3 treatment groups). A dose-related trend was observed among these treatment groups.
- LYMPHs in all treatment groups were lower (up to 45% lower for rats in the 250 mg/m^3 treatment groups). A dose-related trend was observed among these treatment groups.

There were several statistically significant changes in clinical chemical parameters measured in the serum from rats exposed to TiO2. When compared to the controls over the course of the study:
- The bilirubin content in female rats were greater and were more pronounced in the 50 and 250 mg/m^3 treatment groups at the 18-months evaluation (90 and 117%, respectively).
- Calcium concentrations in all treatment groups were generally lower (up to 7% less).
- Phosphorus concentrations were lower in male (up to 36% lower) and higher in female rats (up to 61%). These effects were consistent in males throughout the study but observed in females at only the 3-, 15- and 18-month evaluations.

Other haematological, clinical chemical or urological parameters were within the expected range of biological variability for this species.

Exposure of rats to TiO2 resulted in changes in mean absolute and/or relative weights of several organs over the course of the study. The organs affected were as follows:
- clear dose- and time-dependent lung weight increases were observed)
- Mean absolute and relative lung weights for rats in the 250 mg/m^3 treatment group were significantly greater than controls throughout the study. The range was from approximately 1.52- to 2.59-fold and from 1.53- to 3.38-fold greater for male and female rats, respectively.
- Mean absolute and relative lung weights for rats in the 50 mg/m^3 treatment group were also greater than controls throughout the study. With exception of the mean absolute male lung weights at six months, these effects were significant at the 6-months and subsequent sacrifices. The lung weights ranged from approx. 1.2- to 1.4-fold and 1.46- to 1.7-fold greater for male and female rats, respectively.
- The lung weights at 10mg/m³ were comparable to those of control group, markedly heavier at 50mg/m³, and were more than two times control lung weights at 250mg/m³.
- Mean absolute and/or relative liver weights of TiO2-exposed male and female rats were generally lower than those of controls over the course of the study.
- These differences ranged from approximately 100 to 66% of the control weights. A clear dose-response was not observed.
- A dose-related thymus weight effect was observed in rats in the 50 and 250 mg/m^3 treatment groups. Mean absolute and relative thymus weights were as high as 1.37-fold greater than controls.
Kidney: The mean absolute and/or relative kidney weights for TiO2 exposed male rats were approximately 77 to 88% of the controls at the 3-, 12- and 24-months sacrifices. A clear dose-response was not demonstrated.

During the gross pathological examinations, TiO2 deposits were observed on skin and the mucosa of the nasal cavity, trachea, bronchus and gastrointestinal tract of rats exposed to this compound. The pleural surfaces of the lungs contained scattered white foci which were present in greater numbers and larger sizes in rats exposed to the higher TiO2 concentrations. Subpleural cholesterol granulomas appeared on the lungs of rats in the 50 and 250 mg/m^3 treatment groups as slightly elevated gray nodules. The lungs of rats in the 250 mg/m^3 treatment groups were white in appearance, voluminous, of rubbery consistency and failed to collapse upon opening the chest cavity at necropsy.
The tracheabronchial lymph nodes were markedly swollen and appeared as chalky masses in all exposure groups. Most of these gross observations were apparent at six months with the severity and frequency of occurrence increasing over time.

All TiO2 exposed groups showed slight increases in the incidences of pneumonia, tracheitis, and rhinitis with squamous metaplasia of the anterior nasal cavity.
The lung reaction was characterised by dust-laden macrophage (dust cell) infiltration in the alveolar ducts and adjoining alveoli with hyperplasia of Type II pneumocytes. Rats at 50 and 250 mg/m³ exposure revealed a dose-dependent dust cell accumulation, a foamy macrophage response, type II pneumocyte hyperplasia, alveolar proteinosis, alveolar bronchiolarisation, cholesterol granulomas, focal pleurisy, and dust deposition in the tracheobronchial lymph nodes. Minute collagenised fibrosis occurred in the in the alveolar walls enclosing large dust cell aggregates. The pulmonary lesions with massive dust accumulation appeared to be the result of an overwhelmed lung clearance mechanism at 250 mg/m³ TiO2 exposure.

Bronchioloalveolar adenomas and cystic keratinising squamous cell carcinoma occurred at 250 mg/m³ TiO2 exposure (the tumours produced were ultimately characterised as primarily benign pulmonary keratin cysts (Warheit and Frame,2006)), while no compound-related lung tumours were found in rats exposed either to 10 or 50 mg/m³.

Effect levels

open allclose all
Dose descriptor:
for carcinogenicity in rats
Effect level:
50 mg/m³ air
Dose descriptor:
non-neoplastic changes
Effect level:
10 mg/m³ air

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Based on the findings at the low dose (alveolar cell hyperplasia, broncho/bronchiolar pneumonia) the concentration of 10 mg/m³ is considered as LOEC for non-neoplastic changes in this study.
Bronchioloalveolar adenomas and cystic keratinising squamous cell carcinoma occurred at 250 mg/m³ TiO2 exposure (the tumours produced were ultimately characterised as primarily benign pulmonary keratin cysts (Warheit and Frame,2006)), while no compound-related lung tumours were found in rats exposed either to 10 or 50 mg/m³. Thus, the concentration of 50 mg/m³ represents the NOEC for carcinogenicity in rats.