Diiron titanium pentaoxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Concentrations tested exceeded the maximum tolerated dose (MTD)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Six-week old female Fischer rats were exposed to 10, 50, or 250 mg/m³ titanium dioxide for 6 hours/day, 5 days/week for 13 weeks with recovery groups held for an additional 0, 4, 13, 26 or 52 weeks post-exposure. A vehicle control group was run concurrently. At each time point TiO2 burdens in the lung and lymph nodes and selected ling responses were examined.

GLP compliance:

not specified

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

Test animals

Species:

rat

Strain:

Fischer 344

Remarks:

CDF (F344)/CrlBR

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Age at study initiation: six weeks
- Housing: stainless-steel cages
- Diet: ad libitum, NIH07 cereal-based diet
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 64-79 °F
- Humidity (%): 40-60

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.44 µm

Geometric standard deviation (GSD):

1.71

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m³ H-1000 stainless steel chambers

- System of generating particulates/aerosols: aerosol generation was accomplished using a dust feeder

- Method of particle size determination:
The particle size distribution of the aerosol was measured at least twice per exposure level (excluding the control chambers) during the courde of the study. Measurements were made using a MOUDI impactor (micro-orifice uniform deposit impactor, model 100, MSP Corporation, MN)

TEST ATMOSPHERE
- Brief description of analytical method used:
Target chamber concentrations of p-TiO2 aerosol were 10, 50 and 250 mg/m3 and the actual particle concentration in each chamber was monitored using a Real Aerosol Monitor (RAM). Mean (± SD) particle concentrations over the exposure period were as follows: 9.6 ± 1.1, 47.7 ± 5.1, and 239.1 ± 19.3 mg/m3. During exposures, particle concentrations were continuously monitored using light scatter (Model RAM-S, Monitoring Instruments for the Environment), and the time-averaged concentration was recorded at least six times over the 6-h exposure period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to the field "Details on inhalation exposure" above.

Duration of treatment / exposure:

- 13 weeks treatment
- Post-exposure recovery period: 0, 4, 13, 26 or 52 weeks of recovery

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/m³ air (nominal)

No. of animals per sex per dose:

65 (only females)

Control animals:

yes

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for the first 17 weeks, and biweekly thereafter

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: end of treatment
- Dose groups that were examined: all
- Number of animals: all
- Parameters checked: Cell differential counts were performed on Wright-Giemsa-stained cytocentrifuge slide prepa- rations. LDH and total protein levels in cell-free fluid from the first two pooled lavages were quantitated spectrophotometrically using a COBAS FARA II automated analyzer.

LUNG BURDEN: Yes
- Time schedule for analysis: end of treatment
- Dose groups that were examined: all
- Number of animals: all
- Parameters checked: minimum detectable concentrations (MDC) of TiO2 in pulmonary tissues

CELL PROLIFERATION
Five days prior to euthanasia, animals were subcutaneously implanted with osmotic pumps (Alza. Palo Alto. CA) containing bromodeoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO). At necropsy, left lungs were pressure-infused intratracheally (2with 10% neutral-buffered formalin. Lungs were fixed for approximately 48 h and then changed to 70% ethanol. Subsequently, the lungs were embedded in paraffin, sectioned and stained for BrdU. Terminal bronchiolar and alveolar cell labeling indices were determined fot each animal.

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes
Paraffin-embedded left lung tissues were sectioned and stained with Masson's trichrome. The trichrome-stained lung sections were evaluated for particle-induced histopathologic changes.

Statistics:

All data were tested for normality and homogeneity of variance. If the hypotheses for these assumptions were rejected (p < 0.01), common transformations (e.g., log, square root, arc sine) were applied and the data retested. Comparisons to controls were made using Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Rats exposed to the high concentration of TiO2 demonstrated a consistent pattern of elevated weights (less than 10%), compared to controls, during the latter half of the recovery period. No obvious cause for this weight elevation was evident.

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:

not examined

Clinical biochemistry findings:

not specified

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 examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The mid- and high-dose rats had a variety or lung lesions associated with retained p-TiO2 particles. Particles and particle-laden alveolar macrophages were most numerous in centriacinar lung regions, although in the high-dose animals there was a more diffuse panacinar distribution at the end of the 13-week exposure period. While the nature of the epithelial lesions was similar in mid- and high-dose animals, there were significant differences in lesion severity. The number of particle-laden macrophages observed individually and in aggregated was much greater in the high-dose animals.
Although most particles were retained intraluminally, a minimal to mild interstitial accumulation of particle-laden macrophages was present in mid- and high-dose rats and remained over the course of the 52-week recovery. Immediately post-exposure, both mid- and high-dose rats had alveolar hypertrophy and hyperplasia of type II epithelial cells surrounding aggregations of particle-laden macrophages. These lesions were generally minimal to mild in mid-dose and mild to moderate in high-dose animals. In both of these exposure concentration groups at this time point, histological evidence of chronic active inflammation was noted by the infiltration of neutrophils. Histological recognition of neutrophil infiltrates diminished in mid-dose rats by four weeks recovery although they persisted throughout the 52-week recovery period in the high-dose rats.
High-dose animals developed more severe alveolar type II cell hypertrophy and hyperplasia and alveolar metaplasia that were associated with septal fibrosis and interstitialization of particles, often within macrophages. These lesions were noted by four weeks postexposure, were prominent at the 26-week time point, and progressed through the 52-week recovery time point. In some instances, the alveolar lumens in lesion areas were characterized by lipoproteinosis and cholesterol cleft development. Lesions in both mid- and high-dose animals were scattered throughout the lung lobes.
Airway lesions in rats consisted of minimal bronchiolar hypertrophy in mid-dose animals that did not progress over time of recovery and mild to moderate bronchiolar hypertrophy and hyperplasia in high-dose animals with progressive bronchiolization in areas with lesions.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LUNG BURDEN:
- Dose-related changes in lung burdens observed; particle burdens decreased in the lung with time postexposure
- the lymph node TiO2 burdens of the animals exposed to 250 mg/m3 demonstrated the greatest increase between 4 and 13 weeks after exposure ended
-CYTOLOGY:
- number of recovered macrophages in high-dose group was significantly elevated
- postexposure recovery resulted in a decline in the number of macrophages recovered by lavage; although these values remained significantly elevated over concurrent controls in rats of the high-dose group at 52 weeks postexposure
- increased proportions of neutrophils , with increasing time after exposure there was a diminution in number of neutrophils in rats of mid-and high dose group, although the number of neutrophils remained significantly elevated over contrls at the 52-week postexposure
-significant elevated levels of lymphocytes, which remained so at the end of the 52 weeks postexposure period
PULMONARY TOXICITY END POINTS:
- persistant elevation of LDH in BALF of mid-and high dose group; those in the mid-dose group had returned to control levels by 26 weeks postexposure
- elevated levels of total protein in BALF of mid and high dose group, remained elevated through 52 weeks postexposure
LUNG CELL REPLICATION:
- increased terminal bronchiolar cell replication in high dose group, returned to control level by 4 weeks postexposure
- increased alveolar cell replication in high dose group, remained through 52 weeks postexposure

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

10 mg/m³ air (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Lung burden, BALF, inflammation

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Titanium dioxide showed adverse pulmonary effects in rats, mice and hamsters after repeated inhalation studies only at concentrations above the maximum tolerated dose (MTD)

Executive summary:

Female rats were exposed to 10, 50, or 250 mg/m3 titanium dioxide (p-TiO2) particles for 6 h per day and 5 days per week for 13 weeks with recovery groups held for an additional 4,13,26, or 52 weeks post-exposure . Beside rats, mice and hamsters were exposed in the same manner using the same concentrations. At each time point p-TiO2 burdens in the lung and lymph nodes and selected lung responses were examined. The responses studied were chosen to assess a variety of pulmonary parameters, including inflammation, cytotoxicity, lung cell proliferation, and histopathologic alterations. Burdens following exposure were greatest in mice. Rats and hamsters had similar lung burdens immediately post-exposure. Particle retention data suggested that pulmonary overload was achieved in both rats and mice at the exposure levels of 50 and 250 mg/m3. Under the conditions of the present study, hamsters were better able to clear particles than were similarly exposed mice and rats. Pulmonary histopathology revealed both species and concentration-dependent differences in p-TiO2-particle retention patterns. Inflammation was noted in all three species at 50 and 250 mg/m3, as evidenced by increases in macrophage and neutrophil numbers and in soluble indices of inflammation in bronchoalveolar lavage fluid. In mice and rats, the BALF inflammatory responses remained elevated relative to controls throughout the entire post-exposure recovery period in the most highly exposed animals. In comparison, inflammation in hamsters eventually disappeared, even at the highest exposure dose, due to the more rapid clearance of particles from the lung. Pulmonary lesions were most severe in rats, where progressive epithelial- and fibroproliferative changes were observed in the 250 mg/m3 group. The study clearly shows that the high and mid doses clearly exceed the maximum tolerated concentration by overwhelming physiological clearance mechanisms.