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Titanium dioxide did not show any adverse effects in 90-day and 28-day oral repeated dose toxicity studies in rats with a NOAEL of 1000 and 24,000 mg/kg bw/day, respectively. No adverse effects were observed in rats and mice orally exposed up to a dose of 3500 mg/kg bw/day over a period of 103 weeks in a carcinogenicity bioassay. Titanium dioxide is not absorbed to any relevant extent through human skin, thus no toxic effects can be expected via the dermal route of exposure. Titanium dioxide showed adverse pulmonary effects in chronic inhalation studies only at concentrations above the maximum tolerated dose (MTD).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-11-08 to 2011-08-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
, adopted 1998-09-21
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 8 weeks
- Housing: animals were housed in pairs in polycarbonate pans that contained bedding with enrichment (i.e., Shepherd’s™ Cob + PLUS™).
- Diet (ad libitum, except when fasted): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26ºC
- Relative humidity: 30-70%
- Photoperiod (hrs dark / hrs light): approximately 12/12
Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
H-29865 was suspended in 0.5% aqueous methylcellulose. Dose suspensions of the test substance were prepared daily and stored at room temperature until used.

Animals were dosed at a dose volume of 10 mL/kg body weight. The amount of test substance each animal received was based on the most recently collected body weight and the suspension concentration. Control animals were dosed with the vehicle at a volume of 10 mL/kg of body weight. Beginning test day 35 through the remainder of the study, the dose volume for several male rats was more than 5 mL. The dose for these rats was administered in 2 aliquots at least 15 minutes apart.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each dosing suspension were collected 2 times during the study: near the beginning and near the end of the study. Analysis of the first sampling during the study verified mixing uniformity and concentration (average of verification samples). The subsequent samplings addressed concentration. Samples were submitted for analysis shortly after preparation.
Samples were prepared in a chemical microwave using nitric and hydrofluoric acid and then analysed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).

Results:
1) Test substance stability analyses
The analysis results show that the test substance was homogeneously mixed (RDSs ≤ 8.9%) and at the targeted concentrations (106% and 89.7% nominal, respectively) for the 10 and 100 mg/mL concentrations. After 11 days of room temperature storage, the samples were resuspended and analysed to confirm homogeneity and concentrations. The results show that the test substance was homogeneously mixed (RDSs ≤ 3.22%) and at the targeted concentrations (97.5% and 100% nominal, respectively), indicating that the test substance was stable in the concentration range from 10 to 100 mg/mL when stored at room temperature for 11 days in the vehicle.

2) Dose analyses
The analysis results from the samples prepared near the beginning of the study show that the test substance was homogeneously mixed (RDSs ≤ 3.13%) and at the targeted concentrations (101% and 100% nominal, respectively) for the 30 and 100 mg/mL dose levels, but not for the 10 mg/mL dose level (69.5% nominal with RSD = 13.6%). The low results for the 10 mg/mL samples (69.5% nominal; RSD = 13.6%) were confirmed by re-analysis from the re-suspended samples (73.2% nominal with RSD = 11.3%).
Further analysis results for samples prepared near the beginning of the study at the concentration of 10 mg/mL confirmed the low result for this dose level (53.9% to 72.8% nominal).
The analysis results for the samples prepared near the end of the study show that the test substance was at the targeted concentration (92.4% nominal) for the 100 mg/mL dose level but lower for the 10 and 30 mg/mL dose levels (64.5% and 71.3% nominal, respectively).
The test substance was not detected in the control samples.
Duration of treatment / exposure:
92 (males) or 93 (females) days
Frequency of treatment:
daily (approximately the same time (± 2 hours))
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
67 mg/kg bw/day (actual dose received)
Dose / conc.:
258 mg/kg bw/day (actual dose received)
Dose / conc.:
962 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosages of 0, 100, 300, and 1000 mg/kg were selected for this study based on a previously conducted 28-day oral gavage study with H-27201 and H-27203 (please refer to Section 7.5.1 Repeated dose toxicity: oral: k_Mayer_2006_28 days). No test substance-related effects were observed on any in-life, clinical pathology, or anatomic pathology parameter in male rats dosed at 24,000 mg/kg for 28 consecutive days.

- Rationale for animal assignment: animals of each sex were selected for use on study based on adequate body weight gain and freedom from any ophthalmology abnormalities or clinical signs of disease or injury. The selected animals were distributed by computerized, stratified randomization into study groups as designated in the Study Design so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations: moribund, mortality, abnormal behaviour and/or appearance
An additional cage-site evaluation was conducted daily approximately 2 hours (± 1 hour) postdosing to detect acute clinical signs of systemic toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at every weighing (excluding weights on days of neurobehavioural evaluations and necropsy)

BODY WEIGHT: Yes
- Time schedule for examinations: once a week and on the day of sacrifice.
Animals were also weighed during quarantine.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
The amount of food consumed by each animal over each weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. Only spillage greater than 25 grams is recorded and used in the calculation for food consumption. Cage food consumption was divided by the number of animals in the cage to calculate individual animal food consumption. From these measurements, mean daily food consumption over the interval was determined.

FOOD EFFICIENCY:
From the food consumption and body weight data, the mean daily food efficiency was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: test day -3 (baseline examination) and test day 88 (prior to the final sacrifice)
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 14 (test day 93/94 for males and females, respectively)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia (only when blood was taken for the coagulation parameters)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean corpuscular (cell) volume, mean corpuscular (cell) haemoglobin, mean corpuscular (cell) haemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, prothrombin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14 (test day 93/94 for males and females, respectively)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, cholesterol, triglycerides, glucose, total protein, albumin, globulin, calcium, inorganic phosphorus, sodium, potassium, chloride, and bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: day before collection of samples for clinical pathology evaluation, the animals were placed in metabolism cages.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, at least 15 hours and urine was collected
- Parameters examined: quality, colour, clarity, volume, specific gravity, pH, glucose, ketone, bilirubin, blood, urobilinogen, protein, and microscopic urine sediment examination

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during acclimation (baseline) and during week 13 on all animals
- Dose groups that were examined: all animals
- Battery of functions tested:
1. Abbreviated functional observational battery assessment
Sensory and motor function assessments were conducted by evaluating grip strength (fore- and hind limb grip), responses to approach/touch, sharp auditory stimulus, and tail pinch, and pupillary constriction. During motor activity testing the experimenter also looked for polyuria and diarrhoea.
2. Motor activity:
Duration of movement and number of movements were measured.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

After 92 or 93 days dosing (male and females, respectively), all rats were sacrificed and necropsied for evaluation of subchronic toxicity.
Rats sacrificed by design were fasted for at least 15 hours prior to their scheduled euthanasia. Rats were euthanized by exsanguination while under isoflurane anaesthesia. A complete necropsy was performed on each rat. The necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
The following organs were weighed from all animals included in the scheduled necropsy. Paired organs were weighed together. Group mean values and organ weight ratios (% body weight and % brain weight) were calculated. The weighed organs were the following: adrenal glands, brain, heart, kidneys, liver, spleen, thymus, ovaries including oviducts, uterus including cervix, testes, and epididymides.
At the time of necropsy, the following organs were collected from all animals and fixed: liver, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, pancreas, kidneys, urinary bladder, lungs, trachea, nose (4 levels), larynx/pharynx, heart, aorta, spleen, thymus, mandibular lymph nodes, mesenteric lymph nodes, bone marrow (collected with the femur and sternum), Peyer’s patches (collected with the intestines), pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain (includes cerebrum, cerebellum, midbrain, and medulla/pons), spinal cord (includes cervical, mid-thoracic, and lumbar sections), sciatic nerve, eyes (with retina and optic nerves), skeletal muscle, femur/knee joint, sternum, testes, epididymides, accessory sex organs (prostate with seminal vesicles and coagulating glands), ovaries (with oviducts), uterus (with cervix), vagina, mammary glands, skin, and gross observations.
All tissues collected from male and female rats in the control (0 mg/kg/day) and high dose (1000 mg/kg/day) groups were processed to slides and evaluated microscopically. Gross lesions from all rats were also processed and examined. Based on the results of the microscopic evaluation, as well as organ weight and clinical pathology results, there was no target organ toxicity in male or female rats administered 1000 mg/kg/day. Therefore, tissues from the animals in the 100 and 300 mg/kg/day groups were not evaluated microscopically.
Statistics:
Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.
Please refer to table 1 in the field "Any other information on materials and methods incl. tables" below.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- no test substance-related clinical signs were observed.
- no deaths occurred.

BODY WEIGHT AND WEIGHT GAIN
- no test substance-related effects were observed on body weights or body weight gains.

FOOD CONSUMPTION AND COMPOUND INTAKE
- no test substance-related effects were observed on food consumption.

FOOD EFFICIENCY
- no test substance-related effects were observed on food efficiency.

OPHTHALMOSCOPIC EXAMINATION
- no test substance-related ophthalmological findings were observed in any male or female group.

HAEMATOLOGY
- no treatment-related changes in group mean haematology parameters and group mean coagulation parameters at test day 93/94 in male or female rats.

CLINICAL CHEMISTRY
- no treatment-related changes in group mean clinical chemistry parameters at test day 93/94 in male or female rats.

URINALYSIS
- no treatment-related changes in group mean urinalysis parameters at test day 93/94 in male or female rats.

NEUROBEHAVIOUR
- no test substance-related effects on neurobehavioral parameters.

ORGAN WEIGHTS
- no test substance-related organ weight changes.

GROSS PATHOLOGY
- no test substance-related gross observations.

HISTOPATHOLOGY: NON-NEOPLASTIC
- no test substance-related adverse changes in histopathology were observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
NOAEL (males & females; nominal concentration): >1000 mg/kg/day
NOAEL (males & females; actual concentration): >962 mg/kg/day
The NOAEL is based on a lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameter in rats dosed up to 1000 mg/kg/day.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005-09-14 to 2006-01-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
limit test dose is 24 times higher than in guideline, only one sex was used
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: between 6 and 8 weeks of age
- Weight at study initiation: 211 to 214 g
- Housing: All animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Each cage rack contained only animals of one sex.
- Diet: All rats were fed PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum.
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Photoperiod: 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in NANOpure water and the dosing suspensions were prepared daily.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to address homogeneity/concentration verification and stability were not done during this study.
Duration of treatment / exposure:
The rats were dosed by intragastric intubation for 29 consecutive days.
Frequency of treatment:
daily
Dose / conc.:
24 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Under the conditions of a previously conducted study, the no-observed-effect level (NOEL)a for H-27201 and H-27203 was 24,000 mg/kg/day for male rats, based on the lack of any adverse effects at this dose.
- Rationale for animal selection: Rats were selected for use on study on the bases of adequate body weight gain and freedom from clinical signs of disease or injury. They were distributed by computerised, stratified randomisation into study groups as designated in the study design, so that there were no statistically significant differences among group body weight means.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily throughout the study
- Cage side observations checked: Detection of moribund or dead rats and abnormal behaviour and/or appearance among rats.
Each rat was individually handled and examined for abnormal behaviour and appearance (careful clinical observations ) daily approximately 1 -2 hours after receiving the final daily dose. Any abnormal clinical signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 0 and weekly thereafter
At every weighing, each rat was individually handled and examined for abnormal behaviour and appearance. Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observation included (but were not limited to ) evaluation of fur, skin, eyes, mucous membranes, occurence of secretions and excretions, autonomic nervous systemactivity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behaviour. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : No data

OPHTHALMOSCOPIC EXAMINATION: see below (sacrifice and pathology)

HAEMATOLOGY: Yes, blood samples for hematology were collected from the orbital sinus/abdominal vena cava of each animal.
- Time schedule for collection of blood: A clinical pathology evaluation was conducted on all surviving rats on test day 29.
- Anaesthetic used for blood collection: Yes, the animals were under carbon dioxide anesthesia.
- Animals fasted: Yes, animals were fasted after 3 p.m. for at least 15 hours.
- Parameters checked: red blood cell count, absolute reticulocyte count, hemoglobin, platelet count, hematocrit, white blood cell count, mean corpuscular (cell) volume, differential white blood cell count, mean corpuscular (cell) hemoglobin, microscopic blood smear examination, mean corpuscular (cell) hemoglobin concentration, red cell distribution width, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes, blood samples for clinical chemistry measurements were collected from the orbital sinus of each animal.
- Time schedule for collection of blood: A clinical pathology evaluation was conducted on all surviving rats on test day 29.
- Animals fasted: Yes, animals were fasted after 3 p.m. for at least 15 hours.
- Parameters checked: aspartate aminotransferase, glucose, alanine aminotransferase, total protein, sorbitol dehydrogenase, albumin, alkaline phosphatase, globulin, total bilirubin, calcium, urea nitrogen, inorganic phosphorus, creatinine, sodium, cholesterol, potassium, triglycerides and chloride

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

IMMUNOLOGY: No data


OTHER: Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were stained with Wright-Giemsa stain, but analysis was not necessary to support experimental findings.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After approximately 28 days on study (test day 29), the surviving rats were sacrificed and necropsied for evaluation of repeated dose toxicity. The order of sacrifice for scheduled deaths was stratified among treatment groups. Rats were euthanised by carbon dioxide anesthesia and exsanguination. Gross examinations were performed on all rats. Final body weights and organ weights were recorded.
The following tissues were collected from all (15/15) rats: digestive system (liver, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, pancreas), urinary system (kidneys, urinary bladder), respiratory system (lungs, trachea, nose, larynx/pharynx), cardiovascular system (heart, aorta), hematopoietic system(spleen, thymus, mandibular lymph node, mesenteric lymph node, bone marrow (collected with the femur and sternum), Peyer's patches (collected from sections of the digestive tract)), endocrine system (pituitary gland, thyroid gland, parathyroid glands, adrenal glands), nervous system (brain (three sections; including cerebrum, cerebellum, medulla/pons), spinal cord (three levels; cervical, mid-thoracic, lumber) sciatic nerve), musculoskeletal system (skeletal muscle, femur/knee joint, sternum), reproductive system male (testes, epididymides, prostate, seminal vesicles) and miscellaneous (skin, eyes (including retina and optic nerve), gross observations.
The following tissues were weighed from rats sacrificed by design at the end of the 28-day repeated dose toxicity study: liver, kidneys, adrenal glands, thymus, brain, spleen, heart, testes, and epididymides. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.

HISTOPATHOLOGY: Yes
All tissues collected from all rats were processed to slides and evaluated microscopically.
Testes, epididymides, and eyes were fixed in modified Davidson’s solution. All other tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist.
Other examinations:
no
Statistics:
Significance was judged at p < 0.05. Statistical comparisons were made between the control group (group I) and H-27201 (group III) and H-27203 (group V). Statistical comparison was performed between the two treatment groups H-27201 (group III) and H-27203 (group V).
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No test substance-related signs were noted on daily or weekly clinical observations during the 28-day oral gavage study. Some non-specific clinical observations were noted in some animals but were not test substance related. These observation which included a neck wound, misshapen ears and hair loss. These types of clinical signs are commonly reported among rats of this sex and age group. One animal, number 305 (H-27201), was found dead at the end of the first week. During the second week, animal number 505 (H-27203) was observed to have breathing noises and was sacrificed. The necropsy report indicated both deaths were attributed to trauma from gavage. The remaining 13 male rats survived until the scheduled sacrifice (test day 29).

BODY WEIGHT AND WEIGHT GAIN
No statistically significant test substance related effects were observed on mean body weight or body weight gains in the two treatment groups when comparison was made between the groups and when each group was compared to the control.

FOOD CONSUMPTION AND FOOD EFFICIENCY
There were no statistically significant differences in food consumption and food efficiency in the two treatment groups when compared against each other, and when compared to the control.

HAEMATOLOGY
There were no statistically significant or treatment-related changes in hematologic parameters in male rats exposed to 24,000 mg/kg TiO2 particles in either group when compared against each other or when compared to the control.
There were no statistically significant or treatment-related changes in coagulation parameters in male rats.

CLINICAL CHEMISTRY
There were no statistically significant or treatment-related changes in clinical chemistry parameters in male rats.

ORGAN WEIGHTS
There were no test substance-related effects on organ weights. All individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration.

GROSS PATHOLOGY
There were no test substance-related deaths. Of the 15 rats on study (5 males/dose group), two died before the terminal sacrifice, both due to dosing accidents. Gross examination demonstrated perforation of the esophagus in both rats.
There were no test substance-related gross observations in the treatment or the control groups. All gross observations at the terminal necropsy were consistent with normal background lesions in rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the two rats that died before the terminal sacrifice revealed several lesions associated with esophageal trauma and morbidity (e.g., rupture-associated inflammation, splenic and thymic lymphoid depletion, bone marrow hyperplasia, centrilobular liver necrosis, esophageal reflux and pulmonary aspiration).
There were no test substance-related adverse microscopic findings in the treatment or the control groups.
The observation of gavage material in macrophages within the mesenteric lymph nodes and Peyer’s patches of rats given the test substance was considered to be the expected disposition of a non-toxic particulate. There was no evidence of an adverse cellular response to the presence of the phagocytised particulates. The observation of these particulates was facilitated by their optical birefringence.
Inflammation in the nose of 3/5 Group III rats and 2/5 Group V rats was the result of gastro-esophageal reflux in all but one rat (Group III). This latter rat had minimal focal inflammation in the turbinate mucosa that was interpreted to be incidental. Three of the remaining four rats with evidence of reflux had esophageal perforations identified either by gross or microscopic examination. The remaining rat had minimal gavage material and inflammation and no identified esophageal lesion. All cases of gastro-esophageal reflux and associated inflammation were interpreted to be dosing related and not test substance related.
All other microscopic findings were consistent with either the normal background lesions in rats of this age and strain or were the result of a dosing accident.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
Male rats were exposed by oral gavage for 29 days to 24,000 mg/kg TiO2 particles H-27201, H-27203, or the vehicle. Under the conditions of this study, the no-observed-effect level (NOEL) for H-27201 and H-27203 was 24,000 mg/kg/day for male rats, based on the lack of any adverse effects at this dose.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
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
Specific details on test material used for the study:
not applicable
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
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)
Dose / conc.:
25 000 ppm
Remarks:
equivalent to 3750 mg/kg/day (recalculated from ppm value (factor (mouse): 0.150)
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
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
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
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
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)
Critical effects observed:
no
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.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 50 male and 50 female Fischer 344 rats 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
Specific details on test material used for the study:
not applicable
Species:
rat
Strain:
Fischer 344
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: 64 days
- Housing: housed in polycarbonate cages covered with stainless steel cage lids and non-woven fiber filter bonnets; rats were initially housed 5/cage, but starting at week 48 the males were divided into groups of 2 or 3/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: 30 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 45 - 55 %
- Air changes: 12/hour
- Photoperiod (hrs dark / hrs light): 12/12
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.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
7 days/week (ad libitum)
Dose / conc.:
25 000 ppm
Remarks:
analytical value; equivalent to 1250 mg/kg/day (recalculated from ppm value (factor (older rat): 0.050)
Dose / conc.:
50 000 ppm
Remarks:
analytical value; equivalent to 2500 mg/kg/day (recalculated from ppm value (factor (older rat): 0.050)
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 rats 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 rats 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
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, 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
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
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
- clinical signs observed in the dosed groups were generally comparable to those of the control group and included alopecia, sores, and lacrimating, protruding, and/or pale eyes.
- from weeks 88 through 104, hunched appearance and thinness were noted more frequently in the dosed males and females than in their respective controls (not test-item related).
- urine stains were noted on the dosed rats of each sex (not test-item related).
- animals in all of the dosed groups had white faeces.

MORTALITY
- result of the Tarone test for dose-related trend in mortality is not significant in either sex.
- male rats. 36/50 animals (72%) of the 50000 ppm dose group, 37/50 animals (74%) of the 25000 ppm dose group and 31/50 animals (62%) of the matched controls were alive at week 104.
- females rats: 34/50 animals (68%) of the 50000 ppm dose group, 36/50 animals (72%) of the 25000 ppm dose group and 36/50 (72%) of the matched controls were 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 rats.

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 rats 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: observed in male rats only; incidence 8% (high) vs 0% in control and 2% in low dose; no effects in females (0-4%);
historical controls (HC)* for extramedullary haematopoiesis rat (18-24 month): 7.5 %
Conclusion: the finding in male rats is most probably not related to treatment with the test item but spontaneous in nature, because the incidence in males is generally low. It was observed in females only with a very low incidence. Information from a publication on the pathology of aging male Fischer rats indicates a percentage for haematopoiesis of 7.5% which is very close to the rate observed in this study (8%). Overall, it is not very likely that the finding of haematopoiesis in spleen observed in male Fischer rats is related to treatment with the test item.

- kidney chronic inflammation: male rat only; incidence 90% and 86% in low dose and high dose vs 59% in control (no dose relation); no effects in female rats (38-53%)
HC rats (chronic interstitial nephritis used as correlate for chronic inflammation): 62.5 % (18-24 months), 70.2 % (24-30 months)
Conclusion: the higher incidence of chronic inflammation in male rats observed in the low and high dose groups is not dose-related, although higher than in control males. In general, the incidence of this lesion is high in aged Fischer rats (18-30 months) based on published data (62.5-70.2%). However, the incidences observed in male rats may reflect a chronic infection in these animals and is most probably not related to treatment with the test item.

- seminal vesicle (SV) and testis atrophy: SV rat: incidence 12% (low) and 20% (high) vs 0% in controls
Testis rat: incidence 10% (low) and 14% (high) vs 6% in control
HC seminal vesicles and testis: 1.7% and 12.4%
HC seminal vesicles and testis: 2.3% (24-30%) and 80-100% (18-30 months)
Conclusion: the incidence observed in seminal vesicles of male rats is indeed higher than described in two publication on historical control data observed in studies (1.7%) conducted by the National Cancer Institute or described in a publication about pathology in aging Fischer rats (2.3%). However, the toxicological relevance of this observation is not clear. In addition, the findings in testes are within the HC in the low dose group and only slightly above the HC data (12.4%) in the high dose group. In general, the findings observed in seminal vesicle and testis of Fischer rats should not be considered as related to treatment with the test item.

- liver necrosis: rats (0-2%) not affected

- uterus/endometrium cystic hyperplasia: female rats (0-6%)

HISTOPATHOLOGY: NEOPLASTIC
Male rats:
- pheochromocytomas of the adrenal medulla (matched control: 7/49 (14 %); 25000 ppm dose: 9/49 (18 %); 50000 ppm dose: 14/50 (28 %)) and fibromas of the subcutaneous tissue (matched control: 1/49 (2 %); 25000 ppm dose: 5/50 (10 %); 50000 ppm dose 5/50 (10 %) were observed with slightly greater frequency in dosed groups. The number of neoplasms was compatible with incidences of these tumours in historical-control rats of this age and strain. Thus, these lesions are not considered to be related to administration of the test chemical.
- three keratoacanthomas of the skin were observed in the 50000 ppm dose group, but none in the other two groups studied. Although the result of the Fisher exact test for direct comparison of the incidence in the 50000 ppm group with that in the control group is not significant, the result of the Cochran-Armitage test for positive dose-related trend in the incidence of these tumors is significant (P = 0.038).
- significant results in the negative direction are observed in the incidence of leukemia, in which the incidence in the control group exceeds the incidences in the dosed groups.

Female rats:
-endometrial stromal polyps were observed more frequently in dosed groups (matched control: 6/50 (12 %); low dose: 15/50 (30 %); high dose 10/49 (20 %)) than in control groups, but the incidence of lesions is comparable with that in historical controls. Thus, these lesions are not considered to be related to administration of the test chemical.
- C-cell adenomas or carcinomas of the thyroid occurred at incidences that were dose related (P = 0.013), but were not high enough (P = 0.043 for direct comparison of the 50000 ppm dose group with the control group) to meet the level of P = 0.025 required by the Bonferroni criterion (controls 1/48, 25000 dose 0/47,50000 ppm dose 6/44). Thus, these tumors of the thyroid were not considered to be related to the administration of the test chemical.
- Fisher exact comparison of the incidence of endometrial stromal polyps of the uterus/endometrium in the 25000 ppm dose females with that in the corresponding controls indicates a P value of 0.045, which is above the 0.025 level required for significance when the Bonferroni inequality criterion is used for multiple
comparison. The incidence of these tumors in the 50000 ppm dose group is not significant when compared with that in the control group, and the result of the Cochran-Armitage test for dose-related trend also is not significant.

Inflammatory, degenerative, and hyperplastic lesions that occurred were similar in number and kind to those naturally occurring lesions found in aged Fischer 344 rats.

*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
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
Dose descriptor:
NOEL
Effect level:
2 500 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 0.050 (older rats)
Critical effects observed:
no
Conclusions:
NOEL (systemic toxicity; rats): 50000 ppm (equivalent to 2500 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 in rats 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: observed in male rats only; incidence 8% (high) vs 0% in control and 2% in low dose; no effects in females (0-4%);
historical controls (HC)* for extramedullary haematopoiesis rat (18-24 month): 7.5 %
Conclusion: the finding in male rats is most probably not related to treatment with the test item but spontaneous in nature, because the incidence in males is generally low. It was observed in females only with a very low incidence. Information from a publication on the pathology of aging male Fischer rats indicates a percentage for haematopoiesis of 7.5% which is very close to the rate observed in this study (8%). Overall, it is not very likely that the finding of haematopoiesis in spleen observed in male Fischer rats is related to treatment with the test item.

- kidney chronic inflammation: male rat only; incidence 90% and 86% in low dose and high dose vs 59% in control (no dose relation); no effects in female rats (38-53%)
HC rats (chronic interstitial nephritis used as correlate for chronic inflammation): 62.5 % (18-24 months), 70.2 % (24-30 months)
Conclusion: the higher incidence of chronic inflammation in male rats observed in the low and high dose groups is not dose-related, although higher than in control males. In general, the incidence of this lesion is high in aged Fischer rats (18-30 months) based on published data (62.5-70.2%). However, the incidences observed in male rats may reflect a chronic infection in these animals and is most probably not related to treatment with the test item.

- seminal vesicle (SV) and testis atrophy: SV rat: incidence 12% (low) and 20% (high) vs 0% in controls
Testis rat: incidence 10% (low) and 14% (high) vs 6% in control
HC seminal vesicles and testis: 1.7% and 12.4%
HC seminal vesicles and testis: 2.3% (24-30%) and 80-100% (18-30 months)
Conclusion: the incidence observed in seminal vesicles of male rats is indeed higher than described in two publication on historical control data observed in studies (1.7%) conducted by the National Cancer Institute or described in a publication about pathology in aging Fischer rats (2.3%). However, the toxicological relevance of this observation is not clear. In addition, the findings in testes are within the HC in the low dose group and only slightly above the HC data (12.4%) in the high dose group. In general, the findings observed in seminal vesicle and testis of Fischer rats should not be considered as related to treatment with the test item.

- liver necrosis: rats (0-2%) not affected

- uterus/endometrium cystic hyperplasia: female rats (0-6%)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two key study available (28- and 90-day repeated dose toxicity study in rats) which are reliable without (RL=1) or with minor restrictions (RL=2). The overall quality of the database is therefore high.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female NMRI mice were exposed 18 hours/day, 5 days/week for 13.5 months to ultrafine titanium dioxide (P25, Degussa; MMAD: 0.80 µm) and were subsequently kept in clean air for 9.5 months. The type of inhalation was whole body and the average particle exposure concentrations for the test substance was 10 mg/m³. A control group receiving clean air was run concurrently. The following observations were performed: clinical signs, mortality, body weight, lung wet weight, lung retention of inhaled particles, and histopathology.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
mouse
Strain:
other: NMRI (Crl:NMRI BR)
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 7 weeks ± 3 days
- Housing: housed in groups of 8 - 10 per cage; kept in wire mesh cages during the exposure period, and in Makrolon cages (18.7 x 21 x 15 cm) during the subsequent clean air period. Softwood bedding (H3/4) was used.
- Diet (ad libitum): "1324 N spec. prepared" (Altromin, Lage, Germany)
- Water (ad libitum): drinking water

ENVIRONMENTAL CONDITIONS
- Temperature: 23 - 25 °C
- Relative humidity: 50 - 70%
- Photoperiod: 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
0.8 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: special whole-body exposure chambers (6 or 12 m³) of the horizontal flow type (Heinrich et al., 1985)*.
The mice were exposed together with Wistar rats in the same exposure chamber (for information on the rats please refer to Section 7.7 Carcinogenicity: NANO_s_Heinrich_1995_rats).

- System of generating particulates/aerosols: aerosols of the test item were generated by a dry dispersion technique using a screw feeder and a pressurized air dispersion nozzle. The mass median aerodynamic diameter of the aerosol was about 1.5 µm. In order to increase the deposition efficiency of the test aerosol in the deep lung, the particle size distribution was shifted toward smaller particles in the submicrometer regime by removing the coarse particles using a cyclone (50% cut-off diameter ≈ 1 mm for a flow rate of 100 m³/h).

- Method of particle size determination: the mass median aerodynamic diameter (MMAD) and the geometric standard deviation of the particles in the exposure chambers were measured every month (n = 24) with a 10-stage Berner impactor (LPI 0.01525; range 15 nm to 16 µm).

*Reference:
- Heinrich, U., Muhle, H., Koch, W., and Mohr, U. 1985. Long-term inhalation studies with rodents. In Safety evaluation and regulation of chemicals 2, ed. F. Homburger, pp. 239 - 250. Basel: Karger.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The particle concentration in the exposure chamber was determined continuously, using aerosol photometers (Koch et al., 1986)*. For calibration of the photometer, the aerosol concentration in each exposure chamber was determined gravimetrically at weekly intervals (Heinrich et al., 1986)*.
The following mean particle mass exposure concentrations were measured:
7.2 mg titanium dioxide/m³ for the first 4 months, followed by 14.8 mg titanium dioxide/m³ for 4 months and 9.4 mg titanium dioxide/m³ for 5.5 months

To compare the different exposure concentrations, the cumulative particle exposure (g/m³ x h) was calculated by multiplying the mean particle mass exposure concentration by the actual exposure time per day and subsequently summarizing for the whole exposure period: 51.5 g/m³ x h.

*References:
- Koch, W. Lödding, H., Oenning, G., and Muhle, H. 1986. The generation and measurement of dry aerosols in large scale inhalation experiments., J. Aerosol Sci. 19: 1453 - 1457.
- Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, Z., Pott, F., and Stöber, W. 1986. Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions. J. Appl. Toxicol. 6: 383 - 395.
Duration of treatment / exposure:
13.5 months
Frequency of treatment:
18 hours/day, 5 days/week
Dose / conc.:
10 mg/m³ air (analytical)
Remarks:
Standard deviation: 2.9
No. of animals per sex per dose:
Treatment group (total: 160 female mice):
- carcinogenicity: 80 mice
- histology (serial sacrifice): 40 mice
- Particle mass/lung (serial sacrifice): 40 mice

Control group (total: 160 female mice):
- carcinogenicity: 80 mice
- histology (serial sacrifice): 40 mice
- Particle mass/lung (serial sacrifice): 40 mice
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: after a total exposure time of 13.5 months, the exposure for all groups was stopped and the animals were kept in clean air for another 9.5 months, at the most. The total experimental time for all groups was 23 months.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: every fourth week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified
HAEMATOLOGY: Not specified
CLINICAL CHEMISTRY: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsies of dead or moribund animals were done 7 days/week.
The lung wet weight and lung retention of inhaled particles were determined at different time points during the study. The lung wet weight was determined at 3, 6, 12, 18, and 21 months after the start of exposure. Titanium dioxide samples were determined by atomic absorption spectroscopy after ashing the lungs.

For the histopathological investigations, the organs of scheduled or moribund sacrifices were fixed in 10% neutral buffered formalin or lto­ Karnovsky fixative (Ito & Karnovsky, 1968)*. The tissues were embedded in Paramat-Wax, sectioned at 5 µm, and stained with hematoxylin and eosin (Lilly-Meyer). Histopathological investigations of the following organs were conducted for all animals: nasal and paranasal cavities (four sections; localization according to Popp & Monteiro-Riviere, 1985*), larynx, trachea, and lung (five sections; localization: the left lobe, right caudal lobe, and right middle lobe were sectioned longitudinally, and the right cranial lobe and accessory lobe were sectioned transversely to main bronchus). Graduation of the findings was done with four grades: very slight, slight, moderate, and high.

*References:
- Ito, S., and Karnovsky, M. J. 1968. Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39:168a-l69a.
- Popp, J. A., and Monteiro-Riviere, N. A. 1985. Macroscopic, microscopic, and ultrastructural anatomy of the nasal cavity, rat In ILSI monographs on pathology of laboratory animals. Respiratory system, eds. T.C. Jones, U. Mohr, and R. D. Hunt, pp. 3-10. New York: Springer.
Statistics:
Differences between groups were considered casewise as statistically significant for p < .05. Body weight and data of lung weight and lung retention of particles were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance, the means of the treatment groups were compared with the means of the control group, using Dunnett's modification of the t-test. For comparison of histopathological data, Fisher's exact test was used.
Survival data of the animals of the carcinogenicity study were analyzed by the Kaplan-Meier method (Kaplan & Meier, 1958)* using the Lifetest program (SAS Institute, Inc., 1985). For animals with significantly different survival times, the tumor rates were compared within three time periods (days 200-450, 450-600, >600) using the prevalence method of Hoel and Walburg (1972)*.

*References:
- Kaplan, E. L., and Meier, P. 1958. Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53: 457-481.
- Hoel, D. G., and Walburg, H. E. 1972. Statistical analysis of survival experiments. ]. Natl. Cancer Inst. 49:361-372.
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- mortality rate was 33% in the TiO2 group compared to 10% in the clean air control group 13.5 months after the start of exposure.
- mortality rate of 50% was reached 17 months from birth in the TiO2 group and 20 months in the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- after 8 months in the TiO2 group the body weight of the mice was significantly lower compared to the clean air control group.
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 specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- measurements after 3 and 12 months of exposure to TiO2 (0.3 g, 0.9 g) showed a substantial increase in lung wet weight compared to the controls (0.2 g, 0.2 g), processing with study duration.
- in the recovery phase, after 13.5 months of exposure, a slight decrease in lung wet weight was found in the TiO2 (0.7 g) exposed groups.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS/MORTALITY/BODY WEIGHT
- after 4 months of exposure the TiO2 particle concentrations were increased from 7.0 mg/m³ to 15 mg/m³. Because of some signs of toxicity (individual loss of body weight, bad general condition) and an increased mortality, the particle concentration was reduced after 4 months of exposure from 15 mg/m³ to 10 mg/m³.
- during the last months of exposure there was no significant difference in the body weight between the control and exposed group.

HISTOPATHOLOGY: NEOPLASTIC
- only the lung tumor types adenomas and adenocarcinomas were observed in mice
- percentages of adenomas/adenocarcinomas were 11.3%/2.5% for the TiO2 group and 25%/15.4% for the clean air group
- lung tumor rates (adenomas and adenocarcinomas) of TiO2-exposed (13.8%) animals were not significantly different from the tumor rate of the control animals (30%).

PARTICLE LUNG BURDEN
- particle lung burden of the mice found after 3, 6, and 12 months of exposure was 0.8, 2.5, 5.2 (TiO2) mg/lung. Expressed as milligrams particles per gram clean air control lung (wet weight of control lung 0.2 g), the particle lung loads after 1 year of exposure to TiO2 were 26 mg.
Remarks on result:
other: see remarks
Remarks:
Due to the anomalous study design (frequency, no dose response paradigm, out-dated criteria for tumour classification) an effect level cannot be derived.
Critical effects observed:
not specified
Conclusions:
This study by Heinrich et al. (single exposure concentration: 7.2 mg/m³ 1-4 months, 14.8 mg/m³ 5-8 months, 9.4 mg/m³ 9-13.5 months, 18h/d, 5d/w) was noted as a reliability 3 study because it was a satellite group used for another study. The study included only female mice and did not have a dose response paradigm. In addition, the mice were exposed for 18 hours/day, 5 days per week for 13.5 months. The tumour response of mice was not significantly different from controls. Moreover, the evaluation of tumours (including malignant tumours) was assessed and did not consider the two international lung pathology workshops – which have reassessed the criteria for describing malignant vs. benign tumours.
Consequently, the diagnosis should have changed after reconsideration of the revised criteria.

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female Wistar rats were exposed 18 hours/day, 5 days/week for 2 years to ultrafine titanium dioxide (P25, Degussa; MMAD: 0.80 µm) and were subsequently kept in clean air for 6 months. The type of inhalation was whole body and the average particle exposure concentrations for the test substance was 10 mg/m³. A control group receiving clean air was run concurrently. The following observations were performed: clinical signs, mortality, body weight, lung wet weight, lung retention of inhaled particles, alveolar clearance measurement, biochemical/cytological examinations of lung lavage fluid, and histopathology.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Crl:(WI)BR)
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 7 weeks ± 3 days
- Housing: housed 2 per cage; kept in wire mesh cages during the exposure period, and in Makrolon cages (18.7 x 21 x 15 cm) during the subsequent clean air period. Softwood bedding (H3/4) was used.
- Diet (ad libitum): "1324 N spec. prepared" (Altromin, Lage, Germany)
- Water (ad libitum): drinking water

ENVIRONMENTAL CONDITIONS
- Temperature: 23 - 25 °C
- Relative humidity: 50 - 70%
- Photoperiod: 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
0.8 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: special whole-body exposure chambers (6 or 12 m³) of the horizontal flow type (Heinrich et al., 1985)*.
The rats were exposed together with NMRI mice in the same exposure chamber (for information on the mice please refer to Section 7.7 Carcinogenicity: NANO_s_Heinrich_1995_mice).

- System of generating particulates/aerosols: aerosols of the test item were generated by a dry dispersion technique using a screw feeder and a pressurized air dispersion nozzle. The mass median aerodynamic diameter of the aerosol was about 1.5 µm. In order to increase the deposition efficiency of the test aerosol in the deep lung, the particle size distribution was shifted toward smaller particles in the submicrometer regime by removing the coarse particles using a cyclone (50% cut-off diameter ≈ 1 mm for a flow rate of 100 m³/h).

- Method of particle size determination: the mass median aerodynamic diameter (MMAD) and the geometric standard deviation of the particles in the exposure chambers were measured every month (n = 24) with a 10-stage Berner impactor (LPI 0.01525; range 15 nm to 16 µm).

*Reference:
- Heinrich, U., Muhle, H., Koch, W., and Mohr, U. 1985. Long-term inhalation studies with rodents. In Safety evaluation and regulation of chemicals 2, ed. F. Homburger, pp. 239 - 250. Basel: Karger.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The particle concentration in the exposure chamber was determined continuously, using aerosol photometers (Koch et al., 1986)*. For calibration of the photometer, the aerosol concentration in each exposure chamber was determined gravimetrically at weekly intervals (Heinrich et al., 1986)*.
The following mean particle mass exposure concentrations were measured:
7.2 mg titanium dioxide/m³ for the first 4 months, followed by 14.8 mg titanium dioxide/m³ for 4 months and 9.4 mg titanium dioxide/m³ for 16 months

To compare the different exposure concentrations, the cumulative particle exposure (g/m³ x h) was calculated by multiplying the mean particle mass exposure concentration by the actual exposure time per day and subsequently summarizing for the whole exposure period: 88.1 g/m³/h

*References:
- Koch, W. Lödding, H., Oenning, G., and Muhle, H. 1986. The generation and measurement of dry aerosols in large scale inhalation experiments., J. Aerosol Sci. 19: 1453 - 1457.
- Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, Z., Pott, F., and Stöber, W. 1986. Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions. J. Appl. Toxicol. 6: 383 - 395.
Duration of treatment / exposure:
24 months
Frequency of treatment:
18 hours/day, 5 days/week
Dose / conc.:
10 mg/m³ air (analytical)
Remarks:
Standard deviation: 2.9
No. of animals per sex per dose:
Treatment group (total: 288 female rats):
- carcinogenicity: 100 rats
- histology (serial sacrifice): 80 rats
- DNA adducts (24 months): 14 rats (from Gallagher et al., 1994)*
- Particle mass/lung (serial sacrifice): 66 rats
- Alveolar lung clearance: 28 rats

Control group (total: 408 female rats):
- carcinogenicity: 220 rats
- histology (serial sacrifice): 80 rats
- DNA adducts (24 months): 14 rats (from Gallagher et al., 1994)*
- Particle mass/lung (serial sacrifice): 66 rats
- Alveolar lung clearance: 28 rats

*Reference:
- Gallagher, J., Heinrich, U., George, M., Hendee, l., Phillips, D.H., and Lewtas, J. 1994. Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon black and titanium dioxide particles. Carcinogenesis 15(7):1291-1299.
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: following the exposure period, the rats were removed from the inhalation chambers and kept under clean air conditions for an additional 6 months.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: every fourth week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified
HAEMATOLOGY: Not specified
CLINICAL CHEMISTRY: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
A biochemical and cytological examination of lung lavage fluid was carried out for the rats. Bronchoalveolar lavagate was obtained by a twofold lavage with 4 mL saline. The lavagate was analyzed for cytological and biochemical parameters (lactate dehydrogenase, β-glucoronidase, total protein, hydroxyproline, total number of leukocytes, differential cell count).
Sacrifice and pathology:
GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsies of dead or moribund animals were done 7 days/week.
The lung wet weight and lung retention of inhaled particles were determined at different time points during the study. The lung wet weight was determined 3, 6, 12, 18, 22, and 24 months after starting the exposure. Titanium dioxide samples were determined by atomic absorption spectroscopy after ashing the lungs.

Alveolar lung clearance measurements by means of a 59Fe tracer aerosol (59Fe2O3, MMAD 0.35 µm) were also carried out. The radioactively labeled test aerosol was inhaled by the rats for about 30 minutes at 3, 12, and 18 months after the start of the experiment. The activity in the thorax was measured externally twice a week over a 100-day period. The decay-corrected activity data of days 15-100 were analyzed for each animal.

For the histopathological investigations, the organs of scheduled or moribund sacrifices were fixed in 10% neutral buffered formalin or lto­ Karnovsky fixative (Ito & Karnovsky, 1968)*. The tissues were embedded in Paramat-Wax, sectioned at 5 µm, and stained with hematoxylin and eosin (Lilly-Meyer). Histopathological investigations of the following organs were conducted for all animals: nasal and paranasal cavities (four sections; localization according to Popp & Monteiro-Riviere, 1985*), larynx, trachea, and lung (five sections; localization: the left lobe, right caudal lobe, and right middle lobe were sectioned longitudinally, and the right cranial lobe and accessory lobe were sectioned transversely to main bronchus). Graduation of the findings was done with four grades: very slight, slight, moderate, and high.

Tumors were classified according to the International Classification of Rodent Tumours (IARC. 1992)*.

*References:
- Ito, S., and Karnovsky, M. J. 1968. Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39:168a-l69a.
- Popp, J. A., and Monteiro-Riviere, N. A. 1985. Macroscopic, microscopic, and ultrastructural anatomy of the nasal cavity, rat In ILSI monographs on pathology of laboratory animals. Respiratory system, eds. T.C. Jones, U. Mohr, and R. D. Hunt, pp. 3-10. New York: Springer.
- IARC. 1992. International classification of rodent tumours. Part I: The rat. IARC Sci. Publ. no. 122. Lyon: International Agency for Research on Cancer.

Statistics:
Differences between groups were considered casewise as statistically significant for p < .05. Body weight and data of lung clearance, lung weight, lung retention of particles, and lung lavage were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance, the means of the treatment groups were compared with the means of the control group, using Dunnett's modification of the t-test. For comparison of histopathological data, Fisher's exact test was used.
Survival data of the animals of the carcinogenicity study were analyzed by the Kaplan-Meier method (Kaplan & Meier, 1958)* using the Lifetest program (SAS Institute, Inc., 1985). For animals with significantly different survival times, the tumor rates were compared within three time periods (days 400-700, 700-800, >800) using the prevalence method of Hoel and Walburg (1972)*.

*References:
- Kaplan, E. L., and Meier, P. 1958. Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53: 457-481.
- Hoel, D. G., and Walburg, H. E. 1972. Statistical analysis of survival experiments. ]. Natl. Cancer Inst. 49:361-372.

Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- after 24 months of exposure, the mortality found was 60% in the TiO2 group compared to 42% in the clean air control group.
- at the end of the 130 week experimental time (exposure time and clean air period), the mortality reached 90% in the TiO2 group and 85% in the control group.
- compared to the controls, the mean lifetime of the rats exposed to TiO2 was significantly shortened.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- body weight of the exposed animals was significantly lower from day 400 (TiO2) compared to control.
- at the end of the 2-year exposure, the body weight of the animals exposed to TiO2 (body weight: 365 g) was significantly lower compared to the control rats (body weight: 417 g).
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 specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- exposure to TiO2 led to a substantial increase in lung wet weight, processing with study duration.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- moderate to high grade bronchioloalveolar hyperplasia was observed in the TiO2 (99/100 rats) group.
- very slight to slight interstitial fibrosis in the lungs was found after 6 months of exposure.
- slight to moderate interstitial fibrosis in the lungs was observed in all animals exposed for 2 years.
- particle-laden macrophages and particles in the alveolar region were also observed in the lungs of all exposed rats.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- lung tumors were found in serial sacrificed animals after 18 months of exposure to TiO2 (5/20 rats; p≤0.05). The following tumor were observed:
benign keratinizing cystic squamous-cell tumor: 2/20 rats
squamous-cell carcinoma: 3/20 rats (sometimes together with adenocarcinoma and benign keratinizing cystic squamous-cell tumor)
adenocarcinoma: 2/20 rats
- lung tumors were found in serial sacrificed animals after 24 months of exposure to TiO2 (4/9 rats; p≤0.05). The following tumor were observed:
benign keratinizing cystic squamous-cell tumor: 2/9 rats
squamous-cell carcinoma: 2/99 rats (sometimes together with benign squamous-cell tumor)
adenocarcinoma: 1/9 rats

- after an exposure time of 24 months followed by 6 months of clean air, lung tumor rates of 32% were observed in rats exposed to TiO2. 8 animals showed 2 tumors in their lungs.
- The following tumour types were observed after an experimental time of 30 months (24 months TiO2-exposure plus 6 months clean air):
benign keratinizing cystic squamous-cell tumor: 20/100 rats
squamous-cell carcinoma: 3/100 rats
adenoma: 4/100 rats
adenocarcinoma: 13/100 rats
Number of rats with tumors: 32/100 (19/100 rats: count without benign keratinizing cystic squamous-cell tumors)
1/217 control animal (clean air exposure) showed adenocarcinoma

- lung tumor rate increased with increasing particle exposure concentration.
- lung tumor incidences of the TiO2 exposed group was significantly increased compared to the control group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE
- the differential cell count and the concentration of lactate-dehydrogenase, β-glucuronidase, OH-proline, and total protein in bronchoalveolar lavage (BAL) showed clear exposure-related effects in the exposure group even after 24 months of exposure.
Details on results:
MORTALITY:
- the various exposure groups did not differ significantly in their mean lifetime among themselves.

HISTOPATHOLOGY: NEOPLASTIC
- no lung tumors were observed in the TiO2 satellite group of 20 animals each after 6 and 12 months of exposure.

PARTICLE LUNG BURDEN
- during the second year of exposure, the particle lung load of the TiO2 exposed animals increased by only 13%.
- the retained particle mass in the lung-associated lymph nodes (LALN) of the TiO2-exposed rats after 22 months amounted to about 14%.
- expressed as milligrams particles per gram clean air control lung (wet weight of control lung 1.2 g), the particle lung loads after 1 year of exposure to TiO2 were 29 mg.

ALVEOLAR LUNG CLEARANCE
- the alveolar clearance rate was already significantly compromised after inhalation of TiO2 after 3 months of exposure.
- after 18 months of exposure to TiO2 and 3 months of recovery time without particle exposure, no reversibility of the alveolar lung clearance damage could be detected.

Remarks on result:
other: see remarks
Remarks:
Due to the anomalous study design (frequency, no dose response paradigm, out-dated criteria for tumour classification) an effect level cannot be derived.
Critical effects observed:
not specified
Conclusions:
This study by Heinrich et al. (single exposure concentration: 7.2 mg/m³ 1-4 months, 14.8 mg/m³ 5-8 months, 9.4 mg/m³ 9-24 months, 18h/d, 5d/w) was noted as a reliability 3 study because it was a satellite group used for another study. The study included only female rats and did not have a dose response paradigm. In addition, the rats and mice were exposed for 18 hours/day, 5 days per week for 24 months. The evaluation of tumours (including malignant tumours) was assessed and did not consider the two international lung pathology workshops – which have reassessed the criteria for describing malignant vs. benign tumours – particularly with reference to the cystic keratinizing pulmonary squamous cell lesions that are unique to the rat.
Consequently, the diagnosis should have changed after reconsideration of the revised criteria.
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-06-04 to 1981-07-09
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 100 male and 100 female Crl:CD(SD)BR rats each were exposed to titanium dioxide (10, 50, and 250 mg/m³). The test item was administrated via whole body inhalation for 6 hours/day, 5 days/week for 24 months. A concurrent control group was run concurrently. The following parameters were assessed:clinical signs, mortality, body weights, haematology, clinical chemistry, urinalysis. gross pathology, and histopathology.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: Crl:CD(SD)BR
Details on species / strain 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.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age: 3 weeks
- Housing: housed pairwise in stainless steel wire-mesh cages.
- Diet (ad libitum): Purina Laboratory Chow Checkers '5001
- Water: ad libitum
- Acclimation period: approx. 17 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2 °C
- Relative humidity: 50 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12
- rooms had laminar flows of filtered and recirculated air
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.54 - <= 1.93 µm
Remarks on MMAD:
GSD: 2.52 - 3.14
Mean respirable fraction of TiO2: 93.7% or greater (values for each determination ranged from 84.0 to 99.95 %)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers made of material not reactive with TiO2
- Chamber volume: 3.85 m³
- 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 and humidity: 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

TEST ATMOSPHERE
- Particle size distribution: 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.

TEST ATMOSPHERE
- Brief description of analytical method used: 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see above ("Details on inhalation exposure")
Duration of treatment / exposure:
24 months
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
10.55 mg/m³ air (analytical)
Remarks:
SD: 2.12 mg/m³
Dose / conc.:
50.68 mg/m³ air (analytical)
Remarks:
SD: 6.65 mg/m³
Dose / conc.:
250.1 mg/m³ air (analytical)
Remarks:
SD: 24.70 mg/m³
No. of animals per sex per dose:
100 male rats / 100 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: results from previous inhalation studies.
- Rationale for animal assignment: rats of each sex were divided by computerised, stratified randomisation into groups of 100 males and groups of 100 females such that the mean of body weights of each group of rats within a sex were approximately equal.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily throughout the study
- Cage side observations checked: morbundity/mortality, abnormal behaviour and appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once weekly during the first 3 months and at least every other week during the remainder of the study

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly during the first 3 months of the study followed by approx. once every other week for the remainder of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE : No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: approx. 3, 6, 12, 15 and 18 months after the study's initiation
- How many animals: 10 males / 10 females (same rats were evaluated at each interval throughout the study)
- Parameters checked: basophil count, eosinophil count, erythrocyte count, haematocrit (Ht), haemoglobin, leukocyte count, lymphocyte count, mean cell haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, monocyte count and neutrophil count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: approx. 3, 6, 12, 15 and 18 months after the study's initiation
- How many animals: 10 males / 10 females (same rats were evaluated at each interval throughout the study)
- Parameters checked: alanine aminotransferase activity, alkaline phosphatase activity, bilirubin, calcium, phosphorus, total protein and urea nitrogen.

URINALYSIS: Yes
- Time schedule for collection of urine: approx. 3, 6, 12, 15 and 18 months after the study's initiation.
- Parameters checked: volume, osmolality, pH, bilirubin, blood, protein, sugar, urobilinogen, appearance and sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Gross and histopathological examinations were conducted on 5 rats/sex/treatment group after 3 and 6 months exposure as well as 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 of tissue permitting), were examined grossly and histopathologically.

All rats were sacrificed and the respiratory tract was prepared for fixation.
All other tissues were removed for gross examination and weighed (lungs, trachea, heart, liver, stomach (3 and 12 months sacrifice only), kidneys, spleen, testes, pituitary, brain, thymus and adrenals) at each sacrifice. The tissues were fixed in either Bouin's (nasal cavitiy (turbinates), trachea, luings, oesophagus, kidneys, sternal bone marrow, testes, epididymides, mammary gland,pituitary, thyroid - parathyroids, adrenals, eyes, bone (sternum, femur, and vertebrate, ear (zymbal gland), and skin (neck)) or formalin at the 6- through 24-month sacrifices. At the 3-month sacrifice only, adipose tissue and ovaries were fixed in Bouin's fixative and the adrenals, oesophagus, mammary and Zymbal's glands were fixed in formalin.

Representative specimens of the following organs and tissues were taken from all rats: heart, thoracic aorta, nasal cavity (turbinates), trachea, lungs, liver, pancreas, small intestine (duodenum, jejunum, and ileum), tongue, oesophagus, stomach, salivary glands, large intestine (cecum and colon), rectum, kidneys, bladder, sternal bone marrow, spleen, lymph nodes (cervical, mesenteric and tracheobronchial), thymus, testes, epididymes, prostate, seminal vesicles, uterus, ovaries, mammary gland, pituitary, adrenals, thyroid - parathyroids, brain, spinal cord, sciatic nerve, skeletal muscle, bone (sternum, femur, and vertebrate), eyes, Harderian's gland, exorbital lacrimal glands, ear (Zymbal's gland), skin (neck), adipose tissue, and all gross lesions
Other examinations:
not specified
Statistics:
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 pairwise 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.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
There were several changes in haematological parameters related to exposure of rats to TiO2. When compared to the controls over course of this study:
- haematocrits (both sexes) and heamoglobins (males only) for rats in the 250 mg/m³ treatment groups were greater (up to 12 and 11%, respectively).
- mean cell volumes and mean cell haemoglobins in male rats from all exposed groups were greater (up to 14%); there were only minimal differences among these groups.
- neutrophil counts in all treatment groups were greater (up to 80% in male and 117% in female rats in the 250 mg/m³ treatment groups). A dose-related trend was observed among these treatment groups.
- lymphocyte counts in all treatment groups were lower (up to 45% lower for rats in the 250 mg/m³ treatment groups). A dose-related trend was observed among these treatment groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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:
- bilirubin content in female rats were greater and were more pronounced in the 50 and 250 mg/m³ 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.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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:
Lungs:
- clear dose- and time-dependent lung weight increases were observed
- mean absolute and relative lung weights for rats in the 250 mg/m³ 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/m³ 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.
markedly heavier at 50 mg/m³, and were more than two times control lung weights at 250mg/m³.

Thymus:
- A dose-related thymus weight effect was observed in rats in the 50 and 250 mg/m³ treatment groups. Mean absolute and relative thymus weights were as high as 1.37-fold greater than controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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/m³ treatment groups as slightly elevated gray nodules. The lungs of rats in the 250 mg/m³ 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.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The respiratory tract was the primary deposition site for TiO2 as well as the primary site in which a tissue response to this compound was observed. Over the course of this study, the following dose-related effects were observed:
- increased incidence of rhinitis with accompanying squamous metaplasia in the anterior nasal cavity;
- greater numbers of TiO2 –laden alveolar macrophages (dust cells), and of aggregated, foamy alveolar macrophages;
- deposits of TiO2 particles within the lymph nodes where the relative severity for tracheobronchial > peribronchial and perivascular > mesenteric lymph nodes.
- alveolar proteinosis in approximately 51 and 97% of the rats in the 50 and 250 mg/m³ treatment groups, respectively, but it was not found in all other groups;
- markedly increased incidences of cholesterol granulomas that contained large numbers of collagen fibers in the lungs of rats in the 50 and 250 mg/m³ treatment groups (approximately 75 and 97% of these rats, respectively; 13% or less for rats in all other groups);
- markedly increased incidences of collagenized fibrosis within alveoli of rats in the 50 and 250 mg/m³ treatment groups (in approximately 60 and 99% of these rats, respectively; 14% less for rats in all other groups);
- greater incidences of focal pleurisy associated with subpleural cholesterol granulomas and focal dust cell infiltration in the lungs of TiO2-exposed rats;
- bronchiolarization of alveoli adjacent to terminal bronchioles, which occurred more frequently in female than male rats
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(for a detailed analysis please see study record under the endpoint: carcinogenicity)
Other effects:
not examined
Details on results:
CLINICAL SIGNS
Exposure to TiO2 resulted in no abnormal clinical sign in any exposed group.
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.
- 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.

MORTALITY
- exposure to TiO2 resulted in no excess mortality in any exposed group.
- 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.

BODY WEIGHT AND WEIGHT CHANGES:
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/m³ was consistently lower than those for rats exposed to 10 or 50 mg/m³. The differences in the mean body weight gain among all treatment groups parallelled the mean body weight effects.

HAEMATOLOGY:
- erythrocyte counts for rats in the 250 mg/m³ treatment groups were slightly greater (up to 9%; not statistically significant).
- leukocyte counts in all treatment groups were greater (up to 80% for the high-dose group). The values for rats in the 250 mg/m³ treatment group were generally higher than those of the other treatment groups.

URINALYSIS
Urological parameters were within the expected range of biological variability for this species.

ORGAN WEIGHTS
Lungs:
- lung weights at 10mg/m³ were comparable to those of control group

Liver:
- 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.

Kidney:
- mean absolute and/or relative kidney weights for TiO2 exposed male rats were approx. 77 to 88% of the controls at the 3-, 12- and 24-months sacrifices. A clear dose-response was not demonstrated.

Significant but transient effects were observed for other organ weights over the course of this study. These effects were observed for other organ weights over the course of this study. These effects were either related to body weight differences or were not considered to be biologically significant.

HISTOPATHOLOGY-NEOPLASTIC
Exposures to titanium dioxide at concentrations of 10, 50, or 250 mg/m³ produced lung tumors (bronchioalveolar adenoma and squamous cell carcinoma) only at the highest concentration.

To further characterise the bronchoalveolar lesions, in 1992, a group of pathologists from North America and Europe examined lung lesions produced by para-aramid RFP (respirable fibre-shaped particles) and titanium dioxide. This panel diagnosed the lesion as a "proliferative keratin cyst" (PKC). Additionally, the pathologists agreed that the lesion was not a malignant neoplasm and is most likely not neoplastic. A minority opinion was that the lesion is probably a benign tumour (Carlton 1994; Levy 1994). Another subsequent international pathology workshop was convened to develop standardized histological criteria for classifying pulmonary keratin lesions (Boorman et al., 1996). As a consequence, most of the lesions that had originally been diagnosed as “cystic keratinizing squamous cell carcinomas” were re-classified by the consensus panels as non-tumorous “proliferative keratin cysts” (Warheit and Frame, 2006).
In the aftermath of two international pathology workshops designed, in part, to establish histological criteria for classifying pulmonary keratin lesions, these lesions were evaluated by four pathologists using current diagnostic criteria. Microscopic review of 16 proliferative squamous lesions, previously diagnosed as cystic keratinizing squamous cell carcinoma in the lungs of rats from the 2-year inhalation study was performed. Unanimous agreement was reached as to the diagnosis of each of the lesions. Two of the lesions were diagnosed as squamous metaplasia and 1 as poorly-keratinizing squamous cell carcinoma. Most of the remaining 13 lesions were diagnosed as non-neoplastic pulmonary keratin cysts (Warheit and Frame, 2006).
Consequently, the diagnosis of many of these lesions has changed after the development of revised criteria.

There were several effects associated with exposure to TiO2 which were not clearly dose- related. These include:
- hyperplasia of the Type II pneumocytes lining alveolar walls;
- increased incidences of chronic tracheitis; and
- Increased incidences of broncho/bronchiolar pneumonia.
Over the course of the study, most of the inhaled TiO2 particles that were observed within the alveoli were phagocytized by dust cells. These particles were retained primarily within the alveolar ducts and adjoining alveoli of rats in the 10 and 50 mg/m³ treatment groups which also had some dust-free alveoli in the peripheral acini. However, for rats in the 250 mg/m³ treatment group, the number of dust cells was markedly greater than that for rats in other treatment groups. The dust cells were distributed throughout the alveoli with larger concentrations in the alveolar duct region. This region served as a focal point for several of the tissue responses previously listed, e.g. collagenized fibrosis, bronchiolarization, squamous cell metaplasia and tumor formation. Within each treatment group, the lesions gradually increased in frequency and/or severity with time on test. By the end of this study, rats in the 50 and 250 mg/m³ treatment groups had irreversible effects whereas all of the lesions observed in rats exposed to 10 mg/m³ were considered to be reversible.
Although most of the biological effects associated with TiO2 exposure occurred within the respiratory tract, other tissues were also affected. The incidences of degenerative retinopathy in exposed rats was increased significantly compared to those of controls and were greater among female than male rats; however, a dose-response relationship was not observed. Female rats exposed to TiO2 also had significantly greater incidences of endometritis than the controls (dose-related).
Exposure of rats to TiO2 resulted in a dose-related transmigration of dust particles from the lung through the lymhatics to the lymph nodes, liver, and spleen. The accumulation of TiO2 in the tracheobronchial, cervical, and mesenteric lymph nodes did not result in a significant tissue response. In the spleen, dust particles aggregates were primarily located in the lymphnode tissue of the white pulp. In the liver, TiO2 accumulated in the portal riads and Kupfter cells. There were no cellular responses to these deposits in either the spleen or liver.

A wide variety of spontaneous neoplastic and non-neoplastic lesions that were considered to be age related occurred with similar incidence and severity among control and all TiO2-exposed groups

REFERENCES
- Carlton, W.W. (1994) "Proliferative Keratin Cyst," a Lesion in the Lungs of Rats Following Chronic Exposure to Para-aramid Fibrils. Fundam. Appl. Toxicol. 23, 304-307
- Levy L.S. (1994). Squamous Lung Lesions Associated with Chronic Exposure by Inhalation of Rats to p-Aramid Fibrils (Fine Fiber Dust) and to Titanium Dioxide: Findings of a Pathology Workshop. In: Mohr, U. (Ed.): Toxic and carcinogenic effects of solid particles in the respiratory tract, ILSI Press, 473-478
Boorman, G.A: et al. (1996). Classification of Cystic Keratinizing Squamous Lesions of the Rat Lung: report of a Workshop. Toxicol. Pathol. 24, 564-573
Dose descriptor:
NOAEC
Effect level:
10.6 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Effect level:
50.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Dose-related increases in pleurisy, slight collagenized fibrosis associated with cholesterol granulomas, alveoli bronchiolarization, pneumonia, and alveolar cell hyperplasia were observed. The changes observed at the 10 mg/m³ concentration were minimal in severity in comparison to similar effects observed in the controls (except that the alveolar cell hyperplasia was not observed in controls). The degree of pulmonary fibrosis seen at the two higher levels was slight. In subsequent experimentation (Warheit et al., 1997; summarized below) using these same dosing rates for four weeks, it was demonstrated that high dose animals had substantially increased lung clearance times and sustained inflammation under particle overload conditions.

All concentration used in the Lee et al. study clearly exceeded the MTD, since lung overload conditions were attained even at the lowest concentration of 10 mg/m³

A dosimetric analysis of the 2-year rat inhalation study by Lee et al. shows that all three TiO2 exposure concentrations resulted in significant lung
particle overload, i.e., an impaired alveolar macrophage-mediated particle clearance function. Per g lung weight, the retained normalized lung burden observed in the study of 28 mg/g exposed lung and 39 mg/g control lung for the 50 mg/m3-exposed rats (shown at the beginning of this analysis, p.7) is obviously greatly exceeding a retained lung burden of 1 mg/g lung which - according to Morrow (1988) - signals the beginning of lung overload in rats.
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
only one concentration tested
Principles of method if other than guideline:
It is highlighted that the study was conducted in compliance with OECD 453. However, titanium dioxide was not selected as primary test item (which was carbon black), but as negative control substance. Consequently, only one high titanium dioxide concentration was used and no dose-response relationship can be derived for non-neoplastic lesions as well as no NOAEC be identified.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
The study was done using Fischer-344 rats due to their wide use by the National Toxicology Program and the extensive data base available on their health status and background tumor incidence.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH
- Age: 4 weeks old
- Housing: individually housed in metal wire mesh cages (18.7 x 21 x 15 cm) in horizontal air flow chambers throughout the study.
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: whole-body exposure chambers were used
- System of generating particulates/aerosols: a dry aerosol dispersion technique was used. The aerosol generator consisted of a commercially available feeding system connected to a two-stage pressurised air ejector.
To maximize mixing and uniform distribution of the aerosol, the chamber inlets were equipped with diffusers and perforated plates.
- Temperature, humidity, and air flow: chambers were maintained at 23.5 ± 1 °C and 40 - 60 % relative humidity with an air flow of 3.8 m³/min.

- Method of particle size determination: particle size distribution in each chamber was measured 13 times during the study using Berner impactor.

TEST ATMOSPHERE
- Brief description of analytical method used: at the inlet side of the chamber photometric determination of the aerosol concentration was done and gravimetric samples were collected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on analytical verification of doses or concentrations" above.
Duration of treatment / exposure:
24 months
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
5 mg/m³ air (analytical)
Remarks:
SD: 0.7 mg/m³ air
No. of animals per sex per dose:
144 males / 144 females (total: 288 animals)
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Post-exposure period: 1.5 months
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on arrival, 2 and 4 weeks subsequently, and thereafter at 6-month intervals

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks during the first 14 weeks and then once every 4 weeks

FOOD CONSUMPTION:
- Time schedule for examinations: weekly during the first 13 weeks and then once every 3 months.

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 data

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for examinations: 6, 12, 18 and 25.5 months
- Parameters checked: leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for examinations: 6, 12, 18 and 25.5 months

URINALYSIS: Yes
- Time schedule for examinations: 6, 12, 18 and 25.5 months

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified
Sacrifice and pathology:
Rats sacrificed during and after scheduled exposure were anesthetized and killed. The abdominal cavity was opened and the diaphragm was cut allowing the lungs to collapse. Organs (brain, liver, kidneys, adrenals, and gonads) were weighed. Tissues and organs were fixed and processed for routine histology. Bones were decalcified. The larynx, trachea, oesophagus, thymus, heart, and lungs were removed. The larynx and upper part of the trachea were separated and placed in formalin. The lungs scheduled for histopathology were fixed. Complete histopathological examination of organs, tissues, and gross lesions of animals of the serial sacrifices and of all animals of the “basic study” was performed (please refer to the table 1 in the field “Any other information on materials incl. tables” below).

Bronchoalveolar lavage: the method of Henderson et al. (1987)* was used with minor modifications. Following preparation of the lungs, they were lavaged with saline without massage. The cell concentration was determined using a counting chamber. The lavagate was centrifuged and the supematant used for the determination of the biochemical parameters (lactic dehydrogenase (LDH), β-glucuronidase, total protein). Cytoslides were prepared for differential cell count.

*Reference:
- Henderson, R.F., Mauderly, J. L., Pickrell, J. A., Hahn, R. F., Muhle, H., and Rebar, A. H. (1987). Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329-342.
Statistics:
Parametric data were examined by analysis of variance (ANOVA) followed by Dunnett test to compare various treatment groups with controls. Survival data were analysed by the Kaplan-Meier method using the lifetest program of SAS. For necropsy and tumour occurrance data, simple tests for homogeneity of contingency tables (using qui square statistics or Fisher's exact method) were used.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS
Inhalation of the test material did not cause overt signs of toxicity.
Clinical appearance of the rats was found to be within normal limits. Measured virologic, bacteriologic, and parasitologic parameters were also within normal limits or negative during the study.

MORTALITY:
The mean survival in the basic study (100 animals, 50 of each gender) was 40% at the termination of the experiment. The data analyzed by Kaplan-Meier method, separately for each gender, indicated no difference among various exposure groups.

BODY WEIGHT AND WEIGHT GAIN
Body weight development appeared to be normal when compared to concurrent and historical control.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was normal in the TiO2-exposed groups.

HAEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS
Clinical laboratory test results were essentially within normal range. The results were consistent with healthy animals.

ORGAN WEIGHTS
No changes in organ weights were observed at the terminal sacrifice.

HISTOPATHOLOGY: NON-NEOPLASTIC
- TiO2 group: extent of particle-laden macrophages increased with exposure time.
- a small but statistically insignificant incidence of fibrosis was seen in the TiO2 and air-only control groups as shown below:
Control (mild degree of fibrosis): 1.2 (date of sacrifice: 21 - 25.5 months)
TiO2 (moderate degree of fibrosis): 35.7 (date of sacrifice: 21 months); 19.1* (date of sacrifice: 21 - 25.5 months)
TiO2 (minimal degree of fibrosis): 16.7 (date of sacrifice: 15 months); 4.5 (date of sacrifice: 21 - 25.5 months)
TiO2 (mild degree of fibrosis): 1.1 (date of sacrifice: 21- 25.5 monthsmonths)
(*p <0.001; approx. 14 animals/group were examined at each serial sacrifice point and about 90 animals/ group were evaluated at 21 - 26 months of the study)
- no significant difference from air-only control in the extent of various upper respiratory system lesions was noted regarding the TiO2 group.

HISTOPATHOLOGY: NEOPLASTIC
- two adenomas and one adenocarcinoma (3/100 animals) were observed in the air-only control group while one tumour of each type was detected in the TiO2 group (2/100 animals).

PARTICLE RETENTION
TiO2 accumulated progressively in the lungs of the rats. The mean quantity of TiO2 retained was 2.72 mg/lung.

BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
The number of lavagable leukocytes at 15 months of exposure was unaffected by treatment. In the lavage fluid a substantial amount of fragments of macrophages were observed. The results from the TiO2 exposure showed a significant decrease in macrophages at 15 months. This cytologic pattern persisted throughout the rest of the study as the results at 21, 24, and 25.5 months were quite similar. A linear relationship was observed between the fraction of polymorphonuclear leukocytes (PMN) in the lavagate and the retention half-time of the polystyrene tracer (Bellmann et al., 1991)* at TiO2 concentration.
The levels of cytoplasmic and lysosomal enzymes and total protein in lavage fluid were comparable to those of air-only controls in the TiO2 groups.
Please also refer to table 1 in the field "Any other information on resutls incl. tables" below.

*Reference:
- Bellmann, B., Muhle, H., Creutzenberg, O., Dasenbrock, C., Kilpper, R., Mackenzie, J., Morrow, P., and Mermelstein, R. (1991). Lung clearance and retention of toner, utilizing a tracer technique during chronic inhalation exposure in rats. Fundam. Appl. Toxicol. 17,300-3 13.
Remarks on result:
not determinable
Remarks:
since titanium dioxide was not selected as primary test item but as negative control substance, only one high titanium dioxide concentration was used. Consequently, no dose-response relationship can be derived for non-neoplastic lesions and no NOAEC be identified.
Critical effects observed:
not specified

Table 1: a) Cytology results in bronchoalveolar lavage fluid and b) mean levels of controls and levels normalized to control of LDH,β-glucuronidase, and protein in lavagate

 

Exposure (months)

15

21

24

24 + 6 weeks clean air

Number of lavaged leukocytes (10³ cells/mL)

Control

100

133

221

194

TiO2

102

156

211

265

Macrophages (%)

Control

97.2

97.0

98.0

97.4

TiO2

90.8*

93.2

95.7

92.8

PMN (%)

Control

1.1

1.9

0.5

0.8

TiO2

4.9

4.2

2.3

4.3

Lymphocytes

Control

1.7

1.2

1.6

1.9

TiO2

4.4*

2.7

2.1

3.0

LDH

Control (U/liter

25

35

39

33

Control

1.00

1.00

1.00

1.00

TiO2

1.16

0.71

1.26

1.15

β-glucuronidase

Control (U/liter

0.15

0.20

0.18

0.15

Control

1.00

1.00

1.00

1.00

TiO2

1.00

0.70

1.89

1.33

Protein

Control (U/liter

108

114

146

144

Control

1.00

1.00

1.00

1.00

TiO2

1.04

0.99

1.86

1.04

Conclusions:
Inhalation of titanium dioxide showed no signs of overt toxicity. Body weight, clinical chemistry values, food consumption, and organ weights were normal. Fibrosis was present in the controls at a comparable rate to that of titanium dioxide exposed rats, being minimal to mild and not statistically significantly different from controls. There were no significant increases in lung tumours vs. control rats exposed for up to 24 months by whole body inhalation to titanium dioxide in this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female NMRI mice were exposed 18 hours/day, 5 days/week for 13.5 months to ultrafine titanium dioxide (P25, Degussa; MMAD: 0.80 µm) and were subsequently kept in clean air for 9.5 months. The type of inhalation was whole body and the average particle exposure concentrations for the test substance was 10 mg/m³. A control group receiving clean air was run concurrently. The following observations were performed: clinical signs, mortality, body weight, lung wet weight, lung retention of inhaled particles, and histopathology.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
mouse
Strain:
other: NMRI (Crl:NMRI BR)
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 7 weeks ± 3 days
- Housing: housed in groups of 8 - 10 per cage; kept in wire mesh cages during the exposure period, and in Makrolon cages (18.7 x 21 x 15 cm) during the subsequent clean air period. Softwood bedding (H3/4) was used.
- Diet (ad libitum): "1324 N spec. prepared" (Altromin, Lage, Germany)
- Water (ad libitum): drinking water

ENVIRONMENTAL CONDITIONS
- Temperature: 23 - 25 °C
- Relative humidity: 50 - 70%
- Photoperiod: 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
0.8 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: special whole-body exposure chambers (6 or 12 m³) of the horizontal flow type (Heinrich et al., 1985)*.
The mice were exposed together with Wistar rats in the same exposure chamber (for information on the rats please refer to Section 7.7 Carcinogenicity: NANO_s_Heinrich_1995_rats).

- System of generating particulates/aerosols: aerosols of the test item were generated by a dry dispersion technique using a screw feeder and a pressurized air dispersion nozzle. The mass median aerodynamic diameter of the aerosol was about 1.5 µm. In order to increase the deposition efficiency of the test aerosol in the deep lung, the particle size distribution was shifted toward smaller particles in the submicrometer regime by removing the coarse particles using a cyclone (50% cut-off diameter ≈ 1 mm for a flow rate of 100 m³/h).

- Method of particle size determination: the mass median aerodynamic diameter (MMAD) and the geometric standard deviation of the particles in the exposure chambers were measured every month (n = 24) with a 10-stage Berner impactor (LPI 0.01525; range 15 nm to 16 µm).

*Reference:
- Heinrich, U., Muhle, H., Koch, W., and Mohr, U. 1985. Long-term inhalation studies with rodents. In Safety evaluation and regulation of chemicals 2, ed. F. Homburger, pp. 239 - 250. Basel: Karger.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The particle concentration in the exposure chamber was determined continuously, using aerosol photometers (Koch et al., 1986)*. For calibration of the photometer, the aerosol concentration in each exposure chamber was determined gravimetrically at weekly intervals (Heinrich et al., 1986)*.
The following mean particle mass exposure concentrations were measured:
7.2 mg titanium dioxide/m³ for the first 4 months, followed by 14.8 mg titanium dioxide/m³ for 4 months and 9.4 mg titanium dioxide/m³ for 5.5 months

To compare the different exposure concentrations, the cumulative particle exposure (g/m³ x h) was calculated by multiplying the mean particle mass exposure concentration by the actual exposure time per day and subsequently summarizing for the whole exposure period: 51.5 g/m³ x h.

*References:
- Koch, W. Lödding, H., Oenning, G., and Muhle, H. 1986. The generation and measurement of dry aerosols in large scale inhalation experiments., J. Aerosol Sci. 19: 1453 - 1457.
- Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, Z., Pott, F., and Stöber, W. 1986. Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions. J. Appl. Toxicol. 6: 383 - 395.
Duration of treatment / exposure:
13.5 months
Frequency of treatment:
18 hours/day, 5 days/week
Dose / conc.:
10 mg/m³ air (analytical)
Remarks:
Standard deviation: 2.9
No. of animals per sex per dose:
Treatment group (total: 160 female mice):
- carcinogenicity: 80 mice
- histology (serial sacrifice): 40 mice
- Particle mass/lung (serial sacrifice): 40 mice

Control group (total: 160 female mice):
- carcinogenicity: 80 mice
- histology (serial sacrifice): 40 mice
- Particle mass/lung (serial sacrifice): 40 mice
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: after a total exposure time of 13.5 months, the exposure for all groups was stopped and the animals were kept in clean air for another 9.5 months, at the most. The total experimental time for all groups was 23 months.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: every fourth week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified
HAEMATOLOGY: Not specified
CLINICAL CHEMISTRY: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsies of dead or moribund animals were done 7 days/week.
The lung wet weight and lung retention of inhaled particles were determined at different time points during the study. The lung wet weight was determined at 3, 6, 12, 18, and 21 months after the start of exposure. Titanium dioxide samples were determined by atomic absorption spectroscopy after ashing the lungs.

For the histopathological investigations, the organs of scheduled or moribund sacrifices were fixed in 10% neutral buffered formalin or lto­ Karnovsky fixative (Ito & Karnovsky, 1968)*. The tissues were embedded in Paramat-Wax, sectioned at 5 µm, and stained with hematoxylin and eosin (Lilly-Meyer). Histopathological investigations of the following organs were conducted for all animals: nasal and paranasal cavities (four sections; localization according to Popp & Monteiro-Riviere, 1985*), larynx, trachea, and lung (five sections; localization: the left lobe, right caudal lobe, and right middle lobe were sectioned longitudinally, and the right cranial lobe and accessory lobe were sectioned transversely to main bronchus). Graduation of the findings was done with four grades: very slight, slight, moderate, and high.

*References:
- Ito, S., and Karnovsky, M. J. 1968. Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39:168a-l69a.
- Popp, J. A., and Monteiro-Riviere, N. A. 1985. Macroscopic, microscopic, and ultrastructural anatomy of the nasal cavity, rat In ILSI monographs on pathology of laboratory animals. Respiratory system, eds. T.C. Jones, U. Mohr, and R. D. Hunt, pp. 3-10. New York: Springer.
Statistics:
Differences between groups were considered casewise as statistically significant for p < .05. Body weight and data of lung weight and lung retention of particles were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance, the means of the treatment groups were compared with the means of the control group, using Dunnett's modification of the t-test. For comparison of histopathological data, Fisher's exact test was used.
Survival data of the animals of the carcinogenicity study were analyzed by the Kaplan-Meier method (Kaplan & Meier, 1958)* using the Lifetest program (SAS Institute, Inc., 1985). For animals with significantly different survival times, the tumor rates were compared within three time periods (days 200-450, 450-600, >600) using the prevalence method of Hoel and Walburg (1972)*.

*References:
- Kaplan, E. L., and Meier, P. 1958. Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53: 457-481.
- Hoel, D. G., and Walburg, H. E. 1972. Statistical analysis of survival experiments. ]. Natl. Cancer Inst. 49:361-372.
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- mortality rate was 33% in the TiO2 group compared to 10% in the clean air control group 13.5 months after the start of exposure.
- mortality rate of 50% was reached 17 months from birth in the TiO2 group and 20 months in the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- after 8 months in the TiO2 group the body weight of the mice was significantly lower compared to the clean air control group.
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 specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- measurements after 3 and 12 months of exposure to TiO2 (0.3 g, 0.9 g) showed a substantial increase in lung wet weight compared to the controls (0.2 g, 0.2 g), processing with study duration.
- in the recovery phase, after 13.5 months of exposure, a slight decrease in lung wet weight was found in the TiO2 (0.7 g) exposed groups.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS/MORTALITY/BODY WEIGHT
- after 4 months of exposure the TiO2 particle concentrations were increased from 7.0 mg/m³ to 15 mg/m³. Because of some signs of toxicity (individual loss of body weight, bad general condition) and an increased mortality, the particle concentration was reduced after 4 months of exposure from 15 mg/m³ to 10 mg/m³.
- during the last months of exposure there was no significant difference in the body weight between the control and exposed group.

HISTOPATHOLOGY: NEOPLASTIC
- only the lung tumor types adenomas and adenocarcinomas were observed in mice
- percentages of adenomas/adenocarcinomas were 11.3%/2.5% for the TiO2 group and 25%/15.4% for the clean air group
- lung tumor rates (adenomas and adenocarcinomas) of TiO2-exposed (13.8%) animals were not significantly different from the tumor rate of the control animals (30%).

PARTICLE LUNG BURDEN
- particle lung burden of the mice found after 3, 6, and 12 months of exposure was 0.8, 2.5, 5.2 (TiO2) mg/lung. Expressed as milligrams particles per gram clean air control lung (wet weight of control lung 0.2 g), the particle lung loads after 1 year of exposure to TiO2 were 26 mg.
Remarks on result:
other: see remarks
Remarks:
Due to the anomalous study design (frequency, no dose response paradigm, out-dated criteria for tumour classification) an effect level cannot be derived.
Critical effects observed:
not specified
Conclusions:
This study by Heinrich et al. (single exposure concentration: 7.2 mg/m³ 1-4 months, 14.8 mg/m³ 5-8 months, 9.4 mg/m³ 9-13.5 months, 18h/d, 5d/w) was noted as a reliability 3 study because it was a satellite group used for another study. The study included only female mice and did not have a dose response paradigm. In addition, the mice were exposed for 18 hours/day, 5 days per week for 13.5 months. The tumour response of mice was not significantly different from controls. Moreover, the evaluation of tumours (including malignant tumours) was assessed and did not consider the two international lung pathology workshops – which have reassessed the criteria for describing malignant vs. benign tumours.
Consequently, the diagnosis should have changed after reconsideration of the revised criteria.

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Female Wistar rats were exposed 18 hours/day, 5 days/week for 2 years to ultrafine titanium dioxide (P25, Degussa; MMAD: 0.80 µm) and were subsequently kept in clean air for 6 months. The type of inhalation was whole body and the average particle exposure concentrations for the test substance was 10 mg/m³. A control group receiving clean air was run concurrently. The following observations were performed: clinical signs, mortality, body weight, lung wet weight, lung retention of inhaled particles, alveolar clearance measurement, biochemical/cytological examinations of lung lavage fluid, and histopathology.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Crl:(WI)BR)
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 7 weeks ± 3 days
- Housing: housed 2 per cage; kept in wire mesh cages during the exposure period, and in Makrolon cages (18.7 x 21 x 15 cm) during the subsequent clean air period. Softwood bedding (H3/4) was used.
- Diet (ad libitum): "1324 N spec. prepared" (Altromin, Lage, Germany)
- Water (ad libitum): drinking water

ENVIRONMENTAL CONDITIONS
- Temperature: 23 - 25 °C
- Relative humidity: 50 - 70%
- Photoperiod: 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
0.8 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: special whole-body exposure chambers (6 or 12 m³) of the horizontal flow type (Heinrich et al., 1985)*.
The rats were exposed together with NMRI mice in the same exposure chamber (for information on the mice please refer to Section 7.7 Carcinogenicity: NANO_s_Heinrich_1995_mice).

- System of generating particulates/aerosols: aerosols of the test item were generated by a dry dispersion technique using a screw feeder and a pressurized air dispersion nozzle. The mass median aerodynamic diameter of the aerosol was about 1.5 µm. In order to increase the deposition efficiency of the test aerosol in the deep lung, the particle size distribution was shifted toward smaller particles in the submicrometer regime by removing the coarse particles using a cyclone (50% cut-off diameter ≈ 1 mm for a flow rate of 100 m³/h).

- Method of particle size determination: the mass median aerodynamic diameter (MMAD) and the geometric standard deviation of the particles in the exposure chambers were measured every month (n = 24) with a 10-stage Berner impactor (LPI 0.01525; range 15 nm to 16 µm).

*Reference:
- Heinrich, U., Muhle, H., Koch, W., and Mohr, U. 1985. Long-term inhalation studies with rodents. In Safety evaluation and regulation of chemicals 2, ed. F. Homburger, pp. 239 - 250. Basel: Karger.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The particle concentration in the exposure chamber was determined continuously, using aerosol photometers (Koch et al., 1986)*. For calibration of the photometer, the aerosol concentration in each exposure chamber was determined gravimetrically at weekly intervals (Heinrich et al., 1986)*.
The following mean particle mass exposure concentrations were measured:
7.2 mg titanium dioxide/m³ for the first 4 months, followed by 14.8 mg titanium dioxide/m³ for 4 months and 9.4 mg titanium dioxide/m³ for 16 months

To compare the different exposure concentrations, the cumulative particle exposure (g/m³ x h) was calculated by multiplying the mean particle mass exposure concentration by the actual exposure time per day and subsequently summarizing for the whole exposure period: 88.1 g/m³/h

*References:
- Koch, W. Lödding, H., Oenning, G., and Muhle, H. 1986. The generation and measurement of dry aerosols in large scale inhalation experiments., J. Aerosol Sci. 19: 1453 - 1457.
- Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, Z., Pott, F., and Stöber, W. 1986. Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions. J. Appl. Toxicol. 6: 383 - 395.
Duration of treatment / exposure:
24 months
Frequency of treatment:
18 hours/day, 5 days/week
Dose / conc.:
10 mg/m³ air (analytical)
Remarks:
Standard deviation: 2.9
No. of animals per sex per dose:
Treatment group (total: 288 female rats):
- carcinogenicity: 100 rats
- histology (serial sacrifice): 80 rats
- DNA adducts (24 months): 14 rats (from Gallagher et al., 1994)*
- Particle mass/lung (serial sacrifice): 66 rats
- Alveolar lung clearance: 28 rats

Control group (total: 408 female rats):
- carcinogenicity: 220 rats
- histology (serial sacrifice): 80 rats
- DNA adducts (24 months): 14 rats (from Gallagher et al., 1994)*
- Particle mass/lung (serial sacrifice): 66 rats
- Alveolar lung clearance: 28 rats

*Reference:
- Gallagher, J., Heinrich, U., George, M., Hendee, l., Phillips, D.H., and Lewtas, J. 1994. Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon black and titanium dioxide particles. Carcinogenesis 15(7):1291-1299.
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: following the exposure period, the rats were removed from the inhalation chambers and kept under clean air conditions for an additional 6 months.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: every fourth week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified
HAEMATOLOGY: Not specified
CLINICAL CHEMISTRY: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
A biochemical and cytological examination of lung lavage fluid was carried out for the rats. Bronchoalveolar lavagate was obtained by a twofold lavage with 4 mL saline. The lavagate was analyzed for cytological and biochemical parameters (lactate dehydrogenase, β-glucoronidase, total protein, hydroxyproline, total number of leukocytes, differential cell count).
Sacrifice and pathology:
GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsies of dead or moribund animals were done 7 days/week.
The lung wet weight and lung retention of inhaled particles were determined at different time points during the study. The lung wet weight was determined 3, 6, 12, 18, 22, and 24 months after starting the exposure. Titanium dioxide samples were determined by atomic absorption spectroscopy after ashing the lungs.

Alveolar lung clearance measurements by means of a 59Fe tracer aerosol (59Fe2O3, MMAD 0.35 µm) were also carried out. The radioactively labeled test aerosol was inhaled by the rats for about 30 minutes at 3, 12, and 18 months after the start of the experiment. The activity in the thorax was measured externally twice a week over a 100-day period. The decay-corrected activity data of days 15-100 were analyzed for each animal.

For the histopathological investigations, the organs of scheduled or moribund sacrifices were fixed in 10% neutral buffered formalin or lto­ Karnovsky fixative (Ito & Karnovsky, 1968)*. The tissues were embedded in Paramat-Wax, sectioned at 5 µm, and stained with hematoxylin and eosin (Lilly-Meyer). Histopathological investigations of the following organs were conducted for all animals: nasal and paranasal cavities (four sections; localization according to Popp & Monteiro-Riviere, 1985*), larynx, trachea, and lung (five sections; localization: the left lobe, right caudal lobe, and right middle lobe were sectioned longitudinally, and the right cranial lobe and accessory lobe were sectioned transversely to main bronchus). Graduation of the findings was done with four grades: very slight, slight, moderate, and high.

Tumors were classified according to the International Classification of Rodent Tumours (IARC. 1992)*.

*References:
- Ito, S., and Karnovsky, M. J. 1968. Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39:168a-l69a.
- Popp, J. A., and Monteiro-Riviere, N. A. 1985. Macroscopic, microscopic, and ultrastructural anatomy of the nasal cavity, rat In ILSI monographs on pathology of laboratory animals. Respiratory system, eds. T.C. Jones, U. Mohr, and R. D. Hunt, pp. 3-10. New York: Springer.
- IARC. 1992. International classification of rodent tumours. Part I: The rat. IARC Sci. Publ. no. 122. Lyon: International Agency for Research on Cancer.

Statistics:
Differences between groups were considered casewise as statistically significant for p < .05. Body weight and data of lung clearance, lung weight, lung retention of particles, and lung lavage were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance, the means of the treatment groups were compared with the means of the control group, using Dunnett's modification of the t-test. For comparison of histopathological data, Fisher's exact test was used.
Survival data of the animals of the carcinogenicity study were analyzed by the Kaplan-Meier method (Kaplan & Meier, 1958)* using the Lifetest program (SAS Institute, Inc., 1985). For animals with significantly different survival times, the tumor rates were compared within three time periods (days 400-700, 700-800, >800) using the prevalence method of Hoel and Walburg (1972)*.

*References:
- Kaplan, E. L., and Meier, P. 1958. Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53: 457-481.
- Hoel, D. G., and Walburg, H. E. 1972. Statistical analysis of survival experiments. ]. Natl. Cancer Inst. 49:361-372.

Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- after 24 months of exposure, the mortality found was 60% in the TiO2 group compared to 42% in the clean air control group.
- at the end of the 130 week experimental time (exposure time and clean air period), the mortality reached 90% in the TiO2 group and 85% in the control group.
- compared to the controls, the mean lifetime of the rats exposed to TiO2 was significantly shortened.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- body weight of the exposed animals was significantly lower from day 400 (TiO2) compared to control.
- at the end of the 2-year exposure, the body weight of the animals exposed to TiO2 (body weight: 365 g) was significantly lower compared to the control rats (body weight: 417 g).
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 specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- exposure to TiO2 led to a substantial increase in lung wet weight, processing with study duration.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- moderate to high grade bronchioloalveolar hyperplasia was observed in the TiO2 (99/100 rats) group.
- very slight to slight interstitial fibrosis in the lungs was found after 6 months of exposure.
- slight to moderate interstitial fibrosis in the lungs was observed in all animals exposed for 2 years.
- particle-laden macrophages and particles in the alveolar region were also observed in the lungs of all exposed rats.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- lung tumors were found in serial sacrificed animals after 18 months of exposure to TiO2 (5/20 rats; p≤0.05). The following tumor were observed:
benign keratinizing cystic squamous-cell tumor: 2/20 rats
squamous-cell carcinoma: 3/20 rats (sometimes together with adenocarcinoma and benign keratinizing cystic squamous-cell tumor)
adenocarcinoma: 2/20 rats
- lung tumors were found in serial sacrificed animals after 24 months of exposure to TiO2 (4/9 rats; p≤0.05). The following tumor were observed:
benign keratinizing cystic squamous-cell tumor: 2/9 rats
squamous-cell carcinoma: 2/99 rats (sometimes together with benign squamous-cell tumor)
adenocarcinoma: 1/9 rats

- after an exposure time of 24 months followed by 6 months of clean air, lung tumor rates of 32% were observed in rats exposed to TiO2. 8 animals showed 2 tumors in their lungs.
- The following tumour types were observed after an experimental time of 30 months (24 months TiO2-exposure plus 6 months clean air):
benign keratinizing cystic squamous-cell tumor: 20/100 rats
squamous-cell carcinoma: 3/100 rats
adenoma: 4/100 rats
adenocarcinoma: 13/100 rats
Number of rats with tumors: 32/100 (19/100 rats: count without benign keratinizing cystic squamous-cell tumors)
1/217 control animal (clean air exposure) showed adenocarcinoma

- lung tumor rate increased with increasing particle exposure concentration.
- lung tumor incidences of the TiO2 exposed group was significantly increased compared to the control group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE
- the differential cell count and the concentration of lactate-dehydrogenase, β-glucuronidase, OH-proline, and total protein in bronchoalveolar lavage (BAL) showed clear exposure-related effects in the exposure group even after 24 months of exposure.
Details on results:
MORTALITY:
- the various exposure groups did not differ significantly in their mean lifetime among themselves.

HISTOPATHOLOGY: NEOPLASTIC
- no lung tumors were observed in the TiO2 satellite group of 20 animals each after 6 and 12 months of exposure.

PARTICLE LUNG BURDEN
- during the second year of exposure, the particle lung load of the TiO2 exposed animals increased by only 13%.
- the retained particle mass in the lung-associated lymph nodes (LALN) of the TiO2-exposed rats after 22 months amounted to about 14%.
- expressed as milligrams particles per gram clean air control lung (wet weight of control lung 1.2 g), the particle lung loads after 1 year of exposure to TiO2 were 29 mg.

ALVEOLAR LUNG CLEARANCE
- the alveolar clearance rate was already significantly compromised after inhalation of TiO2 after 3 months of exposure.
- after 18 months of exposure to TiO2 and 3 months of recovery time without particle exposure, no reversibility of the alveolar lung clearance damage could be detected.

Remarks on result:
other: see remarks
Remarks:
Due to the anomalous study design (frequency, no dose response paradigm, out-dated criteria for tumour classification) an effect level cannot be derived.
Critical effects observed:
not specified
Conclusions:
This study by Heinrich et al. (single exposure concentration: 7.2 mg/m³ 1-4 months, 14.8 mg/m³ 5-8 months, 9.4 mg/m³ 9-24 months, 18h/d, 5d/w) was noted as a reliability 3 study because it was a satellite group used for another study. The study included only female rats and did not have a dose response paradigm. In addition, the rats and mice were exposed for 18 hours/day, 5 days per week for 24 months. The evaluation of tumours (including malignant tumours) was assessed and did not consider the two international lung pathology workshops – which have reassessed the criteria for describing malignant vs. benign tumours – particularly with reference to the cystic keratinizing pulmonary squamous cell lesions that are unique to the rat.
Consequently, the diagnosis should have changed after reconsideration of the revised criteria.
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-06-04 to 1981-07-09
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 100 male and 100 female Crl:CD(SD)BR rats each were exposed to titanium dioxide (10, 50, and 250 mg/m³). The test item was administrated via whole body inhalation for 6 hours/day, 5 days/week for 24 months. A concurrent control group was run concurrently. The following parameters were assessed:clinical signs, mortality, body weights, haematology, clinical chemistry, urinalysis. gross pathology, and histopathology.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: Crl:CD(SD)BR
Details on species / strain 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.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age: 3 weeks
- Housing: housed pairwise in stainless steel wire-mesh cages.
- Diet (ad libitum): Purina Laboratory Chow Checkers '5001
- Water: ad libitum
- Acclimation period: approx. 17 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2 °C
- Relative humidity: 50 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12
- rooms had laminar flows of filtered and recirculated air
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.54 - <= 1.93 µm
Remarks on MMAD:
GSD: 2.52 - 3.14
Mean respirable fraction of TiO2: 93.7% or greater (values for each determination ranged from 84.0 to 99.95 %)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers made of material not reactive with TiO2
- Chamber volume: 3.85 m³
- 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 and humidity: 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

TEST ATMOSPHERE
- Particle size distribution: 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.

TEST ATMOSPHERE
- Brief description of analytical method used: 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see above ("Details on inhalation exposure")
Duration of treatment / exposure:
24 months
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
10.55 mg/m³ air (analytical)
Remarks:
SD: 2.12 mg/m³
Dose / conc.:
50.68 mg/m³ air (analytical)
Remarks:
SD: 6.65 mg/m³
Dose / conc.:
250.1 mg/m³ air (analytical)
Remarks:
SD: 24.70 mg/m³
No. of animals per sex per dose:
100 male rats / 100 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: results from previous inhalation studies.
- Rationale for animal assignment: rats of each sex were divided by computerised, stratified randomisation into groups of 100 males and groups of 100 females such that the mean of body weights of each group of rats within a sex were approximately equal.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily throughout the study
- Cage side observations checked: morbundity/mortality, abnormal behaviour and appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once weekly during the first 3 months and at least every other week during the remainder of the study

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly during the first 3 months of the study followed by approx. once every other week for the remainder of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE : No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: approx. 3, 6, 12, 15 and 18 months after the study's initiation
- How many animals: 10 males / 10 females (same rats were evaluated at each interval throughout the study)
- Parameters checked: basophil count, eosinophil count, erythrocyte count, haematocrit (Ht), haemoglobin, leukocyte count, lymphocyte count, mean cell haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, monocyte count and neutrophil count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: approx. 3, 6, 12, 15 and 18 months after the study's initiation
- How many animals: 10 males / 10 females (same rats were evaluated at each interval throughout the study)
- Parameters checked: alanine aminotransferase activity, alkaline phosphatase activity, bilirubin, calcium, phosphorus, total protein and urea nitrogen.

URINALYSIS: Yes
- Time schedule for collection of urine: approx. 3, 6, 12, 15 and 18 months after the study's initiation.
- Parameters checked: volume, osmolality, pH, bilirubin, blood, protein, sugar, urobilinogen, appearance and sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Gross and histopathological examinations were conducted on 5 rats/sex/treatment group after 3 and 6 months exposure as well as 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 of tissue permitting), were examined grossly and histopathologically.

All rats were sacrificed and the respiratory tract was prepared for fixation.
All other tissues were removed for gross examination and weighed (lungs, trachea, heart, liver, stomach (3 and 12 months sacrifice only), kidneys, spleen, testes, pituitary, brain, thymus and adrenals) at each sacrifice. The tissues were fixed in either Bouin's (nasal cavitiy (turbinates), trachea, luings, oesophagus, kidneys, sternal bone marrow, testes, epididymides, mammary gland,pituitary, thyroid - parathyroids, adrenals, eyes, bone (sternum, femur, and vertebrate, ear (zymbal gland), and skin (neck)) or formalin at the 6- through 24-month sacrifices. At the 3-month sacrifice only, adipose tissue and ovaries were fixed in Bouin's fixative and the adrenals, oesophagus, mammary and Zymbal's glands were fixed in formalin.

Representative specimens of the following organs and tissues were taken from all rats: heart, thoracic aorta, nasal cavity (turbinates), trachea, lungs, liver, pancreas, small intestine (duodenum, jejunum, and ileum), tongue, oesophagus, stomach, salivary glands, large intestine (cecum and colon), rectum, kidneys, bladder, sternal bone marrow, spleen, lymph nodes (cervical, mesenteric and tracheobronchial), thymus, testes, epididymes, prostate, seminal vesicles, uterus, ovaries, mammary gland, pituitary, adrenals, thyroid - parathyroids, brain, spinal cord, sciatic nerve, skeletal muscle, bone (sternum, femur, and vertebrate), eyes, Harderian's gland, exorbital lacrimal glands, ear (Zymbal's gland), skin (neck), adipose tissue, and all gross lesions
Other examinations:
not specified
Statistics:
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 pairwise 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.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
There were several changes in haematological parameters related to exposure of rats to TiO2. When compared to the controls over course of this study:
- haematocrits (both sexes) and heamoglobins (males only) for rats in the 250 mg/m³ treatment groups were greater (up to 12 and 11%, respectively).
- mean cell volumes and mean cell haemoglobins in male rats from all exposed groups were greater (up to 14%); there were only minimal differences among these groups.
- neutrophil counts in all treatment groups were greater (up to 80% in male and 117% in female rats in the 250 mg/m³ treatment groups). A dose-related trend was observed among these treatment groups.
- lymphocyte counts in all treatment groups were lower (up to 45% lower for rats in the 250 mg/m³ treatment groups). A dose-related trend was observed among these treatment groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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:
- bilirubin content in female rats were greater and were more pronounced in the 50 and 250 mg/m³ 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.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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:
Lungs:
- clear dose- and time-dependent lung weight increases were observed
- mean absolute and relative lung weights for rats in the 250 mg/m³ 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/m³ 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.
markedly heavier at 50 mg/m³, and were more than two times control lung weights at 250mg/m³.

Thymus:
- A dose-related thymus weight effect was observed in rats in the 50 and 250 mg/m³ treatment groups. Mean absolute and relative thymus weights were as high as 1.37-fold greater than controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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/m³ treatment groups as slightly elevated gray nodules. The lungs of rats in the 250 mg/m³ 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.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The respiratory tract was the primary deposition site for TiO2 as well as the primary site in which a tissue response to this compound was observed. Over the course of this study, the following dose-related effects were observed:
- increased incidence of rhinitis with accompanying squamous metaplasia in the anterior nasal cavity;
- greater numbers of TiO2 –laden alveolar macrophages (dust cells), and of aggregated, foamy alveolar macrophages;
- deposits of TiO2 particles within the lymph nodes where the relative severity for tracheobronchial > peribronchial and perivascular > mesenteric lymph nodes.
- alveolar proteinosis in approximately 51 and 97% of the rats in the 50 and 250 mg/m³ treatment groups, respectively, but it was not found in all other groups;
- markedly increased incidences of cholesterol granulomas that contained large numbers of collagen fibers in the lungs of rats in the 50 and 250 mg/m³ treatment groups (approximately 75 and 97% of these rats, respectively; 13% or less for rats in all other groups);
- markedly increased incidences of collagenized fibrosis within alveoli of rats in the 50 and 250 mg/m³ treatment groups (in approximately 60 and 99% of these rats, respectively; 14% less for rats in all other groups);
- greater incidences of focal pleurisy associated with subpleural cholesterol granulomas and focal dust cell infiltration in the lungs of TiO2-exposed rats;
- bronchiolarization of alveoli adjacent to terminal bronchioles, which occurred more frequently in female than male rats
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(for a detailed analysis please see study record under the endpoint: carcinogenicity)
Other effects:
not examined
Details on results:
CLINICAL SIGNS
Exposure to TiO2 resulted in no abnormal clinical sign in any exposed group.
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.
- 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.

MORTALITY
- exposure to TiO2 resulted in no excess mortality in any exposed group.
- 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.

BODY WEIGHT AND WEIGHT CHANGES:
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/m³ was consistently lower than those for rats exposed to 10 or 50 mg/m³. The differences in the mean body weight gain among all treatment groups parallelled the mean body weight effects.

HAEMATOLOGY:
- erythrocyte counts for rats in the 250 mg/m³ treatment groups were slightly greater (up to 9%; not statistically significant).
- leukocyte counts in all treatment groups were greater (up to 80% for the high-dose group). The values for rats in the 250 mg/m³ treatment group were generally higher than those of the other treatment groups.

URINALYSIS
Urological parameters were within the expected range of biological variability for this species.

ORGAN WEIGHTS
Lungs:
- lung weights at 10mg/m³ were comparable to those of control group

Liver:
- 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.

Kidney:
- mean absolute and/or relative kidney weights for TiO2 exposed male rats were approx. 77 to 88% of the controls at the 3-, 12- and 24-months sacrifices. A clear dose-response was not demonstrated.

Significant but transient effects were observed for other organ weights over the course of this study. These effects were observed for other organ weights over the course of this study. These effects were either related to body weight differences or were not considered to be biologically significant.

HISTOPATHOLOGY-NEOPLASTIC
Exposures to titanium dioxide at concentrations of 10, 50, or 250 mg/m³ produced lung tumors (bronchioalveolar adenoma and squamous cell carcinoma) only at the highest concentration.

To further characterise the bronchoalveolar lesions, in 1992, a group of pathologists from North America and Europe examined lung lesions produced by para-aramid RFP (respirable fibre-shaped particles) and titanium dioxide. This panel diagnosed the lesion as a "proliferative keratin cyst" (PKC). Additionally, the pathologists agreed that the lesion was not a malignant neoplasm and is most likely not neoplastic. A minority opinion was that the lesion is probably a benign tumour (Carlton 1994; Levy 1994). Another subsequent international pathology workshop was convened to develop standardized histological criteria for classifying pulmonary keratin lesions (Boorman et al., 1996). As a consequence, most of the lesions that had originally been diagnosed as “cystic keratinizing squamous cell carcinomas” were re-classified by the consensus panels as non-tumorous “proliferative keratin cysts” (Warheit and Frame, 2006).
In the aftermath of two international pathology workshops designed, in part, to establish histological criteria for classifying pulmonary keratin lesions, these lesions were evaluated by four pathologists using current diagnostic criteria. Microscopic review of 16 proliferative squamous lesions, previously diagnosed as cystic keratinizing squamous cell carcinoma in the lungs of rats from the 2-year inhalation study was performed. Unanimous agreement was reached as to the diagnosis of each of the lesions. Two of the lesions were diagnosed as squamous metaplasia and 1 as poorly-keratinizing squamous cell carcinoma. Most of the remaining 13 lesions were diagnosed as non-neoplastic pulmonary keratin cysts (Warheit and Frame, 2006).
Consequently, the diagnosis of many of these lesions has changed after the development of revised criteria.

There were several effects associated with exposure to TiO2 which were not clearly dose- related. These include:
- hyperplasia of the Type II pneumocytes lining alveolar walls;
- increased incidences of chronic tracheitis; and
- Increased incidences of broncho/bronchiolar pneumonia.
Over the course of the study, most of the inhaled TiO2 particles that were observed within the alveoli were phagocytized by dust cells. These particles were retained primarily within the alveolar ducts and adjoining alveoli of rats in the 10 and 50 mg/m³ treatment groups which also had some dust-free alveoli in the peripheral acini. However, for rats in the 250 mg/m³ treatment group, the number of dust cells was markedly greater than that for rats in other treatment groups. The dust cells were distributed throughout the alveoli with larger concentrations in the alveolar duct region. This region served as a focal point for several of the tissue responses previously listed, e.g. collagenized fibrosis, bronchiolarization, squamous cell metaplasia and tumor formation. Within each treatment group, the lesions gradually increased in frequency and/or severity with time on test. By the end of this study, rats in the 50 and 250 mg/m³ treatment groups had irreversible effects whereas all of the lesions observed in rats exposed to 10 mg/m³ were considered to be reversible.
Although most of the biological effects associated with TiO2 exposure occurred within the respiratory tract, other tissues were also affected. The incidences of degenerative retinopathy in exposed rats was increased significantly compared to those of controls and were greater among female than male rats; however, a dose-response relationship was not observed. Female rats exposed to TiO2 also had significantly greater incidences of endometritis than the controls (dose-related).
Exposure of rats to TiO2 resulted in a dose-related transmigration of dust particles from the lung through the lymhatics to the lymph nodes, liver, and spleen. The accumulation of TiO2 in the tracheobronchial, cervical, and mesenteric lymph nodes did not result in a significant tissue response. In the spleen, dust particles aggregates were primarily located in the lymphnode tissue of the white pulp. In the liver, TiO2 accumulated in the portal riads and Kupfter cells. There were no cellular responses to these deposits in either the spleen or liver.

A wide variety of spontaneous neoplastic and non-neoplastic lesions that were considered to be age related occurred with similar incidence and severity among control and all TiO2-exposed groups

REFERENCES
- Carlton, W.W. (1994) "Proliferative Keratin Cyst," a Lesion in the Lungs of Rats Following Chronic Exposure to Para-aramid Fibrils. Fundam. Appl. Toxicol. 23, 304-307
- Levy L.S. (1994). Squamous Lung Lesions Associated with Chronic Exposure by Inhalation of Rats to p-Aramid Fibrils (Fine Fiber Dust) and to Titanium Dioxide: Findings of a Pathology Workshop. In: Mohr, U. (Ed.): Toxic and carcinogenic effects of solid particles in the respiratory tract, ILSI Press, 473-478
Boorman, G.A: et al. (1996). Classification of Cystic Keratinizing Squamous Lesions of the Rat Lung: report of a Workshop. Toxicol. Pathol. 24, 564-573
Dose descriptor:
NOAEC
Effect level:
10.6 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Effect level:
50.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Dose-related increases in pleurisy, slight collagenized fibrosis associated with cholesterol granulomas, alveoli bronchiolarization, pneumonia, and alveolar cell hyperplasia were observed. The changes observed at the 10 mg/m³ concentration were minimal in severity in comparison to similar effects observed in the controls (except that the alveolar cell hyperplasia was not observed in controls). The degree of pulmonary fibrosis seen at the two higher levels was slight. In subsequent experimentation (Warheit et al., 1997; summarized below) using these same dosing rates for four weeks, it was demonstrated that high dose animals had substantially increased lung clearance times and sustained inflammation under particle overload conditions.

All concentration used in the Lee et al. study clearly exceeded the MTD, since lung overload conditions were attained even at the lowest concentration of 10 mg/m³

A dosimetric analysis of the 2-year rat inhalation study by Lee et al. shows that all three TiO2 exposure concentrations resulted in significant lung
particle overload, i.e., an impaired alveolar macrophage-mediated particle clearance function. Per g lung weight, the retained normalized lung burden observed in the study of 28 mg/g exposed lung and 39 mg/g control lung for the 50 mg/m3-exposed rats (shown at the beginning of this analysis, p.7) is obviously greatly exceeding a retained lung burden of 1 mg/g lung which - according to Morrow (1988) - signals the beginning of lung overload in rats.
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
only one concentration tested
Principles of method if other than guideline:
It is highlighted that the study was conducted in compliance with OECD 453. However, titanium dioxide was not selected as primary test item (which was carbon black), but as negative control substance. Consequently, only one high titanium dioxide concentration was used and no dose-response relationship can be derived for non-neoplastic lesions as well as no NOAEC be identified.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
not applicable
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
The study was done using Fischer-344 rats due to their wide use by the National Toxicology Program and the extensive data base available on their health status and background tumor incidence.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH
- Age: 4 weeks old
- Housing: individually housed in metal wire mesh cages (18.7 x 21 x 15 cm) in horizontal air flow chambers throughout the study.
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: whole-body exposure chambers were used
- System of generating particulates/aerosols: a dry aerosol dispersion technique was used. The aerosol generator consisted of a commercially available feeding system connected to a two-stage pressurised air ejector.
To maximize mixing and uniform distribution of the aerosol, the chamber inlets were equipped with diffusers and perforated plates.
- Temperature, humidity, and air flow: chambers were maintained at 23.5 ± 1 °C and 40 - 60 % relative humidity with an air flow of 3.8 m³/min.

- Method of particle size determination: particle size distribution in each chamber was measured 13 times during the study using Berner impactor.

TEST ATMOSPHERE
- Brief description of analytical method used: at the inlet side of the chamber photometric determination of the aerosol concentration was done and gravimetric samples were collected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on analytical verification of doses or concentrations" above.
Duration of treatment / exposure:
24 months
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
5 mg/m³ air (analytical)
Remarks:
SD: 0.7 mg/m³ air
No. of animals per sex per dose:
144 males / 144 females (total: 288 animals)
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Post-exposure period: 1.5 months
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on arrival, 2 and 4 weeks subsequently, and thereafter at 6-month intervals

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks during the first 14 weeks and then once every 4 weeks

FOOD CONSUMPTION:
- Time schedule for examinations: weekly during the first 13 weeks and then once every 3 months.

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 data

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for examinations: 6, 12, 18 and 25.5 months
- Parameters checked: leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for examinations: 6, 12, 18 and 25.5 months

URINALYSIS: Yes
- Time schedule for examinations: 6, 12, 18 and 25.5 months

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified
Sacrifice and pathology:
Rats sacrificed during and after scheduled exposure were anesthetized and killed. The abdominal cavity was opened and the diaphragm was cut allowing the lungs to collapse. Organs (brain, liver, kidneys, adrenals, and gonads) were weighed. Tissues and organs were fixed and processed for routine histology. Bones were decalcified. The larynx, trachea, oesophagus, thymus, heart, and lungs were removed. The larynx and upper part of the trachea were separated and placed in formalin. The lungs scheduled for histopathology were fixed. Complete histopathological examination of organs, tissues, and gross lesions of animals of the serial sacrifices and of all animals of the “basic study” was performed (please refer to the table 1 in the field “Any other information on materials incl. tables” below).

Bronchoalveolar lavage: the method of Henderson et al. (1987)* was used with minor modifications. Following preparation of the lungs, they were lavaged with saline without massage. The cell concentration was determined using a counting chamber. The lavagate was centrifuged and the supematant used for the determination of the biochemical parameters (lactic dehydrogenase (LDH), β-glucuronidase, total protein). Cytoslides were prepared for differential cell count.

*Reference:
- Henderson, R.F., Mauderly, J. L., Pickrell, J. A., Hahn, R. F., Muhle, H., and Rebar, A. H. (1987). Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329-342.
Statistics:
Parametric data were examined by analysis of variance (ANOVA) followed by Dunnett test to compare various treatment groups with controls. Survival data were analysed by the Kaplan-Meier method using the lifetest program of SAS. For necropsy and tumour occurrance data, simple tests for homogeneity of contingency tables (using qui square statistics or Fisher's exact method) were used.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS
Inhalation of the test material did not cause overt signs of toxicity.
Clinical appearance of the rats was found to be within normal limits. Measured virologic, bacteriologic, and parasitologic parameters were also within normal limits or negative during the study.

MORTALITY:
The mean survival in the basic study (100 animals, 50 of each gender) was 40% at the termination of the experiment. The data analyzed by Kaplan-Meier method, separately for each gender, indicated no difference among various exposure groups.

BODY WEIGHT AND WEIGHT GAIN
Body weight development appeared to be normal when compared to concurrent and historical control.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was normal in the TiO2-exposed groups.

HAEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS
Clinical laboratory test results were essentially within normal range. The results were consistent with healthy animals.

ORGAN WEIGHTS
No changes in organ weights were observed at the terminal sacrifice.

HISTOPATHOLOGY: NON-NEOPLASTIC
- TiO2 group: extent of particle-laden macrophages increased with exposure time.
- a small but statistically insignificant incidence of fibrosis was seen in the TiO2 and air-only control groups as shown below:
Control (mild degree of fibrosis): 1.2 (date of sacrifice: 21 - 25.5 months)
TiO2 (moderate degree of fibrosis): 35.7 (date of sacrifice: 21 months); 19.1* (date of sacrifice: 21 - 25.5 months)
TiO2 (minimal degree of fibrosis): 16.7 (date of sacrifice: 15 months); 4.5 (date of sacrifice: 21 - 25.5 months)
TiO2 (mild degree of fibrosis): 1.1 (date of sacrifice: 21- 25.5 monthsmonths)
(*p <0.001; approx. 14 animals/group were examined at each serial sacrifice point and about 90 animals/ group were evaluated at 21 - 26 months of the study)
- no significant difference from air-only control in the extent of various upper respiratory system lesions was noted regarding the TiO2 group.

HISTOPATHOLOGY: NEOPLASTIC
- two adenomas and one adenocarcinoma (3/100 animals) were observed in the air-only control group while one tumour of each type was detected in the TiO2 group (2/100 animals).

PARTICLE RETENTION
TiO2 accumulated progressively in the lungs of the rats. The mean quantity of TiO2 retained was 2.72 mg/lung.

BRONCHOALVEOLAR LAVAGE (BAL) EXAMINATION
The number of lavagable leukocytes at 15 months of exposure was unaffected by treatment. In the lavage fluid a substantial amount of fragments of macrophages were observed. The results from the TiO2 exposure showed a significant decrease in macrophages at 15 months. This cytologic pattern persisted throughout the rest of the study as the results at 21, 24, and 25.5 months were quite similar. A linear relationship was observed between the fraction of polymorphonuclear leukocytes (PMN) in the lavagate and the retention half-time of the polystyrene tracer (Bellmann et al., 1991)* at TiO2 concentration.
The levels of cytoplasmic and lysosomal enzymes and total protein in lavage fluid were comparable to those of air-only controls in the TiO2 groups.
Please also refer to table 1 in the field "Any other information on resutls incl. tables" below.

*Reference:
- Bellmann, B., Muhle, H., Creutzenberg, O., Dasenbrock, C., Kilpper, R., Mackenzie, J., Morrow, P., and Mermelstein, R. (1991). Lung clearance and retention of toner, utilizing a tracer technique during chronic inhalation exposure in rats. Fundam. Appl. Toxicol. 17,300-3 13.
Remarks on result:
not determinable
Remarks:
since titanium dioxide was not selected as primary test item but as negative control substance, only one high titanium dioxide concentration was used. Consequently, no dose-response relationship can be derived for non-neoplastic lesions and no NOAEC be identified.
Critical effects observed:
not specified

Table 1: a) Cytology results in bronchoalveolar lavage fluid and b) mean levels of controls and levels normalized to control of LDH,β-glucuronidase, and protein in lavagate

 

Exposure (months)

15

21

24

24 + 6 weeks clean air

Number of lavaged leukocytes (10³ cells/mL)

Control

100

133

221

194

TiO2

102

156

211

265

Macrophages (%)

Control

97.2

97.0

98.0

97.4

TiO2

90.8*

93.2

95.7

92.8

PMN (%)

Control

1.1

1.9

0.5

0.8

TiO2

4.9

4.2

2.3

4.3

Lymphocytes

Control

1.7

1.2

1.6

1.9

TiO2

4.4*

2.7

2.1

3.0

LDH

Control (U/liter

25

35

39

33

Control

1.00

1.00

1.00

1.00

TiO2

1.16

0.71

1.26

1.15

β-glucuronidase

Control (U/liter

0.15

0.20

0.18

0.15

Control

1.00

1.00

1.00

1.00

TiO2

1.00

0.70

1.89

1.33

Protein

Control (U/liter

108

114

146

144

Control

1.00

1.00

1.00

1.00

TiO2

1.04

0.99

1.86

1.04

Conclusions:
Inhalation of titanium dioxide showed no signs of overt toxicity. Body weight, clinical chemistry values, food consumption, and organ weights were normal. Fibrosis was present in the controls at a comparable rate to that of titanium dioxide exposed rats, being minimal to mild and not statistically significantly different from controls. There were no significant increases in lung tumours vs. control rats exposed for up to 24 months by whole body inhalation to titanium dioxide in this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

 

Human toxicity data (via inhalation)

Human epidemiological data are discussed under the respective endpoint (Chapter 5 of the CSR, Section 7.10 of the IUCLID).

 

Animal toxicity data

Repeated dose toxicity, oral

None of the chronic oral studies in rodents represent a guideline compliant repeated dose toxicity study which can be used for hazard and risk assessment purposes. The endpoint oral repeated dose toxicity is therefore addressed in a weight of evidence approach, taking into account all available information.

Two chronic oral toxicity studies performed with TiO2 are available in the public domain: one study conducted by the National Cancer Institute (NCI; 1979) with pigment grade TiO2, and one with TiO2-coated mica (Bernard, 1990; regarded as supporting information).

In the chronic oral toxicity study (NCI, 1979), F344 rats (m/f, 8w old, 50 animals/group) and B6C3F1 mice (m/f, 36d old, 50 animals/group) were fed a diet containing 2% corn oil and 25,000 or 50,000 ppm titanium dioxide for 103 weeks (7 days per week). 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. Accordingly, the NOEL for systemic toxicity could be established at 50,000 ppm. The study describes several non-neoplastic findings observed both in rats as well as in mice, without however attributing any adversity to these. By putting these into context with historical control data, these effects can however be considered to lack toxicological significance. The findings can be summarised briefly as follows:

Spleen haematopoiesis in rats (F344) and mice (B6C3F1):

·        observed in male rats only; incidence 8% (high) vs 0% in control and 2% in low dose;

·        no effects in females (0-4%) or in mice (6-10% M / 4-8% F);

·        historical controls (HC) for extramedullary haematopoiesis rat (18-24 month): 7.5 % (Hirouchi, 1994; Goodman, 1978; Coleman, 1977)

Conclusion: the finding in male rats is most probably not related to treatment with the test item but spontaneous in nature, because the incidence in males is generally low. It was observed in females only at a very low incidence and was not seen in mice. Information from a publication on the pathology of aging male Fischer rats indicates a percentage for haematopoiesis of 7.5%, which is very close to the rate observed in this study (8%). Overall, it is not very likely that the finding of haematopoiesis in spleen observed in male Fischer rats is related to treatment with the test item.

Kidney chronic inflammation:

·        Male rat only; incidence 90% and 86% in low dose and high dose vs 59% in control (no dose relation); no effects in female rats (38-53%);

·        Male mice only; incidence 8% (low) and 10% (high) vs. 6% in control; females not affected (0-6%)

·        HC male mice (lymphatic infiltration used as correlate for chronic inflammation): 24.5%

·        HC rats (chronic interstitial nephritis used as correlate for chronic inflammation): 62.5 % (18-24 months), 70.2 % (24-30 months)

Conclusion: the higher incidence of chronic inflammation in male rats observed in the low and high dose groups is not dose-related, although higher than in control males. In general, the incidence of this lesion is high in aged Fischer rats (18-30 months) based on published data (62.5-70.2%). However, the incidences observed in male rats may reflect a chronic infection in these animals and is most probably not related to treatment with the test item. The findings observed in male mice can therefore be considered to be within the historical control data of this strain taken from a publication.

Seminal vesicle (SV) and testis atrophy:

·        SV rat: incidence 12% (low) and 20% (high) vs 0% in controls

·        Testis rat: incidence 10% (low) and 14% (high) vs 6% in control

·        HC seminal vesicles and testis: 1.7% and 12.4%

·        HC seminal vesicles and testis: 2.3% (24-30%) and 80-100% (18-30 months)

Conclusion: the incidence observed in seminal vesicles of male rats is indeed higher than described in two publications on historical control data observed in studies (1.7%) conducted by the National Cancer Institute or described in a publication about pathology in aging Fischer rats (2.3%). However, the toxicological relevance of this observation is not clear. In addition, the findings in testes are within the HC in the low dose group and only slightly above the HC data (12.4%) in the high dose group. In general, the findings observed in seminal vesicle and testis of Fischer rats should not be considered as related to treatment with the test item.

Liver necrosis:

·        Male mice only; incidence 16% (high) vs. 0% in control and low dose; females (0-2%) and rats (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); female rats (0-6%)

·        HC 92.1%

Conclusion: the incidence observed in the control, low and high dose groups is within the range of historical control data.

 

In another chronic feeding toxicity study in rats (Bernard, 1990, reported under the endpoint carcinogenicity), TiO2-coated mica (28% TiO2, 72% mica; flat platelets 10-35 µm) was administered for 103 weeks (vehicle: Purina rat chow supplemented with 1% corn oil) at dietary levels of 0, 1.0, 2.0 and 5.0%. There were no effects at any dose on body weight and survival, no changes in haematological or clinical chemistry parameters, no treatment-related histopathological observations. The only observations commented on by the study authors were (i) reduced mean absolute and relative thyroid weights for high dose males and mid-dose females which were obviously without dose-relation, and (ii) an increased incidence of adrenal medullary hyperplasia in high dose males which however did not progress to either benign or malignant pheochromocytoma, such effect being absent in females. A high incidence of mononuclear cell leukaemia was found in all groups of both sexes is considered an age-related effect of doubtful relevance. The NOEL from this study can be established above the highest dietary exposure level of 5% (corresponding to 1.4% TiO2).

 

In a 90-d repeated dose oral toxicity study (Carpenter, 2011) [OECD TG 408], four groups of Crl:CD(SD) rats (10/sex/group) were dosed with rutile-type titanium dioxide particles by gavage at dosages of 0, 100, 300 or 1,000 mg/kg/day for approximately 90 days (92 days for males and 93 days for females). Body weights, food consumption, and detailed clinical observations were evaluated on a weekly basis. Ophthalmological and neurobehavioral assessments were performed prior to the start of dosing and near the end of the exposure period. Clinical pathology endpoints (haematology, coagulation, clinical chemistry, and urinalysis) were evaluated at the end of the exposure period. After approximately 90 days of dosing, the rats were sacrificed and given a gross and microscopic pathological examination. No test substance-related deaths occurred and no clinical, ophthalmological or neurobehavioral observations were attributed to exposure to the test substance. No test substance-related effects on body weight or nutritional parameters were observed. There were no adverse or test substance-related effects on clinical pathology parameters, organ weights, gross pathology, or microscopic pathology in male or female rats. The NOAEL for male and female rats was above 1,000 mg/kg/day, the highest dosage tested. The NOAEL is based on a lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameter in rats dosed up to 1,000 mg/kg/day.

 

In another 28-d repeated dose oral toxicity limit dose study (Mayer et al., 2006) according to OECD 407 in Crl:CD(SD)IGS BR rats, titanium dioxide was administrated via gavage to 5 males/dose at dose levels of 0 and 24,000 mg/kg bw/day. The titanium dioxide was suspended in NANO pure water. Two types of titanium dioxide were used (H-27201 and H-27203, no data on test substance are available). Mortality and clinical signs were observed daily in all animals. Individual body weights of both sexes were measured once a week during the dosing period. Food consumption was recorded. Ophthalmoscopic examination, haematology, clinical chemistry, organ weights, gross pathology and histopathology were examined. No death was observed. There were no treatment related signs noted on daily or weekly clinical observations during the 28-day oral gavage study. Some non-specific clinical observations were noted in some animals but were not test substance related. These observations included a neck wound, misshapen ears and hair loss. These types of clinical signs are commonly reported among rats of this sex and age group. One animal, number 305 (H-27201), was found dead at the end of the first week. During the second week, animal number 505 (H-27203) was observed to have breathing noises and was sacrificed. The necropsy report indicated both deaths were attributed to trauma from gavage. There were no statistically significant differences in body weight change, food consumption, haematology, and clinical chemistry. There were no test substance-related effects on organ weights. There were no test substance-related gross observations in the treatment or the control groups. All gross observations and microscopic findings at the terminal necropsy were consistent with normal background lesions in rats of this age and strain. Under the conditions of this study, the NOEL for titanium dioxide was 24,000 mg/kg bw/day for male rats, based on the lack of any adverse effects at this dose.

 

The study reported by Blevins et al. (2019) has been conducted to verify the findings of Bettini et al. (see discussion below). Male rats received food containing E171 (supplied by the Titanium Dioxide Manufacturers Association) for 7 and 100 days at doses of 0, 40, 400 and 5000 ppm. Animals exposed for 100 days were pre-treated with a single i.p. dose of 180 mg/kg bw dimethylhydrazine dihydrochloride (DMH*2HCl) or vehicle only, 7 days before study start. Animals were checked daily for moribundity and mortality, detailed clinical observations were performed during study weeks 8 and 13. Body weights and food consumption were determined weekly. Water consumption was determined three times during the study.

At the end of the 7-day study, whole blood was collected and leukocytes were either phenotyped by FACS or PBMCs were isolated and activated to induce cytokine production ex vivo. Plasma was collected for cytokine quantification. Inflammatory cytokines within the gastrointestinal tract were determined by collecting and homogenizing specified segments of the gastrointestinal tract. Peyer´s Patches were removed along the jejunum and ileum and leukocytes were isolated for phenotyping and activated to induce cytokine production ex vivo. Rat spleens were excised, weighed and cut. One half of the spleen was made into single cell suspension and used for phenotyping via FACS or for cytokine quantification after ex vivo activation.

At the end of the 100-day study, all blood and tissue samples for determination of immunological parameters by FACS were collected, processed and analysed as described in the 7-day study (see above). For evaluation of aberrant crypt foci (ACF), three specified sections from the colon were removed. For evaluation of histopathology and goblet cells, a transverse segment from the colon was removed. The spleen, liver and one testis were removed and weighed. Sections from the liver, lung, spleen, duodenum, jejunum and ileum were examined histopathologically.

Although this study is not in accordance with the respective guideline for sub-chronic toxicity studies (OECD 408), the findings of this study clearly demonstrate that food-grade titanium dioxide did not induce systemic toxicity or any kind of inflammatory response after dietary administration for 7 or 100 days.

Animals exposed to 5000 ppm E171-E (equivalent to approx. 373 mg/kg bw/day (7-days study) or approx. 236-300 mg/kg bw/day (100-days study)) or lower concentrations did not show any sign of systemic toxicity or histopathological changes in gastrointestinal tract, spleen, liver, lung and testes. Body weight, food and water consumption were comparable to those of control animals. No changes in the percentage of CD103+ DC or activated CD4+ T helper cells in peripheral blood, spleen, or Peyer´s patches were observed. Further, E171 did not reduce the percentage of anti-inflammatory T reg cells in the periphery and Peyer´s patches. Cytokine levels in plasma, small intestine and colon were not altered after 7 or 100 days of E171 exposure. Ex vivo analysis of isolated and activated lymphocytes from peripheral blood, spleen, and Peyer´s Patches of rats treated with E171 did not result in induced cytokine production compared lymphocytes obtained from control animals. E171 treatment administered after DMH did not result in increases in ACF or ABC. No change in the number of ACF and ABC were observed due to E171 exposure alone in the absence of DMH. And lastly, dietary exposure to E171, with or without pre-treatment with DMH, for 100 days had no effect on the length of the colonic glands examined or the number of goblet cells/unit. 

The intention of this study was not to be guideline compliant but to recapitulate the findings of recent publications (i.e. Bettini et al. 2017, Urrutia-Ortega et al. 2016) reporting that food-grade titanium dioxide induces immunologic and carcinogenic effects. Therefore, the study design was similar to that of Bettini et al., treating animals for 7 and 100 days with food-grade titanium dioxide with and without pre-treatment of the intestinal genotoxic carcinogen DMH. In contrast to Bettini et al., who reported that food-grade titanium dioxide induces aberrant crypt foci and inflammatory responses, no effects at all were observed in this study. Even at high doses, no effect on the immune parameters or tissue morphology such as ACF or ABC formation could be seen. Due to absence of toxic effects after 100 days of dietary E171 exposure the NOAEL for male rats is considered to be 5000 ppm (236-300 mg/kg bw/day).

 

Hansen 2020 (reported in section 7.8.1 Toxicity to Reproduction):

An extended one generation reproduction (EOGRT) study was commissioned by the Titanium Dioxide Manufactures Association (TDMA) in response to a request by the European Food Safety Authority (EFSA) as set forth in their Scientific Opinion of June 2016 (EFSA, 2016) on the re-evaluation of titanium dioxide (E171) as a food additive.

During the fully guideline compliant extended one generation reproductive toxicity study (OECD 443) 20 male and 20 female rats were dosed with 0, 100, 300 and 1000 mg/kg bw/day of food-grade Titanium Dioxide E171-E via the diet for 10 weeks before mating. After mating, all males were analysed for general toxicity. After weaning of F1 animals, all parental females were sacrificed and analysed for reproductive and gerneral toxicity and all F1 pups were allocated to Cohort 1A, 1B, 2A, 2B and 3 for the assessment of reproductive/developmental endpoints (cohort 1A and 1B), potential impact on the developing nervous system (Cohort 2A adult rats, Cohort 2B weaning rats) and potential impact on the developing immune system (Cohort 3). Cohort 1B animals were maintained on treatment until postnatal day 90 and bred to obtain the F2 generation. The F2 generation was analysed for developmental toxicity on postnatal day 7.

In addition to that, 10 male and 10 female F0 satellite animals per group were paired and sacrificed after weaning and used for evaluation of aberrant crypt foci. Further F0 satellite animals were used for hormone analysis of beta-estradiol, Testosterone, T3, T4 and TSH (group 1 and 4 only). This analysis was performed twice, pre-dose and at the end of the pre-mating period (after 10 weeks) throughout a 24-hour cycle. Blood sampling was performed every 2 hours during the 24-hour period of the cycle.

Further, blood samples were taken at sacrifice from F0, Cohort 1A, 1B, 2A and 2B animals for the determination of sexual hormones (estradiol, testosterone and estrone) and whole blood and urine samples taken at sacrifice from F0, Cohort 1A, 1B and F2 pups were used for titanium level analysis.

 

No test item-related influence was noted on the general toxicity and the reproductive performance of the parental animals of the F0 Generation as well as on the pre- and postnatal development of the F1 pups. No test item-related changes were noted during the histopathological examination (including a detailed examination of the testis and the epididymides) and the examination of the intestines for aberrant crypt foci (ACF). There were no treatment-related effects on hormone levels (estradiol, estrone and testosterone, plus T3, T4 and TSH) in any of the treatment groups of each cohort compared to controls. During their post-weaning development, the animals of the F1 Generation showed no signs of general toxicity. No test item-related influence was noted on the development of the reproductive system (levels of sexual hormones, time points of sexual maturations, number and length of estrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries). Also no test item-related influence was noted on the reproductive performance of the F1 males and females (fertility index, gestation index, pre-coital time and gestation length) and on the pre- and postnatal development of the F2 pups until sacrifice on lactation day 4 (number of resorptions, stillborns, live born pups and the viability index after birth until lactation day 4-7). No test item-related influence was noted on the neurological function of the young adult male and female animals of cohort 2A. The neurohistopathological examination of the brains from the high dosed adult animals of cohort 2A and from the high dosed recently weaned animals of cohort 2B did not reveal any test item-related effects when compared to their control group. The examination of the lymphpcyte subpopulations in the spleen and the anti KLH IgM serum levels was not successful, thus complete cohort 3 will be repeated in early 2021. Final results on cohort 3 will be provided with the next dossier update. The analysis of titanium in whole blood and urine is also still ongoing and will be reported with the next dossier update.

 

 

Due to various limitations in test design and reporting, the following publications were considered non-reliable.

Tassinari et al. (2014) administered TiO2 nanoparticles (Source: Sigma-Aldrich, stated to be of particle size <25nm) at doses of 0, 1 and 2 mg/kg bw/d to Sprague Dawley rats (m/f) and claim to have observed adverse effects on hormone levels (testosterone, 17ß estradiol and triiodothyronine (T3) and histopathological effects. The following contradictory information in the results raises doubts about the reliability of the study:

(i) Analytical characterisation of the test material (nano TiO2)

Somewhat contradictory information is given by the authors on the composition/purity and particle size distribution: it is stated that TiO2 nanoparticles (Anatase, primary size <25 nm, BET surface area 45–55 m²/g, purity 99%) were obtained from Sigma-Aldrich Company Ltd. (Gillingham, Dorset, UK). However, the reported impurities (Fe, Cr, Ni, Al) themselves add up to 2% (assuming sulfate process, even to 3.3%), rendering the stated purity (99%) questionable; the selected source is perhaps also not exactly the best possible choice for a well-defined test material, since the production process and manufacturer remain obscure. The reported particle size information does not match up: 13 % particles are stated to be below 100nm, whereas elsewhere 87% of particles are said to range from 30-900 nm; the also given mean diameter of up to 1.6 µm also does not appear to match.

(ii) Validity of apparent effects on hormone levels (testosterone and thyroid hormone)

As a general observation on the assay method (DELFIA - Dissociation-Enhanced Lanthanide Fluorescent Immunoassay), no details on analytical protocol, utilised DELFIA kit and measured data for the determination of the biomarkers serum testosterone, estradiol and triiodothryonine whatsoever are given. Only some thoughts on the interpretation of the less than subtle variations in hormone levels are given here:

It has been known for a long time that testosterone levels in rat are subject to circadian variation. For example, in male Sprague–Dawley rats normal serum testosterone levels can vary between 118 to 410 ng/dL, with peak levels approx. between at 10.00 and 13.00h under normal laboratory rearing conditions. In addition, an inter-assay variability of 7% can be assumed (Wilson et al., Experientia 1976, 32/7, 944-945). In the study by Tassinari, the variations for male rats were approx. within this circadian variation; given the (not unexpectedly) very low female T levels, it may be explored whether they are sufficiently above quantification limit to ensure a reliable conclusion. As to the variation in the measurement of testosterone levels, for example Heywood (1980; Irt J Andrology 3, 519-529) reports a 20-fold variability of plasma T levels in male Wistar rats, and Bartke et al. (Endo, 1973 92(4), 1223-1228) undertook a similar analysis, reporting a 2-5 fold variation in the plasma of male CD rats (range: 180-153 ng/dL). On the question of interpretation on whether such variations represent an effect characteristic of endocrine disruption, the nature of the effect and its direction may be considered further: Tassinari et al. report a moderate rise, which is within circadian variation for males and a marginal decrease in females; for an endocrine disruption, we would usually anticipate the opposite. For a discussion on the effect on thyroid hormone levels, please refer to subsection (iii) below.

(iii) Use of statistical methods by Tassinari et al.

On statistical analysis, it is stated by the authors that “[…] Bonferroni correction was adopted as the standard procedure for multiple comparisons” (p. 657) for body weight gain, absolute and relative organ weights and food consumption. For the conducted t-tests, such correction of p-values is not reported. Consequently, if the desired familywise significance level is set to α= 0.05, each individual hypothesis would have to be tested at a significance level of α’=α/m, with m being the number of comparisons. Since 2 dose groups were compared to control, each comparison would have to be done at a significance level of α’ = 0.05 / 2 = 0.025. Since raw data are not given in the publication, the statistical tests (reported as standard student t-tests) were re-calculated based on the reported mean and standard deviation. It is noted that rounding errors may lead to slightly different results. T-tests for testosterone and thyroid hormone serum levels (Figure 4): T-tests were re-calculated for significance indications (SI, given by stars in the figure) in Figure 4. For this purpose, means and standard deviations had to be read off, potentially introducing some inaccuracy in the results. The table, which can be found in the field "Attached background material", compares re-calculated p-values with the significance indication (i.e. p-values are: * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001) as given in Tassinari et al. (2014). As can be seen from the table in the field "Attached background material", each sub-figure in Tassinari (2014, Figure 4) represents two statistical tests. Since both tests partly involve the same data (i.e. the control group), p-values would have to be compared to modified confidence levels. When re-calculating p-values and comparing to the given significance levels it appears that the latter were not modified. Comparing the re-calculated p-values with the modified significance limit of 0.025, only the comparison for both dose groups with control for triiodothyronine for male rates remain statistically significant, although the 2 mg group at one significance level lower than reported. Overall, the effects on testosterone are not statistically different and are also subject to large circadian variation – which is not accounted for in the interpretation by the authors. The postulated effects on thyroid hormone lack statistical significance and dose-relation in females, and the reduction in males correspond to the same level of a mere 5% in both dose groups. Finally ,it must be said that the difference between males and females lacks biological plausibility with respect to sex difference.

 

Urrutia-Ortega, I.M. et al. (2016): According to the authors, food grade TiO2 induced a significant decrease in the number of goblet cells and tumour progression marker COX2, Ki67 and ß-catenin as well as p65-NF-kB were significantly increased in treated animals. However, body and organ weights were not influenced and tumour formation could not be observed. The study design has major methodological deficiencies and data reporting is insufficiently. The study did not include low-dose groups, only one dose group, and only male mice were used. The significance of this study is clearly reduced due to the low number of animals per group. No ophthalmoscopic examination and haematological determination were conducted and only one part of the clinical chemistry examination was performed (data not shown). This study was focused only on isolated organs and therefore no full gross necropsy and histopathological examination (only colon) was conducted. Raw data or any further information about blood urea nitrogen and serum creatinine, both kidney function marker, were not shown in this study. E171 characterization was performed but no concentration determination of E171 dispersion prior intragastrically administration was stated and no justification was given for the dosing regime. The animals were not treated daily but 5 days per week. No appropriate solvent control was included: only animals in treatment group were gavaged. Additionally, food and water consumption data are incomplete.

 

Bettini, S. et al. (2017): The study conducted by Bettini et al. evaluated the toxicity of orally administered food-grade TiO2 and nano TiO2 (P25) after 7 days via gavage as well as of E171 after 100 days via drinking water. According to the author TiO2 was detected in the immune cells of Peyer´s patches (PP) in rats orally exposed for one week to E171. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in T-helper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100- day E171 led to an increase in TNF-a, IL-10 and IL-8 in the colonic mucosa and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model.

 

In contrast to that, no changes in gut permeability could be observed and E171 administration for 100 days did not initiate genotoxicity (Comet assay) nor an increase in MPO activity. Western blotting for caspase-1 did not show cleaved caspase-1 in the colons of E171-treated rats relative to control animals.

 

Due to the following major study restrictions, the study is not considered relevant for human health hazard assessment and therefore disregarded: The study design was not in accordance with any accepted guideline. The study did not include three dose groups, as recommended by OECD 408, only two dose groups were used in the 100 days toxicity study and one dose group in the 7 days toxicity study. The two concentration that were used are not in accordance with the dose setting recommended by the OECD guideline 408 (two or four fold intervals). Additionally, only male mice were used and the significance of this study is clearly reduced due to the low number of animals/tests per group/experiment (in vivo n=10-12, ex vivo n=5). No ophthalmological examination and haematological determination were conducted. No clinical chemistry examination, gross necropsy and histopathology were performed. E171 characterization was performed but no concentration determination or stability data of E171 dispersion prior intragastric administration or oral exposure via drinking water was stated. Further, no data about animal housing conditions, animal diet and body weight or food/water consumption were given.

 

 

Beside of these study design restriction additional general deficiencies need to be mentioned. 

1. First of all the source of the employed E171 is not stated. Despite the author´s efforts in characterising the test item, it is not possible to reconcile their results with those of the producer.

2. The food-grade TiO2 used in this study is not representative for E171. Electron microscopy and stability testing of representative food-grade TiO2 (E171) samples suggests that approximately 36% of the particles are less than 100 nm. In contrast to that, the E171 sample used in this study had a nano particle amount of 44.7 % and clearly exceeded the average nano particle amount of 36%. 

3. NP-105 was used as reference material. This compound is a mixture of anatase and rutile grains (85/15), shows a mean primary particle size of 23 nm, a specific surface area of 50 m²/g and an isoelectric point at pH 6.5. Therefore NM-105 samples clearly distinguish from E171 samples by all parameters taken into account and it is therefore concluded that NM-105 does not appear to be the most suitable reference material for toxicity studies by ingestion. 

4. In the main study E171 was administered via drinking water. Considering the low solubility of TiO2 in water, it may be anticipated that the test item settled in the water bottle, leading to a discontinuous exposure of E171. Additional to that, no data on water consumption throughout the 100 days study are available and therefore no dose administration determination is possible. 

5. Delivery of TiO2 in water can greatly change the nature of the particles themselves, as TiO2 tends to aggregate and agglomerate in water suspension, raising the possibility that delivery of E 171 in an aqueous solution could lead to clumps of TiO2 passing through to the gut environment.

6. E 171 administered in drinking water, requires sonication, a process with unknown effects on the form of TiO2. Agglomeration and particle settling could also occur over time. This would represent a key difference in how TiO2 is seen by the host when consumed in food, where more particles would be part of the food matrix resulting in a more uniform profile of single particles as compared to clumps.

7. Both modes of exposure (via gavage and drinking water) are strikingly different from the typical mode of human exposure in which E 171 is delivered to the GI tract as a constituent of food preparations.

8. According to the author adult male Wistar rats were used for both studies. However, the given body weight range of 175-200 g is in fact characteristic for a pre-adolescent rats at the age of 5.5-6 weeks and therefore clearly too young for this study. 

9. Cytokine concentrations in different tissues were evaluated with cytokine-kits which are not suitable/validated for tissues homogenates. Due to that, results obtained with these kits are questionable. 

10. T-helper cell detection in flow cytometry was conducted by using the cell surface marker CD4 and CD25. Both surface marker are not only characteristic to T helper cells and therefore cannot be used for specific detection of T helper cells. Indeed, age-related changes in %DC are much greater than the changes suggested in this paper to result from exposure to TiO2.

11. Findings for the divergent relative immune cell distribution of the Peyer´s patches in control animals after 7 and 100 days exposure are not explained. It remains unclear why the %DC increased by more than 7-fold between both control groups.

12. It is interesting to compare the effects on the cytokine profile reported by Bettini et al. (2017) with other similar research for example the work by Nogueira et al. (2012). They treated male Bl 57/6 mice orally with 100 mg/kg bw/day of either (i) uncoated Anatase TiO2 (MPTiO2, 260nm), (ii) KRONOS 171 titanium dioxide E 171) or (iii) uncoated self-synthesised nanoparticles (NPTiO2, 66nm) for 10 consecutive days. Immediately after the last treatment the duodenum, jejunum and ileum were extracted for assessment of cytokines and inflammatory cells. Cytokines were evaluated via ELISA and CD4+ (Th and Tregs) and CD8+ (cytotoxic T cells) T cells, natural killer cells, and dendritic cells were evaluated in the fixed samples by immunohistochemistry.

- Nogueira et al. (2012) measured a statistically significant increase in T CD4+ (Th and Tregs) cells in the duodenum, jejunum and ileum of mice treated with MPTiO2 and NPTiO2, compared to the control group (Fig. 2). No significant difference was observed between the MPTiO2 and NPTiO2 groups. Mice treated with MPTiO2 or NPTiO2 showed no increase in T CD8+ (cytotoxic T cells), natural killers, or dendritic cells in the small intestine.

- The Bettini et al. (2017) results showed that both NM-105 and E171 induced a significant increase in DC frequency in PP (Fig. 3a) after 7 days of oral exposure. Regarding Tregs, the NM-105 had no effect on Peyer’s Patches (PP) after 7 days of oral exposure (Fig. 3b) while E171 led to a significant decrease in Tregs and Th (Fig. 3b,d and Fig. 3c, e).

- Nogueira et al. (2012) measured a statistically significant increase in inflammatory cytokines in MPTiO2 and NPTiO2 treated animals, compared to the control group. Specifically, enhanced concentrations of IFN-γ were shown in the ileum for groups receiving MPTiO2 and NPTiO2 (Figure 1c and d), increasing by approx. 1500% and 675% respectively.

- The Bettini et al. (2017) study on the other hand showed that in the PP, NM-105 and E171 both attenuated inflammatory IFN-γ secretion relative to the controls, decreasing by approx. 58% and 48% respectively.

Given the similarity in treatment duration and the titanium dioxide E171 samples used for the treatment by Bettini and Nogueira, the obviously divergent findings in immune cell response as well as in the cytokine levels are disturbing and raise questions as to whether the reported findings are in fact test-substance related or more likely chance findings of uncertain physiological relevance. Considering that the dose administered by Nogueira et al. was even an order of magnitude higher than that of Bettini et al., the findings by Bettini et al. cannot be interpreted as positive evidence of any adverse inflammatory response in the small intestine with relevance for humans.

13. The suggested preneoplastic lesions and subtle alterations of inflammation markers observed in ex-vivo, in-vitro experiments that are discussed in the paper are an interesting model, but as yet there is no agreed link to cancer in humans. The reported slight alterations in inflammatory cytokines also do not appear to follow any consistent pattern. The authors report on levels of 8 different cytokines in colonic mucosa after 100 days of administration of 10 mg/kg bw/d of TiO2, whereby no statistically significant differences were found for IL-6, IL-17, IL-18, IFN-γ and IL-1β; only slight increases for IL-8, IL-10 and TNF-α. In contrast, under conditions of chronic inflammation such levels are usually elevated by one or several orders of magnitude. The low group size does not allow a statistically robust statement regarding any potential carcinogenic effect.

Without attempting to provide an exhaustive overview of the relationship between colorectal cancer (CRC) and interleukin levels, two examples may be discussed at some length here:

(i) IL-6 signalling is involved in sporadic colorectal cancer: serum IL-6 levels were significantly elevated in patients with sporadic colorectal adenoma compared to normal controls (Uchiyama et al., 2012). IL-6 amount of the serum and tumoural tissue in the patients with colorectal cancer correlate significantly with the staging of the tumour and with each other, with IL-6 level rising to as much as 4-fold during progression of colorectal cancer growth (Esfandi et al., 2006). Several experimental and clinical studies have linked interleukin-6 to the pathogenesis of sporadic and inflammation-associated colorectal cancer (CRC), and an increased IL-6 expression has been related to advanced stage of disease and decreased survival in CRC patients (Waldner et al., 2012). In a large meta-analysis of Fourteen studies comprising 1,245 patients, serum IL-6 expression was highly correlated with overall survival rate, tumour invasion, distant metastasis and tumour stage (Wang et al. 2015).

(ii) It is widely accepted that chronic inflammation plays an active role in cancer, and interleukin-17 as a proinflammatory cytokine can promote cancer-elicited inflammation and is generally considered to be a promoter in CRC progression (Wu et al., 2013).

Despite the established link between inflammation, colorectal cancer and elevated IL-6 and IL-17, there are no statistically significant increases of these two interleukins in the work by Bettini et al.

14. The significance of aberrant crypt foci (ACF) has been investigated over the last several decades with literally hundreds of publications with our knowledge not leading to a consistent opinion, leaving the relevance to humans to still be in question. Extensive epidemiological studies on more than 24,000 titanium dioxide workers over several decades who have been inadvertently exposed orally shown no indication whatsoever of any forms of cancer arising from exposure to titanium dioxide.

Aberrant crypt foci (ACF) were first discovered in mice in 1987 (Bird, 1987) and in colonic mucosa of humans suffering from colorectal cancer in 1991 (Pretlow et al., 1991). Since then, they have been widely investigated as a putative precursor for colorectal cancer. ACF are found predominantly in the distal colon and can for example be recognised in mice and rats as early as two to four weeks after dosing with a colon carcinogen. However, based on knowledge obtained during the last decade it can be stated that an unequivocal correlation between occurrence of ACF (neither qualitatively nor quantitatively) and later development of colorectal tumours has not been demonstrated. Moreover, neither the total number nor the crypt multiplicity of the ACF is correlated with the tumour outcome. Even though a lot of knowledge about ACF has accumulated during the last decade, the outcome of the studies is still inconclusive concerning the neoplastic potential of the ACF (Thorup, 1996).

In several recent reviews, the relevance of ACF as a surrogate endpoint for colorectal cancer is explicitly questioned (Lance & Hamilton, 2017; Khare et al., 2009).

Despite the above, ACF in rats are nevertheless proposed by Bettini et al. as pre-neoplastic markers for colon cancer and said to increase with TiO2 exposure in their study. The dose dependency for this effect is at best weak with only modest increments in response being observed after 50-fold increases in dose. Resistance of preneoplastic cells to toxicity from TiO2 relative to normal cells is proposed to modulate this effect, but the cell culture study data presented exhibit inverse relationships between dose and cytotoxicity with greater toxicity being associated with lower TiO2 concentrations, thus lacking plausibility.

15. It is also noteworthy, that in this study animals were exposed to E171 with and without pre-treatment of DMH, but the results with number of ACF or number of aberrant crypts per colon were only reported for animals pre-treated with DMH and not for those without pre-treatment. For those not pre-treated only the number of animals with and without ACF was reported. This raises the question why the results were reported in a different way. And despite of that, as mentioned before, the number of ACF per animal is not a representative parameter, since ACFs are also noted in colons of untreated animals (background level).

16. While DMH is a known, potent, genotoxic intestinal carcinogen in rodents, variability in numbers and types of lesions in the intestine are usually seen in bioassays of DMH and other carcinogens, particularly when a relatively low dose is administered as in this study. There is also considerable variability in the number and size of ACF that are induced by DMH. Further, in the rats without DMH pre-treatment there are typically also a few ACF (background). However, no groups administered only E171 were available in this study. Bettini et al. only evaluated groups pre-treated with DMH.

17. It was not mentioned whether the highly acidic DMH injection solution was neutralized before i.p. injection during pre-treatment. However, they almost certainly did as the acid without neutralization is highly irritating and highly toxic to the rats when injected i.p. Also, they stated that they injected 180mg/kg of DMH. However, they used must have used DMH·2HCI, and at that level of DMH (which would be 398mg/kg DMH·2HCI), there is severe toxicity. Thus, it is likely that they used 180mg DMH·2HCI, which is 81mg/kg DMH.

18. Scientific considerations: generally, the observation that TiO2 particles can be found in Peyer’s Patches of the small intestine has already been reported several times previously elsewhere. This is also neither alarming nor unexpected, since the cellular composition of these patches is specifically designed to allow for a wide range of particulates to be internalised in order to facilitate an antigen recognition and a protective immunological response, as part of a natural innate immune response. Under these conditions, it is also not particularly unexpected that some (not all) cytokines are slightly elevated, with some cells internalising “foreign matter” and processing it. We fail to see why this should be considered an adverse effect, since it constitutes an expected result of a normal innate immune response.

19. Oral uptake: to date, several reputable scientific organisations (EFSA, RIVM) have considered oral uptake negligible based on toxicokinetic investigations reporting a lack of analytically detectable titanium in blood/tissues even after repeated oral administration of high doses of TiO2 (EFSA, 2016; Geraets et al., 2014), which is in strict contrast to Bettini et al. microscopic findings of TiO2 particulates in liver tissue, for example. No true quantitative estimate of uptake is provided with even microscopic detection of particles in the liver demonstrating sparse distribution of presumed TiO2 particles in what is described as a “Ti-rich” area of the liver. The finding of such sparse distribution of particles does not provide evidence that systemic uptake exceeds the extremely limited uptake findings of other studies.

20. Extrapolation from laboratory animals (rats) to humans: the authors do not adequately acknowledge the inherent difficulties in extrapolating any observations of gastrointestinal effects in rats to humans. When particles such as TiO2 are ingested, they “rapidly adsorb proteins from biological fluids forming a protein ‘corona’. This protein corona alters the size, aggregation state and interfacial composition of a nanomaterial, giving it a biological identity that is distinct from its synthetic nature. The biological identity determines the physiological response, including signalling, kinetics, transport, accumulation and toxicity” (EFSA, 2016). Within the environment of the gastrointestinal tract, the composition of this corona likely includes both proteins and bile salts (McCracken et al., 2013). There are highly significant differences in the kinetics of bile salt production, concentration and composition between rats and humans (Tanaka et al., 2012) that would be expected to yield a species-specific modulation of any gastrointestinal impacts observed. Failure to acknowledge such basic impediments to inter-species extrapolation does not provide the reader with an objective contextual framework from which to evaluate the present work. It also questions the relevance of ex vivo, in vitro experiments exposing extracted cells to unmodified TiO2 outside of a biologically relevant environment.

21. Comparing the results of Bettini et al. with other similar research for example the work by Blevins et al, Nogueira et al. or Carpenter et al. the divergent findings in immune cell response as well as in the cytokine levels are disturbing and raise questions as to whether the reported findings by Bettini et al. are in fact test-substance related or more likely chance findings of uncertain physiological relevance.

 

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Chen et al. (2020):

Chen et al. (2020) conducted an animal experiment to investigate the effect of oral exposure to TiO2 NPs on lipid metabolism in Sprague-Dawley rats. The rats were treated with TiO2 NPs (29 ± 9 nm) orally at doses of 0, 2, 10, 50 mg per kg bw daily for 90 days. According to the author, TiO2 caused a significant decrease in body weight of mid and high dose animals from week 8 to 13. However, no effects on food consumption and organ weights were observed. The level of triglycerides decreased and the lipidomic signature changed significantly in the serum of high dose rats. Additionally, 69 lipophilic metabolites were either up or down regulated in high dose animals in comparison to control animals. MDA was shown to be accumulated and SOD activity was decreased. No effects on short chain fatty acids in faeces were observed.

Due to the following major study restrictions, the study is not considered relevant for human health hazard assessment and therefore disregarded:

 

(i)          The study design was not in accordance with the guideline of the 90-day oral toxicity study or any other accepted guideline

(ii)         Only male rats were used. According to the respective guideline, males and females should be used

(iii)        The author stated that they obtained three weeks old rats. The rats were then acclimated for 7 days, which means that the rats must have been 4 weeks at study initiation. However, according to the graphical overview shown in the publication the rats had a body weight of approx. 50 g at study start (week 0). According to publicly available body weight charts a body weight of 50 g corresponds to 3 weeks old rats. Regardless of whether 3 or 4 weeks old rats were used, the animals were clearly too young in both cases and not young adult rats as recommended in the respective guideline

(iv)        The significance of this study is clearly reduced due to the low number of animals/group (n=6). According to OECD guideline No. 408 10 animals per sex are recommended. Thus, the number of animals was too low and an appropriate statistical analysis cannot be performed

(v)         The author reported that clinical signs and mortality were recorded daily. However, details on observed clinical signs were not stated

(vi)        A detailed clinical observation once prior exposure and once a week thereafter was not conducted

(vii)       Sensory reactivity, assessment of grip strength and motor activity were not conducted

(viii)      No ophthalmological examination and haematology were conducted

(ix)        No complete clinical chemistry examination was conducted (selected parameters only)

(x)         Serum total T4, T3 and TSH were not analysed

(xi)        Gross necropsy and histopathology were not performed

(xii)       Only selected organ weights were reported

(xiii)      The applied test item formulations were not analysed with regard to concentration, stability and homogeneity

(xiv)      The test item was obtained from Shanghai Macklin Reagent Co. Ltd., China which seems to be a distributor and not a manufacturer. The selected source is perhaps not exactly the best possible choice for a well-defined test material, since the production process and manufacturer remain obscure and the impurity profile is unknown

(xv)       It was not clearly stated which vehicle was used to apply the test substance. It is assumed to be distilled water as distilled water served as control, but it was not clearly stated in the description of the test item preparation

(xvi)      Individual data on body weight or any other experimental part were not provided

(xvii)     When showing results of effects on metabolites, the author presented results of only control and high dose animals. Thus, it remains unknown whether there was any dose response effect or only high dose animals were affected

(xviii)    The reported effects on MDA level and SOD activity were most likely not biologically relevant. The decrease of SOD activity in high dose animals was minimal compared to control animals and low and mid dose animals were unaffected. The same was true for the MDA level. The author postulated an increase in high dose level. However, the standard deviation was higher compared to control, low and mid dose group and no dose response was observed

(xix)      Historical control data were not provided

Apart from the above-mentioned deviations, it is also noteworthy to mention that the author report effects on body weight gain from week 8 to 13 in animals treated with 50 mg/kg bw/day. The author mentioned in the discussion that this is in line with effects observed in studies of Duan et al. and Hong et al. However, both studies were published from the University of Soochow and used self-synthesized TiO2 nanoparticles with unknown impurity profiles. Apart from that, this working group published a large number of studies in a wide range of journals. Some of the publications of this working group were subjected to an investigation resulting in the retraction of more than four publication until now. The shortcomings identified were incorrect statistics, experimental errors and missing original data. Thus, referring to publications of this University is perhaps not the best choice.

Further, it was not mentioned in this study that the same study design with the same test material was already used in a study published by Chen et al. in 2019 (see section 7.5.1) and in this particular study no effects on body weight were observed in any treatment group within 90 days. Thus, it remains questionable why the test material caused effects on body weight gain at a dose of 50 mg/kg bw/day in a Chen et al. study in 2020 but not in 2019.

 

Chen et al. (2019):

Rats were administered with TiO2 NPs (29 nm) orally at exposure doses of 0, 2, 10, 50 mg/kg daily for 90 days. Changes in the gut microbiota and hepatic metabolomics were analysed to explore the role of the gut-liver axis in the hepatotoxicity induced by TiO2 NPs.

According to the author TiO2 NPs caused slight hepatotoxicity, including clear mitochondrial swelling, after sub-chronic oral exposure at 50 mg/kg. Liver metabolomics analysis showed that 29 metabolites and two metabolic pathways changed significantly in exposed rats. Glutamate, glutamine, and glutathione were the key metabolites leading the generation of energy-related metabolic disorders and imbalance of oxidation/antioxidation. 16S rDNA sequencing analysis showed that the diversity of gut microbiota in rats increased in a dose-dependent manner. The abundance of Lactobacillus_reuteri increased and the abundance of Romboutsia decreased significantly in faeces of TiO2 NPs-exposed rats, leading to changes of metabolic function of gut microbiota. Lipopolysaccharides (LPS) produced by gut microbiota increased significantly.

 

Following contradictory information in the results raises doubts about the reliability of the study:

1. The author mentioned it the introduction that hepatotoxicity of TiO2 NPs has been reported by most studies, and the liver may be the target organ. In this case it is noteworthy that the studies the author was referring to (here and elsewhere), are studies of the University of Soochow. This working group published a large number of studies in a wide range of journals. In each study the same test material, self-synthesized TiO2 NPs, was used. The test item is poorly described and the influence of the other simultaneously administered process chemicals/by-products is not addressed. This raises severe doubts as to whether (i) nano-sized TiO2 particles at all were used in these experiments, and (ii) whether the diverse effects can really be attributed to titanium dioxide. Apart from that, the publications of this working group were subject to an investigation, which resulted in the retraction of more than six publications. Thus, it is questionable why the author was referring to such unreliable references.

2. In the material and method part the author mentioned that the study design refers to OECD Guideline No. 408- Repeated Dose 90-Day Oral Toxicity Study in Rodents. However, this study design is actually not in line or refers to the OECD test guideline No. 408. This study design is of experimental character and was focused on specific hepatotoxicity parameters only. However, other relevant parameters, such as haematology, complete histopathology and clinical chemistry, functional observation battery, organ weights or thyroid hormones, were not reported. Thus, the comparison with such a complex test guideline is questionable.

3. The author stated that the Sprague Dawley rats used in this study were three weeks at study initiation. In contrast to OECD test guideline 408, where animals need to be young adults and not older than 9 weeks at study start, three weeks of age is clearly too young to perform such a complex study.

4. In contrast to the recommendations in OECD guideline 408, only males were used in this study and the number of animals per group (n=6) is too low for an appropriate statistical analysis.

5. With regard to the statistical methods used in this publication it needs to be noted that Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) are powerful statistical modelling tools that provide insights into separations between experimental groups based on high-dimensional spectral measurements from NMR, MS or other analytical instrumentation. However, when used without validation, these tools may lead investigators to statistically unreliable conclusions. This danger is especially real for Partial Least Squares (PLS) and OPLS, which aggressively force separations between experimental groups. As a result, OPLS-DA is often used as an alternative method when PCA fails to expose group separation, but this practice is highly dangerous. Without rigorous validation, OPLS-DA can easily yield statistically unreliable group separation.

6. It also needs to be mentioned, that the author performed several analyses on liver tissue and for each analysis one part of the liver was used. However, the author did not mention which part was used for which analysis and if for each group always the same part of the liver was used for the same analysis.

7. The author stated that 30.0 mg was used to prepare homogenates for liver metabonomic analysis. Considering that the liver weight is approximately 3-4% of the rats body weight (approx. 300 g at this age), the liver weighed 9-12 g. That means that the author used only 0.25-0.35 % of the whole liver. Apart from that, here again, it was not mentioned whether the same part of the liver was used for control and dose animals.

8. The author reported that biochemical parameters were significantly increased or decreased in mid and high dose animals. However, it needs to be noted that almost all parameters were either changed in mid dose animals or in high dose animals, but not in both dose groups. Thus, a dose response could not be observed. And apart from that, parameters were only slightly increased or decreased. According to the author they were statistically significant, but the biological relevance is questionable.

9. The same is true for oxidative stress markers and inflammatory cytokines. The author reported, that GSH, GSSG and GSH/GSSG ratio as well as Il-1a, IL-4 and TNF were significantly decreased or increased. These effects were minor and not dose dependent. Thus, the biological relevance remains questionable.

10. The reported histopathological findings are considered not significant and these findings differ to those reported in other oral studies with TiO2 in which animals were exposed to TiO2 up to 1000 mg/kg bw/day. Apart from that, historical control data of this rat strain and age were not provided, which makes it impossible to support the conclusion of the author.

11. The test material was obtained from Shanghai Macklin Reagent Co. Ltd. in China. Since no further details were provided (e.g. product ID), it is unknown which material was obtained and thus not possible to verify the test item correctly.

12. There is no evidence regarding how the particles transmigrated from the gastrointestinal tract to the liver (via the hepatic-portal system) – even the authors noted:

“However, considering that only approximately 0.02 to 0.1% of TiO2 NPs could be absorbed through the digestive tract and the rest were excreted through faeces (21-24], the proportion of organ damaged caused by direct interaction of TiO2 NPs with cells could be small.”

13. In contrast to OECD test guideline 408, applied doses were not analytically verified. 

 

Summary entry – Publications of the Hong working group, University of Soochow, China

A working group at the Chinese university of Soochow published a large number of studies in a wide range of journals. All studies were conducted in mice using a similar study design and the same test item. The publications suggest the elicitation of adverse effects on different target organs following administration (oral or instillation/inhalation) of self-synthesised titanium dioxide assumed by the authors to be nano-sized to CD-1 mice. However, since the test item is poorly described and the influence of the other simultaneously administered process chemicals/by-products is not addressed, this raises severe doubts as to whether (i) nano-sized TiO2 particles at all were used in these experiments, and (ii) whether the effects can really be attributed to titanium dioxide.

Additionally, none of these studies are in accordance with any internationally agreed repeated dose toxicity guidelines. The food or water consumption was not or only insufficiently recorded throughout the studies. Further examinations such as gross necropsy, clinical biochemistry, complete histopathology and haematology were also not performed. No reports on ophthalmological or clinical and functional observations are stated, and most of the studies did not record the body weight or body weight gain of the animals throughout the study.

For a number of studies there appears to be a mismatch between the given body weight and the assumed age of the animals, e.g. authors state a mean body weight of 23 g for 5 week old male ICR mice. Such values appear grossly implausible given the usual average bodyweight of 30-34g for 5 week old animals (source: growth charts for ICR mice taken from 3 different breeders).

Only one sex was used in these studies, which does not allow an analysis of sex specific differences. Injection volumes for gavage, nasal administration, intraperitoneal injection etc., in addition to the TiO2 suspension concentration were not reported, which does not allow to determine the actual dose received. Additionally, no data on stability and homogeneity of the TiO2 suspension were available.

Most of the studies analysed the effects of at least three TiO2 NP concentrations, which is in accordance to repeated dose toxicity guidelines, however, no rationale for dose selection or results of dose range finding studies were included.

The publications of this working group were subject to an investigation of the University of Soochow, resulting in the retraction of four publications (Hong et al. 2018a/b, Gui, S. et al. 2013; Zhao, X. et al., 2014) by the editor. The shortcomings identified were incorrect statistics, experimental errors and missing original data. However, our detailed evaluation of all reports and the included raw data published by the University of Soochow showed that not only these four retracted publication showed incorrect statistics. The publications in the below reference list state standard deviations of exactly 5 % for all measured data. Standard deviations shown in graphs without raw data are also very similar and appear to suffer from similar flaws. Standard deviations of exact 5 % throughout all experiments covering multiple endpoints in an in vivo system is impossible and puts into question whether these studies represent reliable and genuine research data:

Cui, Y. et al., 2010, Hepatocyte apoptosis and its molecular mechanisms in mice caused by titanium dioxide nanoparticles, J. Hazard. Mat. 183: 874-880 646

Cui, Y. et al., 2012, Gene expression in liver injury caused by long-term exposure to Titanium dioxide nanoparticles in mice, Toxicol. Sci. 128: 171-185 590

Cui, Y. et al., 2010, Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles, Published online 9 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32976 2400

Duan, Y. et al., 2010, Toxicological characteristics of nanoparticulate anatase titanium dioxide in mice, Biomaterials 31 (2010) 894–899 1230

Gui, S. et al., 2013, Intragastric exposure to titanium dioxide nanoparticles induced nephrotoxicity in mice, assessed by physiological and gene expression modifications, Particle and Fibre Toxicology 2013, 10:4. 1194

Gui, S. et al., 2011, Molecular mechanism of kidney injury of mice caused by exposure to titanium dioxide nanoparticles, J. Hazard. Mater. 195: 365-370 698

Hong, J. et al., 2014, Th2 Factors May Be Involved in TiO2 NP-Induced Hepatic Inflammation, J. Agric. Food Chem. 2014, 62, 6871−6878 2548

Hu, R. et al., 2010, Neurotoxicological effects and the impairment of spatial recognition memory in mice caused by exposure to TiO2 nanoparticles, Biomat. 31, 8043-8050 650

Hu, R. et al., 2011, Molecular mechanism of hippocampal apoptosis of mice following exposure to titanium dioxide nanoparticles, J. Hazard. Mat. 191: 32-40 641

Li, N. et al., 2010, Spleen injury and apoptotic pathway in mice caused by titanium dioxide nanoparticles, Toxicology Letters 195 (2010) 161–168 2402

Sang, X. et al., 2012, The chronic spleen injury of mice following long-term exposure to titanium dioxide nanoparticles, J. Biomed. Mat. Res. 4: 894-902 619

Sang, X. et al., 2014, Immunomodulatory effects in the spleen-injured mice following exposure to titanium dioxide nanoparticles, J. Biomed. Mat. Res. 102, 3562-3572 1323

Sang, X. et al., 2013, Toxicological Mechanisms of Nanosized Titanium Dioxide-Induced Spleen Injury in Mice after Repeated Peroral Application, J. Agric. Food Chem. 2013, 61, 5590−5599 2398

Ze, Y. et at., 2014, Neurotoxicity and gene-expressed profile in brain-injured mice caused by exposure to titanium dioxide nanoparticles, J. Biomed. Mat. Res. 102, 470-478 1324

Sheng, L. et al., 2014, Nano-sized titanium dioxide-induced splenic toxicity: A biological pathway explored using microarray technology, J. Hazard. Mat. 278, 180-188 1338

Wang, J. et al., 2011, P38-Nrf-2 Signaling Pathway of Oxidative Stress in Mice Caused by Nanoparticulate TiO2, Biol Trace Elem Res (2011) 140:186–197 2401

Ze, Y. et al., 2014, Neurotoxic characteristics of spatial recognition damage of the hippocampus in mice following subchronic peroral exposure to TiO2 nanoparticles, J. Hazard. Mat. 264, 219-229 1335

Ze, Y. et al. (2013), Molecular mechanism of titanium dioxide nanoparticles-induced oxidative injury in the brain of mice, Chemosphere 92 (2013) 1183–1189 1622

Zhao, X. et al., 2014, Mechanisms of nanosized titanium dioxide-induced testicular oxidative stress and apoptosis in male mice, Part. Fibre Toxicol.11, 47/1-47/27 1301

Zhao, Y. et al., 2013, Nanosized TiO2-Induced Reproductive System Dysfunction and Its Mechanism in Female Mice, PLoS ONE 8(4): e59378 2399

Hong, F. etl al., 2017, Pulmonary fibrosis of mice and its molecular mechanism following chronic inhaled exposure to TiO2 nanoparticles. Environ. Toxicol. (Epub ahead of print) 2393

Hong, F. et al., 2016, Chronic nasal exposure to nanoparticulate TiO2 causes pulmonary tumorigenesis in male mice. Environ. Toxicol. 00:000–000. doi:10.1002/tox.2 2079

Sun, Q. et al., 2012: Pulmonary effects caused by long-term titanium dioxide nanoparticles exposure in mice. Journal of Hazardous Materials 235-236 (2012) 47-53 623

Gao, G. et al. (2012): Ovarian dysfunction and gene-expressed characteristics of female mice caused by long-term exposure to titanium dioxide nanoparticles. Journal of Hazardous Materials (2012) 19-27 634

Sun, Q. et al. (2012): Oxidative damage of lung and its protective mechanism in mice caused by long-term exposure to titanium dioxide nanoparticles. J Biomed Mater Res A 2012:100A:2554-2562 638

Li, B. et al. (2013): Molecular Mechanisms of Nanosized Titanium Dioxide–Induced Pulmonary Injury in Mice. PLoS ONE 8(2): e55563. doi:10.1371/journal.pone.0055563 1196

Gao, G. et al. (2013): Titanium dioxide nanoparticle-induced testicular damage, spermatogenesis suppression, and gene expression alterations in male mice. Journal of Hazardous Materials 258-259 (2013) 133-143 1216

Ze, Y. et al. (2014): TiO2 Nanoparticles Induced Hippocampal Neuroinflammation in Mice. PLoS ONE 9(3): e92230, 2014. 1602

Yu, X. et al. (2014): Changes of serum parameters of TiO2nanoparticle-inducedatherosclerosis in mice. Journal of Hazardous Materials 280 (2014) 364–371, 2014. 1696

In addition to the questionable statistical analysis, it is also noteworthy that although a large number of animals (between 80 and 200) was used for each of these studies, results were given for only 5-10 animals (such as histopathological examination, blood parameter analysis, etc.). It remains unclear why only a fraction of the animals was reported or used in the analysis and for which purpose the remaining animals were used. Mortality data are not available, thus no conclusion can be drawn whether the remaining animals died prematurely. Although the initial number of animals would be sufficient for a robust statistical analysis of the data, the actual number of animals for each experimental part is too low for the statistical analysis of biologically relevant differences between the treatment groups.

The study design of the various publications and the large number of animals create the assumption that experimenters used the identical test animals but reported organ specific findings individually. Since one has to assume that all publications from this working group reporting findings with titanium dioxide exhibit identical shortcomings, they were disregarded for hazard assessment purposes.

 

Summary entry – repeated dose toxicity (oral)

Further references were identified, representing in vivo mechanistic investigations in rats and mice, following repeated oral administration of pigment and nano-sized titanium dioxide. These studies are primarily academic research papers, focussing on isolated organs, tissues or biomolecules, which respond to exposure with titanium dioxide. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to the low number of animals used, missing dose response relationship, contradictory results in comparison with highly reliable guideline studies. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given blow were included in the IUCLID for information purposes only:

Shrivastava, R. et al. (2014): The publication shows significant methodological deficiencies in the experimental setup and documentation (only one dose administered, type of administration (e.g. gavage) not specified and test material is not sufficiently characterised) and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Jia, F. et al. (2014): The study does not comply with any acceptable guideline and was not conducted under GLP directive. The test material is not sufficiently characterised (characteristics and agglomerate status in dispersion / reaction with dispersants not specified) and details on the form of oral administration (e.g. gavage, food) is missing. Individual results (e.g. individual body weight gain, organ weights) were not specified. An uncommon duration of exposure was chosen (OECD recommendation for exposure in repeated dose toxicity studies is 28, 90 days or chronic).

Nogueira, C.M. et al. (2012): Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide and (iii) the methodical setup is not in accordance with any guideline. Additionally, only male mice were used and only selected parameters were analysed. No full histopathology, clinical biochemistry, haematology and gross necropsy were conducted. Despite of that, the number of animals per group (12) is too low for a statistical analysis and only one dose level was used for the assessment of TiO2 toxicity. No justification for the dosing regime is included and 10 days of dosing is clearly too short for the determination of TiO2 short term toxicity. On top of that, no food and water consumption were recorded and no body weight gain was analysed.

Proquin, H. et al. (2018): The study does not comply with any acceptable guideline and was not conducted under GLP directive. The test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether the effects can really be attributed to titanium dioxide. Additionally, only selected parameters were analysed and the dosing regime of 5 days/week for 21 days is not justified. Thus, this dosing regime is clearly too short for the determination of short-term toxicity induced by titanium dioxide. The number of animals analysed at each time point (4 animals) is too low for a statistical analysis. The investigated endpoints are not required for the human health risk assessment. 

Hu, H. et al. (2019): The publication shows significant methodological deficiencies in the experimental setup and documentation (only one dose administered, none GLP, test material is not sufficiently characterised) and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Elnagar, A.M.B. et al.(2018): The publication shows significant methodological deficiencies in the experimental setup and documentation (only one dose administered, test material is not sufficiently characterised, non GLP, uncommon vehicle (gum acacia solution), according to the body weight too young animals) and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Ali, S.A. et al. (2019): The publication shows significant methodological deficiencies in the experimental setup and documentation (test material is not sufficiently characterised and the influence of the other simultaneously administered process chemicals is not addressed which raises doubts as to whether the effects can really be attributed to titanium dioxide, none GLP, exposure duration of 5 days too short, only selected parameters investigated) and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Canli, E. G. et al. (2020): The publication shows significant methodological deficiencies in the experimental setup and documentation (test material is not sufficiently characterised and the influence of the other simultaneously administered process chemicals is not addressed which raises doubts as to whether the effects can really be attributed to titanium dioxide, none GLP, exposure duration of 14 days too short, only selected parameters investigated) and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Yao, L. et al. (2020): The publication shows significant methodological deficiencies in the experimental set up and documentation (test material is not sufficiently characterised, only two doses administered, none GLP, only selected parameters investigated) and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annex VII-X).

Abbasi-Oshaghi, E. et al.(2019): The publication shows significant methodological deficiencies in the experimental setup and documentation (test material is not sufficiently characterised, none GLP, only selected parameters investigated) and the investigated endpoints are not required for building an expert judgment and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annex VII-X).

Chen, Z. et al. (2018): The publication shows significant methodological deficiencies in the experimental setup and documentation (none GLP, only selected parameters investigated) and the investigated endpoints are not required for building an expert judgment and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annex VII-X). The results of the presented 30-day and 90-day study are not always clearly described in the report from each other. It is also unclear to which study (30 or 90 days) the results in figure 3 to 6 belong.

 

Conclusion

Titanium dioxide was tested in two oral carcinogenicity studies in rats and mice, showing no test substance related adverse effects. Titanium dioxide forms tested were anatase with an unspecified size of primary particles and a mineral silicate covered with titanium dioxide. In addition, no adverse effects were seen in 90-day and 28-day oral repeated dose toxicity studies in rats with a NOAEL of 1000 and 24,000 mg/kg bw/day, respectively. Both studies used a pigment grade titanium dioxide in a rutile phase. Due to a complete lack of adverse effects in all repeated dose toxicity studies with oral route of administration reviewed in the weight of evidence approach, it is concluded that no hazard is identified for titanium dioxide in pigment of ultrafine grade for the endpoint repeated dose toxicity (oral).

 

Repeated dose toxicity, dermal

Titanium dioxide was tested in various percutaneous absorption tests which have been reviewed by the Scientific Committee on cosmetic products and non-food products (SCCNFP/0005/98, 2000) and which concluded: “extensive tests for percutaneous absorption, mostly in vitro, indicate that absorption does not occur, either with coated or uncoated material; one experiment found some evidence that a little of the material could be found in the openings of the follicles. [...] The toxicological profile of this material does not give rise to concern in human use, since the substance is not absorbed through the skin. In view, also, of the lack of percutaneous absorption, a calculation of the margin of safety has not been carried out.”

Further experimental testing for repeated dose toxicity via the dermal route is considered dispensable, based on:

(i) the poor percutaneous absorption and the subsequent poor systemic bioavailability of titanium dioxide (see also the toxicokinetic chapter for more information on dermal absorption) and

(ii) the existence of chronic studies via oral and inhalation route of exposure in rats and mice. Both routes are considered the major routes of exposure for humans.

The study by Osmond-McLeod, M.J. et al. (2016) is included as supporting evidence, indicating no adverse effects upon repeated dermal exposure of titanium dioxide. The study evaluated the impact of sunscreen containing titanium dioxide as well as organic substances as active ingredients. Hairless mice were treated once per week for 36 weeks. Mice were sacrificed after 30 treatments and selected organs were histopathologically examined. No titanium dioxide-treatment related effects were observed. Elevated TiO2 levels in liver tissue of treated animals was the only observed treatment related effect and could probably be explained by the fact that all mice per group were treated at the same time in one cage and oral application of TiO2 by grooming or after treatment in their home cages could not be excluded. This study is not in accordance with any dermal toxicity testing guideline but clearly demonstrate that the use of titanium dioxide containing sunscreen is not linked to any adverse effect in a hairless mice model.

 

Some references were identified, representing in vivo mechanistic investigations in rats and mice, following repeated dermal administration of nano-size titanium dioxide. The studies are primarily academic research papers, focussing on isolated organs, tissues or biomolecules, which respond to exposure with titanium dioxide. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to the low number of animals used, missing dose response relationship, contradictory results in comparison with highly reliable guideline studies. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given blow were included in the IUCLID for information purposes only:

Han, W. et al. (2009): In vivo biocompatibility studies of nano TiO2 materials. Ad. Mat. Res. 1: 79-82.

Unnithan, J. et al. (2011): Aqueous synthesis and concentration-dependent dermal toxicity of TiO2 nanoparticles in Wistar rats. Biol.Trace Elem.Res. 143: 1682-1694.

Adachi, K et al. (2013): Subchronic exposure of titanium dioxide nanoparticles to hairless rat skin. Experimental Dermatology, 22: 278 - 283.

 

Conclusion

Based on the above information it is concluded that titanium dioxide is void of any local dermal effects after prolonged exposure. Due to its poor percutaneous absorption, systemic availability is not expected to occur after prolonged dermal exposure. Consequently, it is concluded that no hazard is identified for titanium dioxide in pigment of ultrafine grade for the endpoint repeated dose toxicity (dermal).

 

Repeated dose toxicity, inhalation

Industrially manufactured titanium dioxide can be distinguished by its surface modification, being either hydrophilic or hydrophobic. In order to assess potential differences in effects elicited in the respiratory tract, a comprehensive literature review was performed. Primarily in vivo studies administering titanium dioxide via inhalation or intratracheal instillation were assessed. A firm conclusion could not be drawn due to various shortcomings in the available literature; however, there do not appear to be any significant differences in the inflammatory response following single or repeated administration of hydrophobically coated compared with hydrophilically coated titanium dioxide.

 

Chronic inhalation toxicity studies with pigment-grade titanium dioxide

None of the chronic inhalation in rodents represent guideline compliant repeated dose toxicity studies useful for hazard and risk assessment purposes. The deficiencies are given below in detail. Consequently, all studies are rated as not reliable and are assessed using the weight of evidence approach.

In 2-year chronic inhalation studies, male and female CD rats were exposed to titanium dioxide (pigment-grade) at concentrations of 10, 50, or 250 mg/m³ for six hours a day, five days a week (Lee et al., 1985). The majority of the titanium dioxide was of respirable size (95% of the particles were less than 10 microns and average particle diameter was about 1.5 microns for high dose group). Survival of the titanium dioxide-exposed animals was comparable to that of the control group. Additionally, these titanium dioxide-exposed animals did not have any compound-related clinical signs of toxicity. Histopathological evaluation showed a significant dose-related increase over controls in the incidence of rhinitis, squamous metaplasia, and tracheitis. The incidence of benign lung tumours and proliferative keratin cysts were significantly increased in the group of rats exposed to the high-dose level of 250 mg/m³ of titanium dioxide. Also observed were dose-related increases in pleurisy, slight collagenized fibrosis associated with cholesterol granulomas, alveoli bronchiolisation, pneumonia, and alveolar cell hyperplasia. The changes observed at the 10 mg/m³ concentration were minimal in severity in comparison to similar effects observed in the controls (except that the alveolar cell hyperplasia was not observed in controls). The degree of pulmonary fibrosis seen at the two higher levels was slight. In subsequent experimentation (Warheit et al., 1997; summarised below) using these same dosing rates for four weeks, it was demonstrated that high dose animals had substantially increased lung clearance times and sustained inflammation under particle overload.

For the dosimetric analysis of the Lee et al. 2-year inhalation study the widely accepted Multiple Path Particle Dosimetry (MPPD) model was used which predicts the deposition and clearance of inhaled particles in the rat and human respiratory tract. The model is based on many published peer-reviewed data of respiratory tract structures and of results of inhaled particle deposition and retention kinetics in different mammalian species. This calculation was also done for the other exposure groups and results expressed in the following table:

Table: Retained lung burden after 2-year exposure of male rats (Lee et al. 1985)

Exposure Concentration
[mg/m³]

Measured in study
[mg/lung]

Predicted by model
[mg/lung]

10

26.5

3.8

50

124

19

250

665

95

 

This result clearly shows that all three titanium dioxide exposure concentrations resulted in significant lung particle overload, i.e., an impaired alveolar macrophage-mediated particle clearance function. Per g lung weight, the retained normalized lung burden observed in the study of 28 mg/g exposed lung and 39 mg/g control lung for the 50 mg/m³-exposed rats is obviously greatly exceeding a retained lung burden of 1 mg/g lung which - according to Morrow (1988) - signals the beginning of lung overload in rats.

The accumulation of particles in the lung were caused by overwhelming the normal pulmonary clearance mechanisms of the respiratory system. The disturbance of physiological mechanisms is considered by internationally accepted guidelines to be in exceedance of the maximum tolerated dose (MTD). The guideline OECD 451 for the conduct of carcinogenicity studies, in conjunction with the relevant OECD guidance document 116, highlights on various occasions that inhalation concentrations overwhelming physiological mechanisms are in exceedance of the MTD:

a)     The guidance explicitly states that “inhalation of doses that overwhelm pulmonary clearance may lead to tissue responses that are specific to the species being tested” (section 94, p.54).

b)     “The robustness of a carcinogenicity or chronic toxicity study, in particular the former, is dependent on a demonstration that the dose levels selected in the study are adequate to show an effect or effects of the test substance, without producing either false negative results (because the doses selected were too low) or false positive results (because metabolic/homeostatic mechanisms are overwhelmed, etc.), which may be problematic in assessing risk in humans” (section 101, page 55).

c)      In the selection of the maximum concentration, it should be considered that “disturbances of physiology or homeostasis that would compromise the validity of the study should be considered in the dose-selection process. Examples include hypotension, inhibition of blood clotting, overwhelming normal pulmonary clearance mechanisms, immune system effects, and in some cases hormonal imbalance” (p.63).

d)     “For substances likely to accumulate in the lung over time due to poor solubility or other properties, the degree of lung-overload and delay in clearance needs to be estimated based on adequately designed pre-studies; ideally a 90-day study with post-exposure periods long enough to encompass at least one elimination half-time. The use of concentrations exceeding an elimination half-time of approximately 1 year due to lung-overload at the end of study is discouraged” (section 135, p.71).

As demonstrated above, all concentrations applied in the Lee et al. study clearly exceeded the maximum tolerated dose, since lung overload and overwhelmed lung clearance mechanisms were already achieved at the lowest concentration. Consequently, the Lee et al. study is considered unsuitable for hazard and risk assessment purposes due to an inadequate dose regime, being not compliant with current guidelines. However, due to an absence of adverse effects at the lowest concentration, it is considered that titanium dioxide shows no adverse effects up to the limit concentration, limited by the maximum tolerated dose.

 

In a study reported by Muhle et al. (1991), titanium dioxide was used as a negative control dust in a two-year inhalation study with toner particles. Male and female F-344 rats were exposed (6 hr/day, 5 days/week) to 5 mg/m³ titanium dioxide (rutile form) of 1.1 μm MMAD with a respirable fraction of 78%. The number of titanium dioxide exposed rats with primary lung tumours was equivalent to the control animals and within published tumour frequency rates in this strain, age and sex. Lipid proteinosis, inflammatory cell infiltration and foamy macrophages was not detected in the titanium dioxide exposed animals. A small but statistically insignificant incidence of fibrosis (1.2% in exposed vs. 1.1% in control animals) was seen in the titanium dioxide exposed group (Muhle et al., 1995).

 

Chronic inhalation toxicity studies with ultrafine titanium dioxide

Heinrich et al. (1995) exposed female Wistar rats by whole body inhalation to sub-pigmentary titanium dioxide (80% anatase: 20% rutile) at an average concentration of 10 mg/m³ (single exposure concentration: 7.2 mg/m³ 1-4 months, 14.8 mg/m³ 5-8 months, 9.4 mg/m³ 9-24 months, 18h/d, 5d/w) for 24 months followed by 6 months without exposure. The primary particle size of the titanium dioxide used ranged from 15 to 40 nm with a MMAD of 0.8 mm (agglomerates of sub-pigmentary particles). At the end of 2-year exposures, body weights of exposed animals were significantly lower than controls. Wet lung weights increased substantially during exposure, progressing with study duration. This was reflected in retained test material in the lungs, which increased from 5 mg/lung to 39 mg/lung from 3 months to 24 months of exposure. This value is higher than the retained material in the Lee et al. study (26.5 mg) at the identical exposure concentration of 10mg/m³, which similarly showed impairment of lung clearance and therefore an exceedance of the maximum tolerated dose.

The study is inconsistent with other data and lacks plausibility with respect to the stated lung burdens and clearance-half times, which appear to have been severely underestimated/underreported. Given the weight this study is assigned for classification and in particular to the level of volumetric loading, the above considerations cast considerable doubt on the reliability of the data in this publication. A comparison with results from a 13w study (Bermudez at el.) with the same test material demonstrates this:

(i) despite similar exposure concentrations, Heinrich et al. employed daily exposure durations of 18h instead of 6h (Bermudez); together with the slightly smaller particle size distribution, the resulting pulmonary deposition in Heinrich et al. should have been higher (ca. 5-fold) than in Bermudez et al.

(ii) in contrast to this prediction, the rats in the Heinrich study accumulate only ca.5 mg/lung in the first three months (Bermudez: 33mg/lung), and reach ca. 35 mg/lung only after 12 months, whereafter lung burdens paradoxically do not increase despite progressive impairment of clearance.

(iii) clearance half-times in the Heinrich study do not increase after the 12month interim assessment, although the already clearly impaired clearance should produce rising lung burdens with a concurrent extension of clearance times.

The Heinrich et al. study is considered unsuitable for hazard and risk assessment purposes due to an inadequate dose regime, being not compliant with current guidelines. Further, the use of a single dose-regime and the significantly extended daily exposure duration to 18 hrs per day renders this study of limited relevance for hazard- and risk assessment purposes. The results by Heinrich et al. also lack plausibility because they are an order of magnitude lower than in Bermudez et al after a 3-month exposure period and do not progress substantially beyond the level attained after 12 months. This casts doubt on this study’s reliability for regulatory conclusions. A detailed critique of the study by Heinrich et al. (1995) is provided in the Appendix.

 

Sub-chronic inhalation toxicity studies with pigment-grade and ultrafine titanium dioxide

Bermudez et al. (2002) compared these responses in rats with pulmonary responses in hamsters and mice after sub-chronic inhalation with pigment-grade titanium dioxide. Animals were exposed to 10, 50 or 250 mg/m³ pigmentary titanium dioxide via whole body inhalation for 6 hours/day, 5 days/week for 13 week with recovery groups held for an additional 4, 13, 26 or 52 weeks post-exposure. At each time point, selected lung responses were examined. The responses studied were chosen to assess a variety of pulmonary parameters including: inflammation, cytotoxicity, and epithelial- and fibroproliferative changes. Lung and associated lymph node loads of titanium dioxide increased in a concentration-related manner. Retained lung burdens were greatest in mice following exposure, with rats and hamsters displaying similar lung burdens immediately following exposure. Particle retention data indicate that particle overload in the lungs was reached in both rats and mice at the 50 and 250 mg/m³ concentrations. Inflammation was observed in all three species at the two highest concentrations. This inflammation persisted in rats and mice throughout the post-exposure recovery period at the highest exposure concentration. In hamsters, inflammatory responses were eventually resolved due to the more rapid clearance of particles from the lung. In rats exposed to the highest concentration (250 mg/m³), pulmonary lesions consisted of epithelial proliferative changes manifested by increased alveolar epithelial cell labelling indices, as evidenced by the results of cell proliferation studies. Associated with these proliferative changes in the rat were increased interstitial particle accumulations and alveolar septal fibrosis. Although rats exposed to 50 mg/m³ developed minimal alveolar cell hypertrophy, accumulation of particle-laden macrophages, and inflammation, no alveolar septal fibrosis or relevant cell turnover at alveolar sites were observed at this lower exposure concentration. Similar changes to those seen in rats were not observed in either mice or hamsters.

Bermudez et al. (2004) compared these responses in rats with pulmonary responses in hamsters and mice after sub-chronic inhalation with ultrafine titanium dioxide (Aeroxide P25). Animals were exposed to 0.5, 2.0 or 10 mg/m³ ultrafine titanium dioxide via whole body inhalation 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. The following pulmonary parameters were investigated at each time point: histopathology, cell proliferation, test item body burden, BALF, cytology, lung volumes (0, 13, and 52 weeks post-exposure only), and electron microscopy (0, 13, and 52 weeks post-exposure only). In addition, overt clinical signs, detailed clinical signs, mortality, and body weight were recorded. Retained lung burdens increased in a concentration-related manner in all three species. Mice and rats had similar lung burdens at the end of exposures but hamsters were significantly lower. Retardation of particle clearance in rats and mice at the highest exposure concentration (10 mg/m³) indicated that pulmonary particle overload had been achieved. Lesions of the lungs in rats consisted of foci of alveolar epithelial proliferation of metaplastic epithelial cells (alveolar bronchiolisation) concomitant with circumscribed areas of heavy, particle-laden macrophages. In rats these changes were manifested by increased alveolar epithelial cell labelling indices, as evidenced by cell proliferation studies. Associated with these foci were areas of interstitial particle accumulation and alveolar septal fibrosis. These lesions observed in the rat became more pronounced with time. Mice developed a less severe inflammatory response without the progressive epithelial and fibroproliferative changes.

Data and findings from the Bermudez 90-day interspecies rodent inhalation studies with pigment and ultrafine titanium dioxide provide convincing mechanistic justifications to better understand the distinct differences in cellular responses to particle overload exposures when comparing rats to either mice or hamsters. For both ultrafine and pigment-grade titanium dioxide a higher sensitivity of rats compared to mice and hamsters was shown. At high concentrations of ultrafine and pigment-grade titanium dioxide, alveolar metaplasia and fibro-proliferative changes were only observed in rats, not in mice and hamsters.

In a supporting study by Warheit et al (1997), male rats were exposed to titanium dioxide particles 6 hours/day, 5 days/week for 4 weeks at concentrations of 5, 50, and 250 mg/m³. The animals were evaluated at selected intervals through the 6 months post-exposure period (0 hour, 1 week, and 1, 3, and 6 months). Indices of pulmonary inflammation as well as alveolar macrophage clearance functions (morphology, in vivo and in vitro phagocytosis, and chemotaxis), cell proliferation, and histopathology endpoints were measured at several post-exposure time periods through 6 months. In addition, amounts of TiO2 in lungs and tracheobronchial lymph nodes were measured to allow an evaluation of particle clearance and translocation patterns. Four-week exposures to TiO2 at concentrations of 250 mg/m³ produced lung burdens of 10 mg titanium. Associated with this high dust burden was the development of a persistent pulmonary inflammatory response, which was evident through a period of 180 days postexposure and was manifested by significant numbers of neutrophils recovered in bronchoalveolar lavage fluids from exposed animals. The data presented here supports the notion that subchronic as well as chronic exposures to high concentrations of low toxicity, low solubility dusts in rats produces persistent, generic effects of particle clearance impairment, inflammation and enhanced cellular proliferation.

 

In a series of mechanistic tests, Creutzenberg (2013) investigated the effects of three different ultrafine titanium dioxide samples (TiO2 UV TITAN M262, TiO2 UV TITAN M212 and TiO2 P25 coded in the European nanomaterials repository with NM-103, NM-104 and NM-105) after 28-day nose-only inhalation in Wistar rats. Rats were exposed to aerosol concentrations (low, mid, high) of 3, 12 and 48 mg/m³ for 28 days (6 hours/day, 5 days/week) while concurrent controls inhaled clean air. This dosing scheme was aiming at achieving non-overload, partial overload and complete overload conditions in the low, mid and high dose groups, respectively. Subsequently, endpoints were analysed at day 3, 45 and 94 of the post-exposure period. PMN in BALF as inflammation indicator resulted in the low dose groups for NM-105 in a level similar to clean air controls, whereas NM-103 and NM-104 induced a slight inflammation reaching approx. 10% PMN. Biochemical parameters in the BAL supernatant showed statistically significant increases on day 3 of the recovery period in the NM-103 and NM-104 mid dose groups for LDH and total protein levels. In the high dose groups, all three test items showed statistically significant increases in all three endpoints as compared to clean air control groups. After 45 days of recovery only in the NM-104 mid dose group both significant levels persisted, in the NM-103 mid dose group only total protein was still statistically significantly increased. After 94 days of recovery all mid dose groups had returned to normal levels. Histopathology revealed a similar dose-dependent character of changes between NM-103, NM-104 and NM-105. The distribution of the intra-alveolar particle-laden macrophages changed over time slightly from an initially more disseminated distribution to a more multifocal distribution with a higher concentration of these cells in the vicinity of the alveolar ducts. The interstitial accumulation of particle-laden macrophages was accompanied in a dose-dependent manner by a very slight interstitial fibrosis and an interstitial mononuclear cell infiltration which increased slightly during the time period investigated. In addition, a very slight bronchio-alveolar hyperplasia was observed in the mid- and high-dose groups. This type of hyperplasia is considered to be non-preneoplastic and to represent an “attempt of the lung” to facilitate a more efficient removal of inhaled materials. Within the alveolar a lipoproteinosis and an infiltration of granulocytes were noticed in a dose-dependent way which was still observable after 94 days of recovery. In all other organs than the respiratory tract, treatment-related changes were not observed. Pronounced differences in toxicity were not observed between the three test items in these experiments. The dosing chosen for the inhalation experiments according to the author was aimed to mimic exposure scenarios at workplaces (6 hours/day, 5 days/week for 28 days), with a dosing scheme inducing a non-, partial, and complete lung overload, respectively. Such exposure conditions are neither mimicking human exposure conditions (general dust limits are usually set at 5 mg/m³ and below) nor can exposure conditions at lung overload be considered as being below the maximum tolerated dose. Consequently, the study provides evidence on the similarity of three different ultrafine titanium dioxide samples, but is of limited relevant for hazard and risk assessment purposes.

 

The study by Landsiedel et al. (2014) investigated the pulmonary response after 5-day inhalation exposure with hydrophobically coated ultrafine titanium dioxide in male and female rats. No test item induced histopathological findings were seen in the respiratory tract or adjacent organs (lymph nodes). The BAL analysis showed an increase in PMNC in biochemical parameters, in a dose-dependent manner. Due to the limited study design, exclusively restricted to the respiratory tract (other organs and tissues as well as other parameters were not investigated) and the short exposure duration of only 5 days, the study will not be used for hazard and risk assessment purposes but as supporting mechanistic information.

 

In a study by Creutzenberg et al. (2009, 2012) reports the results of a 21-day inhalation study with TiO2 nanoparticles (Aeroxide P25). Adult Wistar rats were exposed to 2 and 10 mg/m³ TiO2 aerosol via nose-only inhalation. After 21 days of treatment lungs were histopathologically examined and the TiO2 amount in the lungs were determined. Additionally, a haematological examination was conducted. According to the authors analysis of the lungs by histopathology and bronchoalveolar lavage resulted in very slight inflammatory findings 3 days after end of the exposure that were not statistically significant as compared to controls. Furthermore, the authors stated that TiO2- inhalation did not alter levels of polymorphonuclear cells (PMN) in BAL. Lastly, haematology revealed decreases in white blood cells in the high-dose group (overload) at days 3, 28 and 90. The test conditions are insufficiently described and this study was not in accordance with the subacute inhalation toxicity guideline. Only two concentrations for inhalation were used. Additionally, the number of animals per group is not stated and the treatment duration of 21 days is too short for a subacute 28 days inhalation toxicity study. The test item (purity, etc.) the monitoring of exposure conditions is insufficiently described. Beside of that, no body weights and food/water consumption were recorded and clinical observation was not conducted. After sacrifice no clinical pathology assessment, organ weight record, full haematology, clinical chemistry, ophthalmoscopic examination as well as full gross pathology and histopathology were conducted. Because no further unexpected toxic results were observed but the study design is not in accordance with any subacute inhalation toxicity guideline, this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information.

Due to various limitations in test design and reporting, the following publications were considered non-reliable.

 

 

In the study by Gate et al (2017) male Fisher 344 rats 12-13-weeks old and 19 months old were each exposed to 10 mg/m³ of ultrafine titanium dioxide (Aeroxide P25) 6 h/day (2 periods of 3h) , 5 days/week for 4 weeks. The lung burden and clearance of titanium after inhalation in young adult and elderly rats were evaluated. Lungs were collected immediately and 3, 28, 90 and 180 days after the end of the inhalation exposure. The titanium content was measure by ICP-MS. According to the author, Ti quantification showed that the Ti burdens in exposed rat lungs from both groups were significantly higher than those of the corresponding control groups. The clearance of lung Ti was observed to be 54 % and 45 % for young adults and elderly rats, respectively. Additionally, but the data were not shown, the author reported that a strong lung inflammatory response were induced immediately and 3 days after the end of exposure which however decreased over the time. An increase in lactate dehydrogenase and of total proteins in BALF immediately and 3 days after the end of exposure were also observed. This study was not in accordance with any subacute inhalation toxicity guideline, showing a number of shortcomings, such as the use of male rats only, the number of animals per group is not clearly stated but appears to be insufficient for any hazard assessment purpose or for a robust statistical analysis. The study design has major limitations due to the fact that only one dose level was used. The rats used in this study were clearly too old (12-13 weeks, OECD recommendation 5-6 weeks). Basic records of clinical signs were not reported, such as body weight, food/water consumption, clinical observation. After sacrifice no clinical pathology assessment, organ weight record, haematology, clinical chemistry, ophthalmological examination as well as gross pathology and histopathology were conducted. Due to the very limited study design and the lack of reporting details, the study is not considered relevant for risk and hazard assessment purposes and was therefore disregarded. 

 

Bernstein et al. (2020) evaluated the pulmonary toxicity of titanium dioxide (Aspect ratio: 7.04; mean diameter: 0.27 ± 0.17 µm; mean length: 1.57 ± 1.1 µm) aerosol in male Wister rats after sub-chronic inhalation exposure. The test animals were exposed to 0.7 mg/m³ titanium dioxide particles at a frequency of 6 hrs/day, 5 days/week for 13 weeks, via nose-only inhalation. Bronchioalveolar lavage fluid parameters, including levels of macrophages, neutrophils, lymphocytes, eosinophils, and LDH activity, were examined at days 45 and 90 of the exposure period as well as 3- and 9-months post exposure. The level of the cytokines, TNF-α, TGF-β1, and IL-1β, in BALF was determined at days 45 and 90 of the exposure period. The particle burden, histopathological examination, and confocal microscopy analyses of the lung were performed on days 45 and 89/90 of the exposure period as well as 3 months post exposure. Notably, the confocal microscopy analyses for collagen quantification and pathological examination included apart from the lung also the chest wall. According to the authors, the aerosol contained a mean of 2752 particles/cm³. The number of total fibres was 24 per cm³. The lung particle burden was increasing by means of time and was highest at the last post exposure observation on day 180. In contrast, the fibrous fraction in the lungs was gradually decreasing and completely cleared on day 180. The exposure to titanium dioxide did not result in statistically significantly changed body and lung weights, when compared to the air-only control group. Moreover, the BALF parameters examined showed no statistically significant alteration, at any point in time. The histopathological examination of the lung revealed (multi)focal very slight/minimal alveolar/interstitial accumulation of particle-laden macrophages up to the last post exposure observation at day 180. Moreover, the analyses of the lung using Masson’s trichrome stain indicated no significant collagen deposition. Evaluation of the histopathological findings using the Wagner-score revealed scores not significantly different from the air-only control group and indicated only reversible changes. Analysis of the lung via confocal microscopy and 3D reconstruction confirmed the findings obtained via histopathology, i.e. slight persistent accumulation of particle-laden macrophages and absence of significant collagen deposition. Moreover, confocal microscopy revealed no significant collagen deposition in the visceral and parietal pleura. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. The test material was only poorly characterised, since information on crystalline phase, manufacturer, surface treatments, and the number-based particle size distribution are missing. The data presented on particle number per lung is implausible. According to the authors, the number of particles per lung was increased by 32% three months post exposure as compared to the last day of exposure. However, based on lungs clearance kinetics and the data presented by Bermudez et al. (2004)*, a marked decrease in the particle number per lung is anticipated up to the third month post exposure rather than an increase in lung burden. The authors were not able to determine an MMAD. This study was not in accordance with any subacute inhalation toxicity guideline and only male rats were used for the toxicity evaluation. Only one dose level was tested, which precludes evaluation of dose-response relationships. Methodology on lung burden analysis and aerosol generation is insufficiently described. The description on aerosol exposure conditions lack details, since i.a. air flow rates are not specified. The number of animals used for BALF analysis (n = 3 rats per group) and histopathological examination (n = 4 rats per group) was very low, and thus, the robustness of the data is only limited. The number of animals used for lung burden analysis is not specified. After sacrifice no haematology, clinical chemistry, and ophthalmological examination were conducted. Information on food/water consumption, clinical signs, and body weight development are missing. Results on organ weight and histopathology were exclusively specified for the lung.

 

Chézeau, L. et al. (2018) investigated on pulmonary effects in male Fischer 344 rats after short-term inhalation of titanium dioxide NANO4 (Aeroxide P25). A total of thirty male rats was exposed to titanium dioxide NANO4 aerosol at a concentration of 10.17 mg/m³ via nose-only inhalation at a frequency of 6 hrs/day, 5 days/week for 4 weeks. Six rats per group were euthanised and examined immediately after the last exposure or 3, 28, 90, and 180 days post exposure. The examinations were restricted to BALF analyses, determination of the lung weight, histopathology of the lung and lymph nodes, as well as differential protein and gene expression analyses in lung tissue. Additionally, BALF cytokine levels and differential protein expression were examined (Chézeau, L. et al. (2019)). In general, male rats exposed to titanium dioxide NANO4 showed an inflammatory response immediately after the exposure, which is mainly characterised by BALF changes in absence of marked histopathological alterations, which declines during the post exposure period. In titanium dioxide NANO4 exposed rats, all BALF parameters, including LDH activity, total protein concentration, macrophage count, and total cell count, were only transiently statistically significantly increased and were latest at post-exposure day 90 similar to control values. The only exception was the neutrophil count, which was slightly but statistically significantly increased up to post exposure day 180. The lymphocyte count was comparable to control values at all post exposure time-points. The level of 11 out of 29 analysed cytokines in BALF were changed at some point after exposure. The cytokines Cinc-1, Cinc-2α/β, Fractalkine, Il-1ra, Lix, L-Selectin, Timp-1, and VEGF showed increased expression levels immediately after exposure, whereas Thymus chemokine showed a statistically significantly lower level. In contrast, at post exposure day 180, Cinc-2α/β, Fractalkine, Lix, and Vegf showed statistically significantly lower expression levels. Notably, none of these cytokines showed consistently changed expression levels. The absolute lung weight was slightly but statistically significantly increased at post exposure day 0 and 3 (+20.3% and + 13.3%, respectively). However, the lung weight was highly variable in the control group at the different post exposure observations and were found to be considerably lower on day 0 and 3 when compared to the other time-points. Histopathological examination of the lungs of titanium dioxide-exposed test animals revealed particle-laden macrophages in the alveolar and bronchial lumen, proliferation of type II pneumocytes associated with minimal epithelialisation of alveoli, and transient granulomatous inflammation in the alveoli of some animals. On day 180, most of the particle-laden macrophages present in the alveolar lumen were limited to a few large aggregates. In the lymph nodes, slight to moderate diffuse multifocal distribution of particle-laden cells was observed up to post exposure day 180. The gene expression analysis of lung tissue revealed that 350 genes were differentially expressed immediately after the last inhalation exposure to titanium dioxide. The number of differentially expressed genes decreased gradually over time resulting in 92 differentially expressed transcripts on day 180 post exposure. The genes enriched were, according to GO biological process and KEGG, mostly linked to inflammation. Other enriched GO biological processes included among others “response to organic substances” and “locomotory behaviour”. Differentiation between short- and long-term effects on gene expression revealed that genes involved in cell cycle, protein maturation, and circulatory system were mostly enriched immediately after exposure, whereas response to protein stimulus, response to cold, and gland morphogenesis were mostly enriched in the long-term response. The BALF proteome analyses revealed a total of 107, 50 and 45 proteins (UniprotKB identifiers) differentially expressed in exposed rats immediately, 3 and 180 days after the end of exposure, respectively. The most enriched biological processes were “proteolysis”, “response to wounding”, “defense response”, and “response to organic substances”. Levels of inflammatory proteins, members of proteasome, various histones, proteins involved in cytoskeleton organization, were increased up to 3 days. Some of these proteins were linked with Neutrophil Extracellular Trap formation (NETosis). Long-term response was characterised differential expression of proteins involved in “response to drug”, “regulation of blood coagulation”, “response to cytokine stimulus”, and “lipid transport” on post exposure day 180. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. This study was not in accordance with any subacute inhalation toxicity guideline and only male rats were used for the toxicity evaluation. A dose range finder was not performed, and the study included only one dose level, which precludes dose-response relationship evaluations. Moreover, information on clinical observations, body weights, food/water consumption, ophthalmological examination, haematology, clinical chemistry, urinalysis, or gross pathology are missing. The histopathological examinations are restricted to the lung and lymph nodes. Furthermore, the titanium lung burden was not examined. Exposure to an aerosol mass concentration of 10 mg/m³ used in this study possibly resulted in lung overload conditions, which would implicate that the MTD was exceeded. Information on exposure system, including air flow rates, are missing. Moreover, information on the analytical verification of the test concentration. Differential cell counts were not determined. Information on test animals, i.e. age/body weight at study initiation, test group randomisation, and housing conditions, are missing. The results on lung weight and BALF parameters were not presented in a tabulated format. The investigation of differential gene and protein expression provide only mechanistic information which are only of limited value for hazard and risk assessment purposes.

 

The fate and effect of titanium dioxide (P25) in male Wistar rats after sub-acute inhalation was evaluated by van Ravenzwaay et al. (2009). Six male mice were exposed to 100 mg titanium dioxide/mg³ (analytical concentration: 88 mg/m³) for six hours on five consecutive days in a head-nose inhalation system. After euthanasia, gross necropsy was performed and the lung, mediastinal lymph nodes, liver, kidney, adrenals, thymus, spleen and brain were weighed. Histopathological examinations and particle distribution analyses were performed on days 1 and 14 after exposure. Moreover, the BALF analyses were performed 3- and 14-days post-exposure. Further, immunohistochemistry was used to investigate the proportion of pro- and anti-inflammatory macrophages in the alveolar macrophage population (Koltermann-Jülly et al. (2020)). According to the authors, titanium was detectable only in the lung and mediastinal lymph nodes after 1- and 14-days post exposure. However, the most particles were found in the lung. Moreover, the relative and absolute lung weight were transiently increased only immediately after the last exposure. Microscopical investigations of the lung tissue revealed that titanium dioxide NANO4 particles were mainly found in the cytoplasm of alveolar macrophages and extra-cellularly in the lumen of alveoli and bronchi. The BALF analyses revealed transiently increased protein concentration, macrophage and lymphocyte counts as well as increased activity of LDH, ALP, gamma-glutamyltransferase, N-acetyl-beta-glucosaminidase, when compared to the negative control group. These effects not observed 14 days after the last exposure. The total cell count and neutrophil count were increased at both exposure time points (3 and 14 days) but were also characterised by a regressive tendency. Histopathological examination of the lung revealed a transient bronchiolar hyperplasia/hypertrophy and granulocytic infiltration. Histiocytosis and pigment loaded macrophages persisted throughout the post exposure period. Moreover, immunostaining with macrophage markers revealed a strong increase in the pro-inflammatory M1 type immediately after exposure, when compared to the negative control group. At the end of the post exposure period, the proportion of pro-inflammatory M1 macrophages decreased, while the proportion of CD206+ anti-inflammatory M2 macrophages increased, when compared to the first evaluation time point. The proportion of ArgI+ M2 macrophages was slightly higher at both post exposure time point, when compared to negative controls. In the mediastinal lymph nodes, lymphoreticular hyperplasia was observed immediately after exposure. After 14 days, the histopathological findings in the mediastinal lymph nodes were restricted to a number of particle-laden macrophages. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. This study was not in accordance with any subacute inhalation toxicity guideline and only male rats were used for the toxicity evaluation. Only one dose level was tested, which precludes evaluation of dose-response relationships of the effects observed. Information on exposure system, including the air flow rates and environmental conditions, are missing. Body weights and food/water consumption were not recorded, and clinical observation was not conducted. After sacrifice no clinical pathology assessment, haematology, clinical chemistry, and ophthalmological examination were conducted. Results on organ weight and histopathology were nearly exclusively specified for the lung. Information on other organs is missing. The aerosol mass concentration of 100 mg/m³ (measured concentration: 88 mg/m³) was excessively high and resulted in lung overload conditions. Insoluble materials like titanium dioxide deposited in the alveolar region of the lung may accumulate over time with resultant impairment of particle clearance and particle-mediated inflammatory response. Therefore, high titanium dioxide lung burden and relatively slow clearance rates are expected. This in turn initiates the inflammatory response which was observed directly after the titanium dioxide NANO4 exposure, but which was clearly regressed after 14 days, when the lung titanium dioxide burden was lowered. Differential cell counts were not determined. Information on test animals, i.e. body weight at study initiation and test group randomisation, are missing. The organ weights and BALF analysis results were not presented in a tabulated format. Results on lung burden and BALF parameters were not statistically analysed and standard deviations were not specified.

 

The fate and effect of pigmentary titanium dioxide (crystalline phase: rutile; surface area: 6 m²/g; particle size (DLS, ethanol): 200 nm) in male Wistar rats after sub-acute inhalation was evaluated by van Ravenzwaay et al. (2009). Six male mice were exposed to 250 mg nano-TiO2/mg³ (analytical concentration: 274 mg/m³) for six hours on five consecutive days in head-nose inhalation system. An untreated control group was run concurrently. After euthanasia, gross necropsy was performed and the lung, mediastinal lymph nodes, liver, kidney, adrenals, thymus, spleen and brain were weighed. Histopathological examinations and particle distribution analyses were performed on days 1 and 14 after exposure. Moreover, the BALF analyses were performed 3- and 14-days post-exposure. According to the authors, titanium was detectable only in the lung and mediastinal lymph nodes after 1- and 14-days post exposure. However, the most particles were found in the lung. Moreover, the lung weight was transiently increased. Microscopical investigations of the lung tissue revealed that titanium dioxide particles were mainly found in the cytoplasm of alveolar macrophages and extra-cellularly in the lumen of alveoli and bronchi. The BALF analyses revealed a significantly increased protein concentration, macrophage count, neutrophil count, lymphocyte count, and total cell count as well as increased activity of LDH, ALP, gamma-glutamyltransferase, N-acetyl-beta-glucosaminidase, when compared to the negative control group. These effects were partly reversible within the recovery period. The histopathological examination of the lung revealed a histiocytosis, which regressed within the recovery period. The mediastinal lymph nodes were activated, and pigment-loaded macrophages were observed. These findings were still observed at the end of the post exposure period. In single animals, very few particles on the surface or intracellularly in the olfactory epithelium of the nasal cavity were observed. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. This study was not in accordance with any subacute inhalation toxicity guideline and only male rats were used for the toxicity evaluation. Only one dose level was tested, which precludes evaluation of dose-response relationships of the effects observed. Information on exposure system, including the air flow rates and environmental conditions, are missing. Body weights and food/water consumption were not recorded, and clinical observation was not conducted. After sacrifice no clinical pathology assessment, haematology, clinical chemistry, and ophthalmological examination were conducted. Results on organ weight and histopathology were nearly exclusively specified for the lung. Information on other organs is missing. The aerosol mass concentration of 250 mg/m³ (measured concentration: 274 mg/m³) was excessively high and resulted in lung overload conditions. Insoluble materials like titanium dioxide deposited in the alveolar region of the lung may accumulate over time with resultant impairment of particle clearance and particle-mediated inflammatory response. Therefore, high titanium dioxide lung burden and relatively slow clearance rates are expected. This in turn initiates the inflammatory response, which was observed directly after the titanium dioxide exposure, but which was clearly regressed after 14 days, when the lung titanium dioxide burden was lowered. Information on test animals, i.e. body weight at study initiation and test group randomisation, are missing. The organ weights and BALF analysis results were not presented in a tabulated format. Results on lung burden and BALF parameters were not statistically analysed and standard deviations were not specified.

 

Yu, K.-N. et al. (2015) evaluated the effect of titanium dioxide nanoparticles (particle size: 19.3 ± 5.4 nm) on lungs of A/J Jms Slc mice after short-term inhalation. Five mice per sex were exposed to titanium dioxide dose levels of 2.4, 5.0, and 10.1 mg/m³, for 6 hours per day and 5 days per week over a total period of 28 days, via whole body inhalation. After the exposure period, the animals were euthanised and blood was collected for haematology and clinical biochemistry. Histopathological examinations were restricted to the lung. In addition, structural changes in single lung cells were examined via TEM. Moreover, Western blot analysis was performed to investigate on expression changes of markers for cell proliferation (CD31 and PCNA), inflammation (phospho-p38, NF-kB, and VCAM-1), ER stress (Grp78/Bip and CHOP), and autophagy (LC3, p62, and Beclin 1). Moreover, the expression of Grp78/Bip, CHOP, and LC3 was examined in lung sections via immunohistochemistry.

The short-term inhalation of titanium dioxide nanoparticle at three different dose levels did neither affect body weights nor food consumption of exposed mice, when compared to negative control mice exposed to fresh air. The haematological examinations revealed statistically significantly increased red cell distribution width, mean platelet volume, and proportion/number of neutrophils in the intermediate and high dose groups. The proportions of lymphocytes and large unstained cells were statistically significantly altered only the intermediate dose group. Thus, the effect is considered to be not treatment related. The clinical biochemistry revealed a slight but statistically significantly increase in the blood urea nitrogen level and statistically significantly increased levels of triglycerides in the intermediate and high dose groups, when compared to the negative control group. Moreover, the AST level and the ALT level were statistically significantly increased in the high dose group. The creatinine level was found to be statistically significantly decreased only in the intermediate dose group. The high dose group did not show such an effect, and thus, the finding is considered to be of an incidental nature. Histopathological examination of the lungs revealed hyperplasia and haemorrhage in the low dose group, hyperaemia, bronchial atelectasis, and particle-laden macrophages in the intermediate dose group. In the high dose group, bronchial atelectasis and multifocal lymphoid tissue hyperplasia were observed. The TEM analysis revealed a dose-dependent swelling of the endoplasmic reticulum (ER) and mitochondrial disruption. The effect on the ER were substantiated by statistically significantly increased protein expression levels of Grp78/Bip and CHOP, indicating ER stress. Further, Western blot analysis indicated increased cell proliferation in lung tissue, as indicated by statistically significantly increased levels of CD31 and PCNA as well as inflammation in lung tissue, as indicated by statistically significantly increased levels of phospho-p38, NF-kB, and VCAM-1. Moreover, the statistically significant increases of LC3, p62, and Beclin-1 protein expression levels in lung tissue indicated autophagy. The TEM examination combined with EDX analysis revealed penetration of titanium dioxide nanoparticles into lung cells. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. The test material was only poorly characterised and information on the preparation of the test material is not provided. The description of the methodology shows significant deficiencies. The description of the test atmosphere generation is insufficient and restricted to a figure showing the exposure apparatus. The MMAD (0.47 µM) is not within the range recommended by the OECD TG 412 (v. 2009; 1 - 3 µm). Further, the GSD and particle size distribution are not specified. The analysis of the test material concentration in the exposure chamber was only monitored two weeks before start of the exposure but not during exposure. In general, the study is focussed on lung effects and only selected parameters were analysed. Information on clinical signs are not reported. The authors performed no analysis of BALF or lung burden. Further, ophthalmology, urinalysis, and gross pathology were not conducted. The histopathological examination is restricted to analysis of undefined sections of the lung. Moreover, the haematology and clinical biochemistry were restricted to the analysis of selected parameters. The description of the immunofluorescence assay lacks essential details. The description of the test animals lacks details, since information on acclimation period, caging conditions, food/water type and access and test group randomisation are not provided. Moreover, the test animals were younger than recommended (5 vs. 7-9 weeks of age). In addition, the method of euthanasia is not reported. Individual body weights are not reported, and mean body weights are not presented in a tabulated format.

 

Thyssen, J. et al. (1978) investigated on effects in female Sprague-Dawley rats after sub-chronic inhalation exposure to pigment size titanium dioxide. Fifty male and fifty female Sprague-Dawley rats were exposed via inhalation to 15.95 mg/m³ air (analytical concentration) titanium dioxide dust for 6 hours/day, 5 days/week during a period of 12 weeks. The substance was administered using a whole body exposure chamber. A control group with the same number of animals was run concurrently. After 12 weeks of exposure the animals were observed for another 128 weeks. The following parameters were investigated/determined: clinical signs, mortality, body weight, macroscopic examination, and histopathology. The study does not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The study design is not guideline-conform: test items were not sufficiently characterised; only one concentration; concentration caused lung overload; haematology, clinical chemistry, food consumption, ophthalmological examination & organ weights were not determined; histopathology not sufficiently described.

 

In this study by Warheit, D.B. et al. (2005), different titanium dioxide formulations with various surface treatments, ranging from 0–6% alumina (Al2O3) or alumina and 0–11% amorphous silica (SiO2) were tested. The test items were administration by whole body inhalation (exposure duration: 6 hours/day, 5 days/week (total of 20 exposures)) and intratracheal instillation (single administration). In the inhalation study, groups of 25 male rats were exposed to high exposure concentrations of TiO2 particle formulations at aerosol concentrations ranging from 1130–1300 mg/m³ and lung tissues were evaluated by histopathology immediately after exposure, as well as at 2 weeks and 3, 6, and 12 months postexposure. In the intratracheal instillation study, groups of 32 male rats were treated with nearly identical TiO2 particle formulations as in the inhalation study at doses of 2 and 10 mg/kg. Subsequently, the lungs of TiO2-exposed rats were assessed using both bronchoalveolar (BAL) biomarkers and by histopathology/cell proliferation assessment of lung tissues at 24 h, 1 week, 1 and 3 months postexposure. In both studies vehicle control groups were run concurrently. The study does not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The study design is not guideline-conform: males only, one concentration per test item only; concentrations caused lung overload; clinical signs, haematology, clinical chemistry, food consumption, gross pathology & organ weights (incomplete, lungs only) were not determined; incomplete histopathology.

 

In the study by Baggs, R.B. et al. (1997), male Fisher 344 rats were exposed for 6 hours a day, 5 days a week, for 3 months to filtered air (control), ultrafine titanium dioxide (23.5 mg/m³; 20 nm) or crystalline silicon dioxide (positive control;~ 800 nm, 1.3 mg/m³). The substances were administered using a whole body exposure chamber. Groups of 3-4 animals were sacrificed at 6 and 12 months following the completion of exposure. Pulmonary effects of exposure were evaluated using standard haematoxylin and eosin-stain sections and histochemical stains for collagen. The study does not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The study design is not guideline-conform: test items were not sufficiently characterised; males only; only one dose per test item; actual concentration and particle size were not determined; number of animals per group too low; exposure only 12 weeks instead of 13 weeks; clinical observations, body weights, food consumption, clinical chemistry, haematology, ophthalmological examination, and gross pathology were not conducted/determined. Incomplete histopathology (lungs only).

 

In this study by Baggs, R.B. et al. (1997), male Fisher 344 rats were exposed for 6 hours a day, 5 days a week, for 3 months to filtered air (control), pigment-grade titanium dioxide (22.3 mg/m³; 250 nm) or crystalline silicon dioxide (positive control; approx. 800 nm, 1.3 mg/m³). The substances were administered using a whole body exposure chamber. Groups of 3-4 animals were sacrificed at 6 and 12 months following the completion of exposure. Pulmonary effects of exposure were evaluated using standard haematoxylin and eosin-stain sections and histochemical stains for collagen. The study does not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The study design is not guideline-conform: test items were not sufficiently characterised; males only; only one dose per test item; actual concentration and particle size were not determined; number of animals per group too low; exposure only 12 weeks instead of 13 weeks; clinical observations, body weights, food consumption, clinical chemistry, haematology, ophthalmological examination, and gross pathology were not conducted/determined. Incomplete histopathology (lungs only).

 

The study by Xu, J. et al. (2010) was conducted to detect carcinogenic activity of nanoscale titanium dioxide (20 nm). Three groups of female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were given 0.2% N-nitrosobis(2-hydroxypropyl)amine (DHPN; initiate carcinogenesis) in the drinking water for 2 weeks and one group of rats were given drinking water without DHPN. Two weeks later, the rats were divided into the following four groups for test item administration: DHPN alone, DHPN followed by 250 µg/mL TiO2 (0.875 µg TiO2/rat), DHPN followed by 500 µg/mL TiO2 (1.75 µg TiO2/rat), and 500µg/mL TiO2 without DHPN (1.75 µg TiO2/rat). The titanium dioxide suspension was intratracheally administered once every 2 weeks from the end of week 4 to week 16 (a total of seven times). Three days after the last treatment, animals were sacrificed and the following parameters were investigated: elemental titanium analysis in different organs, histopathological examination, and immunohistochemistry. The study utilised non-standard models and a non-physiological route of exposure (intratracheal instillation). The findings from this study would not be considered reliable for classification.

 

Summary entry – Publications of the Hong working group, University of Soochow, China

A working group at the Chinese university of Soochow published a large number of studies over a wide range of journals. All studies were conducted in mice using a similar study design and the same test item. The publications contained in this summary entry indicate adverse effects on lungs following intratracheal or intranasal administration of (assumed) self-synthesised nano titanium dioxide in CD-1 mice. The studies are primarily academic research papers, focussing on isolated organs, tissues or biomolecules, which respond to exposure with titanium dioxide. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. The self-synthesized test item is poorly described and also influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in these experiments, and (ii) whether the effects can really be attributed to titanium dioxide. The unphysiological route of administration via intratracheal or intranasal instillation is not suitable for the hazard assessment of industrial chemicals. In the experiments, excessive high doses were administered to the animals, leading to an unspecific pulmonary response via lung overload. Based on the above, it is concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given blow were included in the IUCLID for information purposes only:

Sun, Q. et al. 2012: Oxidative damage of lung and its protective mechanism in mice caused by long-term exposure to titanium dioxide nanoparticles, J. Biomed. Mat. Res. 100A: 2554-2562

Sun, Q. et al. 2012: Pulmotoxicological effects caused by long-term titanium dioxide nanoparticles exposure in mice, J. Hazard. Mat.235: 47-53

Li, B. et al. 2013: Molecular Mechanisms of Nanosized Titanium Dioxide– Induced Pulmonary Injury in Mice, PLoS ONE 8(2)

Hong, F. et al. 2017: Pulmonary fibrosis of mice and its molecular mechanism following chronic inhaled exposure to TiO2 nanoparticles, Environ. Toxicol

Yu, X. et al. 2014: Changes of serum parameters of TiO2nanoparticle-induced atherosclerosis in mice, J. Hazard. Mater. 280, 364-371

Ze, Y. et al. 2013: Molecular mechanism of titanium dioxide nanoparticles-induced oxidative injury in the brain of mice, Chemosphere 92, 1183-1189

Ze, X. et al. 2014: TiO2 Nanoparticle-Induced Neurotoxicity May Be Involved in Dysfunction of Glutamate Metabolism and Its Receptor Expression in Mice. Environ. Toxicol. 31, 655-662

Ze, Y. et al. 2014: TiO2 Nanoparticles Induced Hippocampal Neuroinflammation in Mice. PLoS ONE 9(3): e92230

Hong, F. et al. 2016: Chronic nasal exposure to nanoparticulate TiO2 causes pulmonary tumorigenesis in male mice. Environ. Toxicol. 00:000–000. doi:10.1002/tox.22393

Hong, F. et al. 2015: Toxicological effect of TiO2 nanoparticle-induced myocarditis in mice

 

 

Summary entry – Repeated dose toxicity (inhalation)

The references contained in these summary entries represent in vivo experiments with mechanistic investigations in repeated dose toxicity (route: inhalation) with very limited value for risk assessment purposes. The study designs are not in accordance with accepted guidelines or the test substance has not been sufficiently characterised, therefore the references are of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to the low number of animals used, missing dose response relationship, contradictory results in comparison with highly reliable guideline studies. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given blow were included in the IUCLID for information purposes only:

Four references (Scuri, M. et al, 2010, Ma-Hock, L. et al., 2009, Rossi, E.M. et al., 2010, and Eydner, M. et al., 2012) on the effect of TiO2 nanoparticles and/or TiO2 microparticles were identified. The test item was administered by whole-body, head-nose or nose-only exposure to mice and rats. In the first reference the authors stated that the in vivo inhalation exposure to titanium dioxide nanoparticles produced upregulation of lung neurotrophins in weanling (2-weeks-old) and newborn (2-days-old) rats but not in adult (12-weeks-old) animals compared to controls. They mentioned that this effect was associated with increased airway responsiveness and upregulation of growth-related oncogene/keratine-derived chemokine in bronchoalveolar lavage fluid. In the second reference the authors mentioned that signs of dose-dependent partial reversible inflammatory response were observed. In the third reference the authors stated that SiO2-coated rutile TiO2 nanoparticles (cnTiO2) was the only sample tested that elicited clear-cut pulmonary neutrophilia. Uncoated rutile and anatase did not induce significant inflammation. Pulmonary neutrophilia was accompanied by increased expression of tumour necrosis factor-alpha (TNF-a) and neutrophil-attracting chemokine CXCL1 in the lung tissue. TiO2 particles accumulated almost exclusively in the alveolar macrophages. In the fourth reference the authors stated that after inhalation of nano-sized and fine TiO2 particles, no marked differences between behaviour and elicited lung effects of nanoscale and fine particles were detectable. Furthermore, they stated that particle deposition was similar in both groups and took place mainly in alveolar macrophages and type-I pneumocytes, which was quantified using the RDI. They mentioned that no particles were found in cell organelles such as mitochondria or nuclei and focally, nanoparticles were detected in a granulocyte located inside a capillary. According to the authors, distribution to other organs via the blood circulation is possible, although only to a minimal extent. A significant difference in the translocation and deposition behaviour of nanoparticles, compared to fine particles, was not observed by the authors. Lastly, the authors stated that a higher toxic potential (e.g. evidence of severe lesions in the lung or significant translocation to other compartments than previously known) of TiO2 nanoparticles compared to fine TiO2 particles was not found.

Four references (Li, Y. et al., 2010, Ambalavanan, N. et al., 2013, Wang, J.X. et al., 2007, and Wang, J. et al., 2008) on the effects of TiO2 nanoparticles were identified. The test item was administered either intratracheal or intranasal to mice at different dose levels and during different time periods. In the first reference, the authors mentioned that the results showed that the instilled test item could induce lung damage, and change the permeability of alveolar-capillary barrier. The test item was able to get access to blood circulation and reach extrapulmonary tissues, then lead to injury at the different level, such as liver and kidney. The authors mentioned that the result indicated that the test item might pass through the blood-brain barrier, and induce the brain injury through oxidative stress response. In the second reference, the authors mentioned that nanoparticles administration causes inflammatory cell infiltrate and inhibits lung development. Furthermore, they stated that nanoparticle administration does not significantly affect lung function or pulmonary vascular remodelling. Lastly, they mentioned that nanoparticle administration increases gene expression and protein amounts of specific cytokines in lung homogenates. Lastly, in the third and fourth reference, the authors stated that the inhaled TiO2 nanoparticles could be translocated to and deposited in murine brain after absorbing by nasal mucosa, and further influence the releases and metabolism of monoaminergic neurotransmitters in brain.

Leppänen, M. et al. (2015): The study design is not guideline conform (e.g. unusual exposure duration, insufficient number of animals tested) and the test material is not sufficiently characterised. The generated information is not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Halappanavar, S. et al. (2001): The study design is not guideline conform (e.g. only one concentration tested, exposure duration too short, MMAD/GSD was not determined and the exposure apparatus not sufficiently described) and the investigations in toxicogenomics have no direct value for fulfilling data requirements under the REACH regulation.

Leroux, M.M. et al. (2020): The study design is not guideline conform (e.g. only one concentration tested, MMAD/GSD were not determined, and the aerosol generation was not described) and the investigations on toxicogenomics have no direct value for fulfilling data requirements under the REACH regulation.

 

Conclusion

The rat is a uniquely sensitive species in its pulmonary responses to sub-chronic or chronic exposures to high doses of inhaled low solubility dusts, leading to inflammation, the development of fibroproliferative effects, and eventually lung tumour formation. Data and findings from three sub-chronic, 90-day interspecies rodent inhalation studies provide convincing mechanistic justifications to better understand the distinct differences in cellular responses to particle overload exposures when comparing rats to either mice or hamsters

The pulmonary effects observed in rats, including inflammatory and fibrotic responses, are also not observed in large mammals such as nonhuman primates and humans. It has also been clearly demonstrated through epidemiology studies of TiO2-exposed workers that there is no causal link between TiO2 exposure and the risk of non-malignant respiratory disease in humans.

 

 

Repeated dose toxicity- other routes

 

Due to various limitations in test design and reporting as well as the non-physiological route of application, the following publications were reported either as full or short entry but considered non-reliable.

 

Wang, J. et al. 2008: The study is composed of two different parts. In the first part the authors evaluated the time-dependent deposition of TiO2 particles in the murine brain. The other part was an evaluation of the antioxidative response, immune responses, and histopathological changes of murine brain tissues after TiO2 treatment. Female CD-1(ICR) mice were intranasally instilled using two different TiO2 particle suspensions (nanoscale TiO2 (rutile, 80 nm) and commercial fine TiO2 (anatase, 155 nm)). Instillations were performed every other day and the animals were sacrificed after 2, 10, 20, and 30 days (first part) or only after 30 days (second part). 

According to the authors, TiO2 levels in murine brains were significantly elevated in most of the brain tissues analysed. After 30 days, TiO2 levels were found to be highest in the hippocampus and olfactory bulb. Biochemical serum parameters remained largely unaffected and were changed only transiently if at all. Antioxidative markers were transiently but significantly increased only after treatment with nanoscale TiO2 at day 10. Lipid peroxidation marker malondialdehyde was significantly increased after treatment with both TiO2 particle types at day 30. Organ weights were not significantly different from controls except for kidneys accompanied by atrophy of the renal glomerulus and occurrence of inflammatory cells in the lumen of Bowman’s capsules. Nissl staining revealed structural changes in olfactory bulbs marked by an increased number of neuron cells and a disturbed pattern of neurons in the olfactory layer. The hippocampus was marked by enlarged and elongated pyramidal cell soma and decreased number of Nissl body were found in the CA1 region. Ultrastructure analysis revealed that the olfactory bulb neurons showed condensation of chromatin and “small amount of increased mitochondria”. Hippocampal nerve cells were degenerating and showed a changed nuclear morphology going along with chromatin condensation. Moreover, the cells showed a changed number and structure of mitochondria as well as "increased rough endoplasmic reticulum and free ribosome". Cytokine level analyses revealed a significant increase only of TNF-α and IL-1β levels in brains and only after treatment with fine TiO2 particles.

The results of this study are difficult to interpret due to the fact that the study design is not compliant with any accepted testing guideline. Further, the study shows some deficiencies in reporting and major shortcomings in study design.

The authors state that about 500 µg TiO2 particles were instilled into the nasal cavity. Based on the density of TiO2, the volume of the administered suspension must have been about 125 µl. However, the nasal cavity volume of 7 weeks old mice is only 32.5 ± 3.2 mm³ *. Growth chart derived data indicate that the mice must have been even younger than 7 weeks and consequently the mice must have had even smaller nasal cavity volumes. Thus, it is unclear what happened to the excessive suspension volume. Hence, this study design is not feasible and renders the study of little relevance for risk assessment purposes.          

The authors tested only one dose, which does not allow to draw conclusions on dose-response relations or effect levels. Moreover, a justification for the dose selected is missing. 

Details on animal husbandry are largely missing. The authors do state not on test individual’s age and caging. Furthermore, justifications for species and strain used are missing. Only one sex was tested and it is specified whether female mice have been nulliparous or pregnant.

Description of materials and methods is insufficient. Statements on the concentration and volume of the administered suspension are missing. Further, stability and homogeneity of the test item in the vehicle are not specified. Information on pH and effects on osmolality are missing. Details on the analysis of TiO2 deposition, enzymatic activities, and histopathological examinations are either unclear or missing.

Furthermore, the method section shows deficiencies in reporting. The authors stated: “All animals were randomly divided into four groups: control group and two experimental groups (80 and 155 nm group).” However, only three groups are listed. The identity of the fourth group is not specified.

The authors state: “Both in vivo and in vitro studies have shown that the physicochemical properties such as the particle size, charge and surface chemistry would endow nanoparticles the ability for their transcytosis or endocytosis across epithelial, endothelial and macrophage cell membranes and preferential location in lysosome or mitochondria”. However, details on the specific surface area and charge are not specified.

Information in the results section are unclear or missing. The authors make no statement on clinical observations, haematological tests, body weight changes during and at the end of study, and consumption of food and water. Gross necropsy and clinical biochemistry were only partially performed.

Individual rat values are missing. In Fig. 1, the controls are displayed only as total means represented by a threshold. Thus, standard deviations are not depicted. Significances are not clearly assigned and not indicated in Fig. 1. Statistical tests were not precisely assigned to the different experiments.

Significance of increased GSH-Px activity after treatment with fine TiO2 at 10 days seems to be by looking at the data and absence of individual mouse data implausible.

Results are partially contradictory. On the one hand, a loss of nerve cells in hippocampal CA1 region was observed. On the other hand, they found am increased neuron cell number in the olfactory bulb. The authors do not state on this phenomenon. Thus, it is unclear whether findings are artefacts or findings with biological relevance.

Reporting in the results sections shows some deficiencies. The authors state: “…small amount of increased mitochondria were observed in the olfactory bulbs…”, and: “…decreased mitochondria and increased rough endoplasmic reticulum and free ribosome…”. However, it is not clear which parameter is changed in these cases.

The authors show elevated TiO2 contents in the different brain structures, but they do not specify whether TiO2 particles can be found in the nerve cells. Moreover, the authors state in the methods section that Ti contents have been analysed in six different brain tissues: olfactory bulbs, cerebral cortex, hippocampus, cerebellum, brain stem, and rest of the brain. However, results of the analysis of the brain stem and rest of the brain are missing.     

The discussion section shows deficiencies in reporting. The authors state: “In the current study, as a response to oxidative stress, proinflammatory cytokines of TNF-α, IL-1β and IL-6 were highly secreted in the brain of mice after exposure to TiO2 particles for 30 days.” However, IL-6 was not significantly changed after TiO2 treatment. Further, the authors state: “With the prolonged time, Ti content in the olfactory bulb continuously increased and the translocation to the cerebral cortex and cerebellum occurred under the diffusion mechanism, even to other organs such as lung and kidneys”. However, TiO2 translocation was neither shown by the results nor correctly referenced. Even more, it is stated: “After exposure for 30 days, no obvious changes were observed in heart, liver, spleen and lung except the kidneys and brain.”

 

Ma, Y. et al. (2019) examined on lung effects in young and adult NIH mice after repeated nasal exposure to TiO2 anatase NPs. Male mice of the young group were 5 weeks old at the treatment start, whereas the adult mice were 10 weeks old. Six mice per group received daily nasal instillations at a dose of 20 mg TiO2 NPs/kg bw over a period of 30 days. A vehicle control group was run concurrently. After the last exposure, the mice were euthanised and weighed, and subsequently, the lungs and hearts were removed. The lung was weighed, and histopathological examinations were performed using HE and Masson’s trichrome stain. Moreover, the authors investigated on cytokine release as well as on global DNA methylation and DNA hydroxymethylation. Additionally, selected genes were analysed for gene and protein expression as well as for their promoter methylation profile. Further RNA sequencing was performed in order to analyse differential gene expression with subsequent KEGG and GO term analyses.

According to the authors, the terminal body weights were not statistically significantly altered in both young and adult mice after TiO2 NP exposure. However, the relative lung weights were statistically significantly increased in young mice of the TiO2 NPs exposure group. The histopathological examination revealed proliferation of type II pneumocytes, accumulation of particle-laden macrophages, and thickened alveolar walls. The effects were found to be more severe in young mice treated with TiO2 NPs. Moreover, the trichrome stain indicated collagen deposition in the alveolar septa of both TiO2 NP treatment groups. The deposition was more pronounced in young mice. Moreover, optical density analysis revealed only in young mice a statistically significant increase in the collagen deposition. Further, the level of cytokines IFN-γ and TNF-α was statistically significantly increased in both young and adult mice treated with TiO2 NPs. Repeated nasal exposure to TiO2 NP resulted in statistically significant global DNA hypomethylation and a statistically significantly lower level of global DNA hydroxymethylation in young mice. In young and adult TiO2 NP-treated mice, the analyses of mRNA level revealed statistically significantly increased expression of IFN-γ and TNF-α, whereas Thy-1 expression was statistically significantly decreased. The same alterations were observed for the protein expression levels of the three different cytokines. Further, the promoter sequence of TNF-α showed a lower methylation level in young mice after TiO2 NP treatment when compared to vehicle control mice. In contrast, Thy-1 promoter methylation was reduced in the same treatment group. The promoter of IFN-γ showed no statistically significant alteration in any exposure group. Moreover, genes related to DNA methylation and hydroxymethylation were predominantly statistically significantly increased. Differential gene expression analysis showed 8043 and 6973 genes to be altered in expression in young and adult mice exposed to TiO2 NPs, respectively. The vast majority of these differentially expressed genes were upregulated. The KEGG pathway analyses revealed that cancer-related and glycerophospholipid metabolism pathways were most enriched in young and adult mice, respectively. The GO term analysis showed that inflammatory response and methylation were also enriched in young mice exposed to TiO2 NPs.

The publication shows several reporting and methodological deficiencies.

The animals were dosed via intranasal instillation, which is a non-physiological route of exposure with only limited value for risk assessment purposes. Only one dose group was included, which precludes dose-response relationship and effect level evaluations. The test material was insufficiently characterised, since information on purity, coating, and surface reactivity are missing. Details on the test animals, including weight at study initiation and during the study, as well as the number of animals caged together, are missing. The dosing volume is not specified. The results shown in Fig 1B. and Fig 1C. showing the effects on collagen deposition in adult mice are not equivalent. Haematology and clinical biochemistry were not performed. Clinical observations and gross necropsy were not performed. Historical control data are not provided. The investigations on gene expression, pathway analyses, DNA methylation state, and cytokine levels are considered to provide only mechanistic information and are no standard endpoints for toxicological evaluations. The only toxicological relevant parameters assessed are body weighty, lung weights, and histopathological examination of the lung.

Based on the shortcomings mentioned above the reference is considered not reliable.

 

 

Summary entry – Repeated dose toxicity (other routes)

The references contained in these summary entries represent in vivo experiments with mechanistic investigations in repeated dose toxicity (other routes) with very limited value for risk assessment purposes. The study designs are not in accordance with accepted guidelines or the test substance has not been sufficiently characterised, therefore the references are of limited relevance for chemicals hazard assessment. Further, the references usually lack significance due to the low number of animals used, missing dose response relationship, contradictory results in comparison with highly reliable guideline studies. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given blow were included in the IUCLID for information purposes only:

Numano, T. et al. (2014): The investigated parameters have no direct value for fulfilling data requirements under the REACH regulation. The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The test substance is not sufficiently characterised and number of animals were lower than foreseen in guidelines, only females used and administration of only one dose does not allow a dose-response related analysis.

Chang, X. et al. (2015): The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The test substance is not sufficiently characterised, number of animals were lower than foreseen in guidelines, unusual exposure conditions were used (e.g. administration of test substance twice weekly and no information is given to the post exposure period.

Stenbäck, F. et al. (1976): The methodical setup is not adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation and only single dose tested is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The test substance is not sufficiently characterised and co-exposure with benzo-a-pyrene is not relevant for chemicals safety assessment of the test substance.

Lauvas, A.J. et al. (2019): The investigated parameters have no direct value for fulfilling data requirements under the REACH regulation. The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The test substance is not sufficiently characterised, only males were used, unusual exposure conditions were used (once weekly for 7 weeks) and administration of only one dose does not allow a dose-response related analysis.

Zhou, Y. et al. (2019): Since the self-synthesized test substance is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide and (iii) the methodical setup is not in accordance with any guideline. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The number of animals per group (n=5) is too low for a statistical evaluation. Important information about the general study design, material and methods and results (individual results, etc.) are missing. 

Apart from that, this publication is part of the publication series of the University of Soochow. The publications of this working group were subject to an investigation of the University of Soochow, resulting in the retraction of several publication. The shortcomings identified were incorrect statistics, experimental errors and missing original data. Thus, due to these major study restrictions this study is disregarded.

Abdelgied, M. et al. (2019): The investigated parameters have no direct value for fulfilling data requirements under the REACH regulation. The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The test substance is not sufficiently characterised and no source is given, only males were used, the number of animals per group (n=5) is too low for an appropriate statistical analysis, unusual exposure conditions were used (every other day) and administration of only two doses does not allow a dose-response related analysis.

Rossi, S. et al. (2019): The test material is insufficiently characterised, since information on the purity, crystalline phase, manufacturer, coatings, particle sizes, and particle size distribution are missing. The investigated parameters, except or heart tissue histopathology, have no direct value for fulfilling data requirements under the REACH regulation. The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The Spontaneously Hypertensive male rats are an unsuitable test system for human risk assessment purposes. Only one dose group was included, which precludes evaluation of dose-response relationships.

Fu, Y. et al. (2014): Most of the investigated parameters have no direct value for fulfilling data requirements under the REACH regulation. The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The description of the test material preparation lacks details. The concentration of the administered suspension is not specified. Only males were used. The TiO2 bulk test material was evaluated at only one dose level.       

Chang, X. et al. (2014): The investigated parameters have no direct value for fulfilling data requirements under the REACH regulation. The methodical setup is not guideline compliant or adequately designed for risk assessment purposes of the test substance. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose toxicity via inhalation. The description of the test material preparation lacks details. The concentration of the administered suspension is not specified. Only males were used. The TiO2 bulk test material was evaluated at only one dose level.

Meena, R. et al. (2012): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. Additionally, no justification for the dosing regime is included and only male mice were used. Further on, only selected organs were used for histopathological examination (only liver and kidney) and no full haematology as well as full clinical biochemistry were conducted. The non-physiological route of administration via intravenous injection is not guideline conform and not suitable for human hazard assessment.

Han, W. et al. (2009): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. Additionally, no justification for the dosing regime is included and the exposure duration (7 days) is too short for the determination of TiO2 short term effects. Despite of that, only male mice were used and the number of animals per group (6) is clearly too low for a statistical analysis. Although different TiO2 particle sizes were analysed, only one dose level was tested, which is not sufficient for the determination of dose level effects. Further on, the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable for human hazard assessment. The experimental procedure and the results are insufficiently reported and no food and water consumption as well as body weight were recorded. No full haematology, clinical biochemistry, full histopathology and full gross necropsy were conducted.

Moon, E.-Y. et al. (2011): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. Additionally, no justification for the dosing regime is included and the exposure duration (7 days) is too short for the determination of TiO2 short term effects. Despite of that, the number of animals per group (5 animals) is too low for statistical analysis and only one dose group was tested. No food and water consumption were recorded and the main endpoints of repeated dose toxicity studies such as haematology, clinical biochemistry, histopathology, etc. were not analysed. Further on, the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable for human hazard assessment.

Miura, N. et al. (2017): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. Additionally, no justification for the dosing regime is included (once per week for 4 weeks) and investigators suspended the acidic P25 in an assumed alkaline DSP solution. No investigations regarding pH effects were performed. No food and water consumption were recorded and the number of animals per group is too low for statistical analysis.

Liu, X. et al. (2017): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized/micro TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. No food and water consumption were recorded and the main endpoints of repeated dose toxicity studies such as haematology, clinical biochemistry, complete histopathology, etc. were not analysed. Further on, the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable for human hazard assessment.

Valentini, X. et al. (2019): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. Additionally, only male rats were used and the number of animals per group (n=5) is too low for a statistical analysis. Further on, the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable for human hazard assessment.

Abdel Aal, S.M. et al.(2020): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described, this raises doubts as to whether the effects can really be attributed to titanium dioxide. Additionally, only male rats were used and the number of animals per group (n=10) is too low for a statistical analysis. Further on, the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable for human hazard assessment.

Rizk, M.Z. et al.(2017): This study is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and insufficient information about the preparation of the administered test material, this raises doubts as to whether the effects can really be attributed to titanium dioxide. Additionally, only male mice were used and the number of animals per group (n=15) is too low for a statistical analysis. Further on, the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable for human hazard assessment.

 

 

References

Boorman G.A. et al. (1996): Classification of cystic keratinizing squamous lesions of the rat lung: report of a workshop, Toxicol Pathol. 24, 564–72

Carlton W.W. (1994): “Proliferative keratin Cyst", a lesion in the lungs of rats following chronic exposure to para-aramid fibrils, Fundamental Appl. Toxicol. 23, 304-307

Coleman G.L. (1977): Pathological Changes During Aging in Barrier-reared Fischer 344 Male Rats, J Gerontol 32 (3):258-278

Goodman D.G., et al. (1978): Neoplastic and Non-neoplastic Lesions in Aging F344 Rats, Tox and Appl Pharmacol 48, 237-248

Hirouchi Y., et al. (1994): Historical Data of Neoplastic lesions in B6C3F1 mice, J. Toxicol Pathol 7: 153-177

Levy, L.S. (1995): Review: The 'Particle Overload' phenomenon and human risk assessment, Indoor and Built Environment 4, 254-262

Morrow, 1988: Possible mechanisms to explain dust overloading of the lungs. Funda. & Appl. Toxicol. 10:369-384.

Justification for classification or non-classification

Repeated dose toxicity, oral

The reference National Cancer Institute (1979) is considered as the key study for repeated dose toxicity via oral application and will be used for classification. Rats were dosed at 3500 mg/kg bw/day orally via feed. Based on the lack of any adverse effects, the no observed adverse effect level (NOAEL) via oral application for titanium dioxide was established at 3500 mg/kg bw/day

(for detailed information please refer to the endpoint study records reported under section carcinogenicity).

The classification criteria according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – repeated exposure, oral are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and the no observed adverse effect level (NOAEL) via oral application is above the guidance value for a Category 1 classification of 10 mg/kg bw/day and above the guidance value for a Category 2 classification of 100 mg/kg bw/day. For the reasons presented above, no classification for specific target organ toxicant (STOT) – repeated exposure, oral is required.

 

Repeated dose toxicity, dermal

Titanium dioxide was tested in various percutaneous absorption tests which have been reviewed by the Scientific Committee on cosmetic products and non-food products (SCCNFP/0005/98, 2000) and which concluded “extensive tests for percutaneous absorption, mostly in vitro, indicate that absorption does not occur, either with coated or uncoated material; one experiment found some evidence that a little of the material could be found in the openings of the follicles. [...] The toxicological profile of this material does not give rise to concern in human use, since the substance is not absorbed through the skin. In view, also, of the lack of percutaneous absorption, a calculation of the margin of safety has not been carried out.”

For the reasons presented above, no classification for specific target organ toxicant (STOT) – repeated exposure, dermal is required.

 

Repeated dose toxicity, inhalation

The rat is uniquely sensitive to lung damage when exposed under conditions of particle overload to poorly soluble low-toxicity particles such as titanium dioxide or others (Levy, 1995). Although particle overload is observed in other experimental species such as the mouse, it is only in the rat that a sequence of events is initiated that leads to fibroproliferative disease, septal fibrosis, hyperplasia and eventually lung tumours. However, similar pathological changes are not observed in other common laboratory rodents, non-human primates or in exposed humans. Detailed epidemiological investigations have shown no causative link between titanium dioxide exposure and the risk of non-malignant respiratory disease in humans.

According to regulation (EC) 1272/2008, a classification for specific target organ toxicity – repeated exposure shall be taken into account only when reliable evidence associating repeated exposure to the substance with a consistent and identifiable toxic effect demonstrates support for the classification. These adverse health effects include consistent and identifiable toxic effects in humans, or, in experimental animals, toxicologically significant changes which have affected the function or morphology of a tissue/organ, or have produced serious changes to the biochemistry or haematology of the organism and these changes are relevant for human health.

The following observations have been made in experimental animals and in human epidemiological studies:

(i) No systemic toxicity was shown to result from chronic inhalation exposure in rats to high concentrations of pigment grade titanium dioxide

(ii) Particle overload is observed for insoluble particles such as titanium dioxide (Levy, 1995), whereby the rat is the most sensitive species studied, and species-specific differences are demonstrated in various mechanistic animal studies (Oberdörster, 1996). It has been demonstrated with reasonable certainty that lung overload conditions are not relevant for human health and, therefore, results based on these data do not justify classification.

(iii) It has also been clearly demonstrated through epidemiological studies of titanium dioxide -exposed workers that there is no causal link between titanium dioxide exposure and the risk of non-malignant respiratory disease in humans

For the reasons presented above, no classification for specific target organ toxicant (STOT) – repeated exposure, inhalation is required.