Diiron titanium pentaoxide

Endpoint summary

Description of key information

Diiron titanium pentaoxide as a single component has not been tested for systemic toxicity after repeated dosing. However, diiron titanium pentaoxide is a constituent of several pigments and it is therefore considered toxicologically more relevant to administer it as a constituent of a typical pigment mixture with a relatively high content. Therefore, a WoE approach was used to investigate the repeated-dose toxicity of diiron titanium pentaoxide using data from pigments which contain diiron titanium pentaoxide and data from the oxides titanium dioxide (TiO2) and iron oxide (Fe2O3) since Diiron titanium pentaoxide is a mixed oxide of titanium dioxide and iron oxide.

Oral:

No adverse effects have been observed in a limit dose 90-day oral feeding study in rats with a mica-based pearlescent pigment containing diiron titanium pentaoxide (13.5%). Based on the amount of diiron titanium pentaoxide in the pigment, a NOAEL of > 550 mg/kg bw/day can be calculated for the substance (reference 7.5.1 -1). Furthermore, administration of Titanium Dioxide coated mica containing diiron titanium pentaoxide (2 -7%) in the diet for up to 2 years to rats at concentrations of 10000, 20000 and 50000 ppm, did not significantly alter survival nor demonstrate toxic effects. No indication of oncogenic potential was elicited by the test material at the designated concentration levels (reference 7 .5.1 -3). Based on the concentration of diiron titanium pentaoxide in this pigment (up to 7%) and the food consumption during this chronic toxicity study a NOAEL of 180 mg/kg bw/day and 230 mg/kg bw/day for male and female rats was calculated for the 2-year study.

Furthermore, a study according OECD TG 407 was performed with TiO2 using male Sprague-Dawley rats. Under the conditions of this study, the NOEL was 24000 mg/kg bw/day for male rats, based on the lack of any adverse effects at this dose (reference 7.5.1 -2).

Inhalation:

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

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Diiron titanium pentaoxide as a single component has not been tested for systemic toxicity after repeated dosing. However, diiron titanium pentaoxide is a constituent of several pigments and it is therefore considered toxicologically more relevant to administer it as a constituent of a typical pigment mixture with a relatively high content. Therefore, a WoE approach was used to investigate the repeated-dose toxicity of diiron titanium pentaoxide using data from pigments which contain diiron titanium pentaoxide and data from the oxides titanium dioxide (TiO2) and iron oxide (Fe2O3) since Diiron titanium pentaoxide is a mixed oxide of titanium dioxide and iron oxide.
A subchronic feeding toxicity study equal to OECD TG 408 was performed to investigate the toxicity of different mica-based pearlescent pigments (13.5 % Diiron titanium pentaoxide). Groups of 40 rats (20 males and 20 females) received the pigments incorporated into feed pellets at the highest internationally recommended concentration of 50000 ppm, for the males and females, respectively. One placebo group (20 males and 20 females) received feed pellets containing mica only at 50000 ppm. In addition, a control group consisting of 80 rats (40 males and 40 females) were fed exclusively with standard commercial feed pellets. Treatment was for 3 months followed by a 2-months recovery period. No treatment related mortalities occurred during the study. A slight increase of body weights was observed in females receiving the second pigment compared to the feed control group 1. Food intake was temporarily or permanently increased in males and females of all groups treated with mica or mica-based pigments due to the 5% mineralic content in the diet. No treatment-related effects were observed in haematology or clinical chemistry. Gross pathology revealed a discoloration of the gut content in the animals of the treatment groups which was not observed in the recovery animals and which is not considered to be an adverse effect. All histopathological findings encountered were considered to have arisen spontaneously. In conclusion, no adverse effects were observed in rats treated with mica pigments at 50000 ppm. Based on the concentration of in this pigment (13.5%) and the food consumption during the subchronic toxicity study the mean daily intake of Diiron titanium pentaoxide can be calculated as 534 mg/kg bw/day and 602 mg/kg bw/day for male and female rats, respectively. As these mean daily intakes of diiron titanium pentaoxide by rats over a period of 90 days did not induce any adverse effects, a NOAEL of > 550 mg/kg bw/day can be deduced for Diiron titanium pentaoxide on the basis of this study.
In a further study the possible toxicity of Titanium Dioxide Coated Mica (contained 2-7% Diiron titanium pentraoxide) and its oncogenic potential was evaluated using a standardized and accepted test regimen by administration in the diet of Fischer 344 rats (50 m and f per dose group) at levels of 0, 10000, 20000 and 50000 ppm for 104 weeks. No dose-related differences in survival were noted. Compound-colored or silver-colored feces were noted for the high-dose animals from Week 2 throughout the remainder of the study. Growth rates through Week 26 were significantly lower for the mid- and high-dose females compared to controls. Mean absolute body weights at Week 26 were significantly lower for the high-dose males and females compared to controls. Although not statistically significant, mean body weights for the compound-treated groups were lower than controls throughout the remainder of the study until Week 126 when the weights were more comparable among groups. Mean total food consumption through Weeks 26 and 50 was significantly greater for the high-dose males and females, respectively, compared to controls. Mean food consumption became more comparable among groups during the second year of the study. At Week 104, ophthalmoscopic examinations revealed a dose-related increased incidence of cataracts in the male rats. Except for one male, all cataracts were unilateral. Statistical analysis revealed a significant linear trend in the incidence of cataracts and a significantly higher incidence of cataracts in the high-dose compared to controls. However, histopathological examination did not support this trend as there was no correlation between dose level and cataract incidence. Compound-colored tint on the walls of the stomach and/or cecum, and silver-colored fecal material in the cecum and/or intestines were observed grossly at necropsy in the mid- and high-dose rats. In the animals that died or were sacrificed in extremis, an increased incidence of adrenal medullary hyperplasia was noted microscopically for the high-dose males compared to controls. This increase was not observed for the females, nor was it observed for the males or females that were sacrificed at Week 130. The incidence of mononuclear cell leukemia was unusually high for animals in all groups compared to historical control data. This increased incidence is thought to be due to the length of the study (130 weeks) and the tendency of the Fischer 344 strain to develop this lesion as they age. Based on these findings, administration of Titanium Dioxide coated mica in the diet of rats at concentrations up to 50000 ppm did not significantly alter survival nor demonstrate toxic effects. Additionally, no indication of oncogenic potential was elicited by the test material at the designated concentration levels. Based on the concentration of diiron titanium pentaoxide in this pigment (up to 7%) and the food consumption during this chronic toxicity study a NOAEL of 180 mg/kg bw/day and 230 mg/kg bw/day for male and female rats was calculated.
Furthermore, a study according to OECD TG 407 was performed with the read-across substance TiO2. Male Sprague-Dawley rats were exposed by oral gavage for 29 days to 24000 mg/kg bw/day TiO2 particles, or the vehicle water. Under the conditions of this study, the NOEL was 24000 mg/kg bw/day for male rats, based on the lack of any adverse effects at this dose.
Based on these available long-term toxicity studies in rodents with the pigment, the read-across substance TiO2 and the physico-chemical properties, it is concluded that diiron titanium pentaoxide is a non-reactive and non-toxic compound. A hazard after repeated exposure for Diiron titanium pentaoxide is not expected. Therefore, and because of animal welfare reasons an additional repeated-dose toxicity study for Diiron titanium pentaoxide is not required since same results are expected.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

This oral toxicity study was performed as a limit test. The study was not performed under GLP regulations.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

yes

Remarks:

ophthalmological examinations have not been performed; Functional : observation battery (FOB) was not performed;

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: mean males: 183 g (157-212); mean females 162 g (145-184)
- Housing: individual housing of animals in Makrolon cages type III
- Diet: ad libitum, Altromin Standard Diet
- Water: ad libitum), tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-31
- Humidity (%): 40 - 75
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
The test material was incorporated into feed pellets (Altromin standard diet TPF No. 1324) Content of test material in feed: 5% (w/w)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days/week

Dose / conc.:

50 000 ppm

Remarks:

nominal in diet

Dose / conc.:

6 750 ppm

Remarks:

based on diiron titanium pentaoxide content

No. of animals per sex per dose:

feed control: 40 animals/sex
placebo group (mica): 20 animals/sex
treatment group: 20 animals/sex

Control animals:

yes

yes, concurrent no treatment

Details on study design:

Groups of 40 rats (20 males and 20 females) received the pigments incorporated into feed pellets at the highest internationally recommended concentration of 50,000 ppm, for the males and females, respectively. One placebo group (20 males and 20 females) received feed pellets containing mica only at 50,000 ppm. In addition, a control group consisting of 80 rats (40 males and 40 females) were fed exclusively with standard commercial feed pellets. Treatment was for 3 months followed by a 2-months recovery period. Hematology, clinical chemistry and urinalysis were performed on half of the animals in each group, 4 and 12 weeks after start of the study and once during the follow-up phase (20 weeks after start of the study). Half of the animals were killed at the end of the 2-months recovery phase. All animals were subjected to gross pathological and histopathological examinations.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4 and 12 (during treatment) and at the end of the recovery period (week 20)
- Animals fasted: Yes
- How many animals: half of each group
- Parameters checked: hemoglobin, red blood cells, reticulocyte count, white blood cells, differential count in %

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 4 and 12 (during treatment) and at the end of the recovery period (week 20)
- Animals fasted: Yes
- How many animals: half of each group
- Parameters checked: sodium, potassium, calcium, inorganic phosphorus, glucose, urea, creatinine, bilirubin, protein, cholesterol, triglceride, glutamic pyruvic transaminase, alkaline phosphatase

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 4 and 12 (during treatment) and at the end of the recovery period (week 20)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, protein, glucose, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

Animals were sacrificed under CO2 anesthesia by incision of the abdominal vessels and exsanguination.

GROSS PATHOLOGY: Yes
Organ weights: Heart, liver, kidneys, spleen, thymus, testes/ovaries, prostate/uterus, adrenals, thyroids, pituitary, brain, eyes

HISTOPATHOLOGY: Yes
Organ weights: Heart, liver, kidneys, spleen, thymus, testes/ovaries, prostate/uterus, adrenals, thyroids, pituitary, brain, eyes

Statistics:

Dunnett's multiple t-test
Bartlett's test

Clinical signs:

effects observed, non-treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

higher in all pigments and placebo control (mica) group. Can be explained by the 5% foreign material in food.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

> 550 mg/kg bw/day (actual dose received)

Based on:

other: content of diiron titanium pentaoxide in test material

Sex:

male/female

Basis for effect level:

other: highest dose tested

Remarks on result:

other: Based on the concentration of diiron titanium pentaoxide (13.5%) and the food consumption the mean daily intake of diiron titanium pentaoxide can be calculated as 534 and 603 mg/kg bw/d for males and females, respectively.

Critical effects observed:

not specified

Conclusions:

No adverse effects have been observed in rats treated orally with diiron titanium pentaoxide as a component of pearlescent pigments for a time period of 90 days. Based on (i) a concentration of the pearlescent pigment in the food of 50000 ppm (ii) the concentration of diiron tiitanium pentaoxide in the pearlescent pigments and (iii) the food consumption of the rats, a NOAEL of > 550 mg/kg bw/day could be derived for diiron titanium pentaoxide.

Executive summary:

A subchronic feeding toxicity study equal to OECD TG 408 was performed to investigate the toxicity of different mica-based pearlescent pigments.

Groups of 40 rats (20 males and 20 females) received the pigments incorporated into feed pellets at the highest internationally recommended concentration of 50,000 ppm, for the males and females, respectively. One placebo group (20 males and 20 females) received feed pellets containing mica only at 50,000 ppm. In addition, a control group consisting of 80 rats (40 males and 40 females) were fed exclusively with standard commercial feed pellets. Treatment was for 3 months followed by a 2-months recovery period. Hematology, clinical chemistry and urinalysis were performed on half of the animals in each group, 4 and 12 weeks after start of the study and once during the follow-up phase (20 weeks after start of the study. Half of the animals were killed at the end of the 2-months recovery phase. All animals were subjected to gross pathological and histopathological examinations.

No treatment related mortalities occurred during the study. A slight increase of body weights was observed in females receiving the second pigment compared to the feed control group 1. Food intake was temporarily or permanently increased in males and females of all groups treated with mica or mica-based pigments due to the 5% mineralic content in the diet. No treatment-related effects were observed in haematology or clinical chemistry. Gross pathology revealed a discoloration of the gut content in the animals of the treatment groups which was not observed in the recovery animals and which is not considered to be an adverse effect. All histopathological findings encountered were considered to have arisen spontaneously. In conclusion, no adverse effects were observed in rats treated with mica pigments at 50000 ppm. Thus, based on the results of this study the NOAEL for Fe2TiO5-containing pigments exceeds 50,000 ppm corresponding to 3,952 and 4,466 mg/kg bw/day.

Based on the concentration of Diiron titanium pentaoxide (13.5%) in this pigment and the food consumption during the subchronic toxicity study the mean daily intake of Diiron titanium pentaoxide can be calculated as 534 mg/kg bw/day and 603 mg/kg bw/day for male and female rats, respectively. As these mean daily intakes of diiron titanium pentaoxide by rats over a period of 90 days did not induce any adverse effects, a NOAEL of > 550 mg/kg bw/day can be deduced for Diiron titanium pentaoxide on the basis of this study.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 higher than recommended in guideline, only one sex used

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

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: housed singly in stainless steel, wire-mesh cages suspended above cage boards
- Diet: ad libitum, PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

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. The rats were dosed at a total volume of 4 mL/100 g of body weight. Control rats were dosed with NANOpure water at a volume of 4 mL/100 g of body weight.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

29 days

Frequency of treatment:

daily

Dose / conc.:

24 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 (only males)

Control animals:

yes, concurrent vehicle

Positive control:

none

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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 0 and weekly thereafter
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: Yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, for at least 15 h
- How many animals: all
- 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
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes, for at least 15 h
- How many animals: all
- 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

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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.

HISTOPATHOLOGY: Yes

Statistics:

Significance was judged at p < 0.05.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two rats died (one from each of the test groups) before the terminal sacrifice, both due to dosing accidents.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic evidence of the test substance was observed in intestinal lymphoid tissue of the treated group but was not considered to be an adverse effect.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

24 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Conclusions:

No adverse effects have been observed after 29-days oral exposure to male rats.

Executive summary:

A study according OECD TG 407 was performed. Male Sprague-Dawley rats were exposed by oral gavage for 29 days to 24000 mg/kg bw/day TiO2 particles, or the vehicle water. Under the conditions of this study, the NOEL was 24,000 mg/kg/day for male rats, based on the lack of any adverse effects at this dose.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 24, 1981 - October 26, 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 6 weeks
- Weight at study initiation: 103.9 to 166.3 g for males and 90.0-125.4 g for females
- Housing: individually housed in elevated wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks

DETAILS OF FOOD AND WATER QUALITY:
The basal diet and water were periodically analyzed for concentrations of specific heavy metals, antobiotics, aflatoxin, pesticides, and nitrosamines.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66-83
- Humidity (%): 28-96
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
Approximately every four weeks, equal amounts of each of the two test pigments were blended together in a 1:1 ratio and stored at room temperature until use. One day prior to scheduled diet preparation, the basal diet (Purina Rodent Laboratory Chow®) was premixed with a sufficient amount of corn oil such that the concentration of corn oil was 1% of the diet. On the scheduled mixing day, the appropriate amount of the blended test materials required for each dietary level was incorporated into the corn oil/basal feed mixture on a weight per weight basis. Fresh diets were prepared and administered weekly. Control rats received the basal diet/1% corn oil blend, only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical results of pretest samples showed the diets to be homogeneously mixed and stable under use conditions. In general, the concentration analyses of test diets revealed acceptable levels of the test materials in the diets for the purpose of this study.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

administered continuously in diet

Dose / conc.:

10 000 ppm

Remarks:

520 mgkg bw/day (males) / 588 mg/kg bw/day (females)

Dose / conc.:

20 000 ppm

Remarks:

1056 mgkg bw/day (males) / 1197 mg/kg bw/day (females)

Dose / conc.:

50 000 ppm

Remarks:

2574 mgkg bw/day (males) / 3291 mg/kg bw/day (females)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high-dose level was selected because it represented the maximum level required by various regulatory agencies and was recommended in OECD Guidelines and this level would not compromise the animals from a nutritional standpoint.

Positive control:

none

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the first 14 weeks and once every four weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Time schedule for examinations: weekly during the first 14 weeks and once every four weeks thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at beginning of study and an additional examination was performed on all study animals at weeks 52 and 104
- Dose groups that were examined: all

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
External surface, All orifices, Cranial cavity, External surface of the brain and spinal cord, Nasal cavity and paranasal sinuses, Thoracic, abdominal, and pelvic cavities and their viscera, Cervical tissues and organs, Carcass

HISTOPATHOLOGY: Yes
All of the preserved tissues from the control and high-dose animals were embedded in Paraplasm, sectioned, stained with hematoxylin and eosin, and examined microscopically. In addition, histological examinations were conducted on all unusual lesions observed in the low- and mid-dose animals, and on all tissues from animals which died or were sacrificed moribund prior to scheduled sacrifice.

Brain, Pituitary, Thoracic spinal cord, Eyes, Mandibular salivary glands, Thyroid, parathyroids, Trachea, Thymus, Esophagus, Heart, Aorta, Liver, Kidneys, Stomach, Nerve (tibial), Duodenum, jejunum, ileum, Colon, cecum, rectum, Mesenteric lymph node, Urinary bladder, Testes with epididymides, Prostate, Seminal vesicles, Ovaries, Uterus, Femur (including joint), Lung, Spleen, Adrenals, Pancreas, Skin

Statistics:

Bartlett's test for homogeneity of variances (Bartlett, 1937) was performed, followed by a one-way classification analysis of variance (ANOVA) (Snedecor and Cochran, 1967) if the variances proved to be homogeneous. If the variances proved to be heterogeneous a log10 transformation was performed, which was followed by Bartlett's test. If the log10 transformation was ineffective in removing variance heterogeneity, a loge transformation of the original data was performed which was followed by Bartlett's test. If homogeneity could not be achieved by transformation, ANOVA of the non-transformed data was completed. If ANOVA of homogeneous data was significant, Scheffe's (1953) multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA of heterogeneous data was significant, Games and Howell's (1976) multiple pairwise comparison procedure was used to compare the group mean values. The incidence of cataracts (unilateral and bilateral, combined) noted in males at Week 104 was analyzed by Cochran-Armitage test for linear trend of proportions (Armitage, 195b; Cochran, 1954). Fishers "Exact" lest (Fisher, 1950) was then used for control vs. compound-treated group comparisons.
The incidence of mononuclear cell leukemia (all animals) was analyzed by Fishers "Exact" test (Fisher, 1950) with Bonferroni adjustment (Miller, 1966) for multiple group comparisons. In addition to the above data, cumulative survival through termination was analyzed by the National Cancer Institute Package (Thomas, Breslow, and Gart, 1977).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Beginning at Week 2 and continuing throughout the study, high-dose animals were observed to have compound-colored or siIver-colored feces. This appeared to be the only treatment-related clinical observation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Analysis of survival through Week 130 revealed statistically significantly lower survival for the Group 2 females compared to control females. No other significant differences between groups and no significant treatment-related nor dose-related trends were noted.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At study initiation, mean body weights were comparable among all male and female groups. Analysis of growth rates through Week 26 showed significantly lower mean growth rates for mid- and high-dose females compared to control females. At Week 26 mean body weights were significantly lower for high-dose males and females compared to the respective control groups. At Week 42, mean body weights were decreased for all male groups compared to weights at Weeks 38 and 46. This weight loss was related to the SDA infection and was not noted in the female groups. Through Week 50, mean body weights were lower for the compound-treated groups compared to control values; however, statistical analyses revealed no significant differences in growth rates or mean body weights at Week 50. Mean body weights continued to be lower for the compound-treated groups compared to control weights throughout the study until Weeks 126 and 130 when the mean weights were more comparable between groups, especially the control and high-dose groups.Statistical analysis of mean body weight data from Week 26 demonstrated a significant decrease in Group 4 animals when compared to controls. Although statistical significance was attained for this parameter, it only accounted for a 3.9% decrease in females and a 4.1% decrease in males, therefore, biological significance of this finding is questionable. The validity of this finding is further reduced by comparison of mean body weight data from Week 22 and Week 30 where only a 2.4% and 2.9% decrease in body weight was seen in females, respectively, and a 3.3% and 4.6% in males, respectively, when high-dose and controls were compared.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Analyses of mean total food consumption through Weeks 26 and 50 revealed significantly higher values for the high-dose males and females compared to the respective control values. Due to the SDA viral infection, mean food consumption values were higher for all groups at Week 42 than at Weeks 38 and 46. During the second year of study, mean food consumption values gradually became more comparable between groups, especially the control and high-dose groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross ophthalmoscopic examination was performed at Week 52 and Week 104. Eye lesions at Week 52 were considered incidental and not related to compound administration. The incidence of cataracts in males as detected by gross ophthalmic exam at Week 104 appeared to be dose-related. However, histopathological examination revealed a high incidence of subcapsular/cortical mineralization and degeneration of lens fibers in all groups. The apparently high incidence rate of cataract formation may be regarded as a sequalla to sialodacryoadenitis (SDA) virus contracted by the rats, and not dose related.

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:

effects observed, treatment-related

Description (incidence and severity):

Findings that were considered attributable to compound administration consisted of a silver-colored (compound-colored) tint on the walls of the stomach and/or cecum, and silver-colored fecal material in the cecum and/or intestines in several mid- and high-dose animals. All remaining gross observations were considered incidental and not attributable to the test material.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A large increase in the frequency of adrenal medullary hyperplasia in the male high-dose group was noted (13/25) when unscheduled deaths were examined compared to controls (8/33). Smaller increases were
present in male low- (13/34) and mid-dose (12/34) rats from the unscheduled deaths. The increase was not noted in the females. Examination of these findings in relation to neoplasia noted in these adrenals did not show any increase in adrenal neoplasia in treated animals, nor was there a large increase when animals with either medullary hyperplasia or neoplasia (eliminating duplications) were examined. When terminal sacrifice animals were examined, the differences between controls (7/17 males) and high-dose (13/25 males) were negligible. Other non-neoplastic changes noted were lesions expected in aged Fischer 344 rats and were not considered compound related.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A high incidence of mononuclear cell leukemia was noted in all groups. As cited in the literature and Hazleton Control Oata, this neoplastic process is expected to be high in Fischer 344 rats (7-18% in untreated control males, 10-27% in untreated control females, 104 wks).

Key result

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Effect level:

2 574 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Effect level:

3 291 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Conclusions:

Based on these findings, administration of Titanium Dioxide coated mica in the diet of rats at concentrations of 10000, 20000 and 50000 ppm did not significantly alter survival nor demonstrate toxic effects. Additionally, no indication of oncogenic potential was elicited by the test material at the designated concentration levels.

Executive summary:

The purpose of this study was to provide evidence of possible toxicity of Titanium Dioxide Coated Mica (contained 2-7% Diiron titanium pentraoxide) and evaluate the oncogenic potential using a standardized and accepted test regimen by administration in the diet of Fischer 344 rats (50 m and f per dose group) at levels of 0, 10000, 20000 and 50000 ppm for 104 weeks. No dose-related differences in survival were noted. Compound-colored or silver-colored feces were noted for the high-dose animals from Week 2 throughout the remainder of the study. Growth rates through Week 26 were significantly lower for the mid- and high-dose females compared to controls. Mean absolute body weights at Week 26 were significantly lower for the high-dose males and females compared to controls. Although not statistically significant, mean body weights for the compound-treated groups were lower than controls throughout the remainder of the study until Week 126 when the weights were more comparable among groups. Mean total food consumption through Weeks 26 and 50 was significantly greater for the high-dose males and females, respectively, compared to controls.

Mean food consumption became more comparable among groups during the second year of the study. At Week 104, ophthalmoscopic examinations revealed a dose-related increased incidence of cataracts in the male rats. Except for one male, all cataracts were unilateral. Statistical analysis revealed a significant linear trend in the incidence of cataracts and a significantly higher incidence of cataracts in the high-dose compared to controls. However, histopathological examination did not support this trend as there was no correlation between dose level and cataract incidence. Compound-colored tint on the walls of the stomach and/or cecum, and silver-colored fecal material in the cecum and/or intestines were observed grossly at necropsy in the mid- and high-dose rats. In the animals that died or were sacrificed in extremis, an increased incidence of adrenal medullary hyperplasia was noted microscopically for the high-dose males compared to controls. This increase was not observed for the females, nor was it observed for the males or females that were sacrificed at Week 130. The incidence of mononuclear cell leukemia was unusually high for animals in all groups compared to historical control data. This increased incidence is thought to be due to the length of the study (130 weeks) and the tendency of the Fischer 344 strain to develop this lesion as they age. Based on these findings, administration of Titanium Dioxide coated mica in the diet of rats at concentrations of 10000, 20000 and 50000 ppm did not significantly alter survival nor demonstrate toxic effects. Additionally, no indication of oncogenic potential was elicited by the test material at the designated concentration levels.

Reference 5

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 114.2 to 159.9 g for males; 91.1 to 117.8 g for females
- Housing: individually housed in elevated wire mesh cages
- Diet: ad libitum, Purina Lab Chow
- Water: ad libitum, tap water
- Acclimation period: two weeks

DETAILS OF FOOD AND WATER QUALITY:
The basal diet and water were periodically analyzed for concentrations of specific heavy metals, antobiotics, aflatoxin, pesticides, and nitrosamines.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
Approximately every four weeks, equal amounts of each of the two test pigments were blended together in a 1:1 ratio and stored at room temperature until use. One day prior to scheduled diet preparation, the basal diet (Purina Rodent Laboratory Chow®) was premixed with a sufficient amount of corn oil such that the concentration of corn oil was 1% of the diet. On the scheduled mixing day, the appropriate amount of the blended test materials required for each dietary level was incorporated into the corn oil/basal feed mixture on a weight per weight basis. Fresh diets were prepared and administered weekly. Control rats received the basal diet/1% corn oil blend, only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical results of pretest samples showed the diets to be homogeneously mixed and stable under use conditions. In general, the concentration analyses of test diets revealed acceptable levels of the test materials in the diets for the purpose of this study.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

administered continuously in diet

Dose / conc.:

10 000 ppm

Remarks:

472 mgkg bw/day (males) / 602 mg/kg bw/day (females)

Dose / conc.:

20 000 ppm

Remarks:

941 mgkg bw/day (males) / 1264 mg/kg bw/day (females)

Dose / conc.:

50 000 ppm

Remarks:

2412 mgkg bw/day (males) 3217 mg/kg bw/day (females)

No. of animals per sex per dose:

10 (satellite groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high-dose level was selected because it represented the maximum level required by various regulatory agencies and was recommended in OECD Guidelines and this level would not compromise the animals from a nutritional standpoint.

Positive control:

none

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for weeks 1-13, monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Time schedule for examinations: for weeks 1-13; monthly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at beginning of study and an additional examination was performed on all study animals at week 52
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- at 26 and 52 weeks
- number of animals: 10/sex/group (satellite groups) at 26 and 52 weeks
- parameters: hemoglobin, hematocrit, erythrocyte count, platelet count, leukocyte count, differential
Blood will be collected by orbital sinus bleeding (nonanesthetized rats) and urine will be collected overnight while animals are housed in individual metabolism cages. Animals will be fasted overnight prior to collection of samples.

CLINICAL CHEMISTRY: Yes
- at 26 and 52 weeks
- number of animals: 10/sex/group (satellite groups) at 26 and 52 weeks
- parameters: urea nitrogen, GPT, GOT, alkaline phosphatase, total protein, albumin, A/G ratio, glucose, total bilirubin
Blood will be collected by orbital sinus bleeding (nonanesthetized rats) and urine will be collected overnight while animals are housed in individual metabolism cages. Animals will be fasted overnight prior to collection of samples.

URINALYSIS: Yes
appearance, specific gravity, protein, pH, ketones, glucose, bilirubin, microscopic examination of sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
External surface, All orifices, Cranial cavity, External surface of the brain and spinal cord, Nasal cavity and paranasal sinuses, Thoracic, abdominal, and pelvic cavities and their viscera, Cervical tissues and organs, Carcass

HISTOPATHOLOGY: Yes
Brain, Pituitary, Thoracic spinal cord, Eyes, Mandibular salivary glands, Thyroid, parathyroids, Trachea, Thymus, Esophagus, Heart, Aorta, Liver, Kidneys, Stomach, Nerve (tibial), Duodenum, jejunum, ileum, Colon, cecum, rectum, Mesenteric lymph node, Urinary bladder, Testes with epididymides, Prostate, Seminal vesicles, Ovaries, Uterus, Femur (including joint), Lung, Spleen, Adrenals, Pancreas, Skin

Organ Weights:
For each sacrificed animal the following organs will be weighed following careful dissection and triircning to remove fat and other contiguous tissue in a uniform manner:
heart, kidneys, liver, testes with epididymides,spleen, ovaries, adrenaIs, pituitary, thyroid, brain

Statistics:

Growth rates, total food consumption, clinical laboratory, and organ weight data of control groups (Groups 1 and 5) will be compared statistically to the data of the treated groups of the same sex by Bartlett's test for homogeneity of variance (Bartlett, 1937). This analysis will be followed by a one-way classification analysis of variance (ANOVA) (Snedecor and Cochran, 1967) if the variances prove to be homogeneous. If the variances prove to be heterogeneous a log10 transformation will be performed, which is followed by Bartlett's test.
If log10 transformation is ineffective in removing variance heterogeneity, a log10 transformation of the original data is performed which is followed by Bartlett's test. If homogeneity cannot be achieved by transformation, ANOVA of nontransformed data will be completed. If ANOVA of homogeneous data is significant, Scheffe's (1953) multiple pairwise comparison procedure will be used to compare the group mean values. If ANOVA of heterogeneous data is significant, Games and Howell (1976) multiple pairwise comparison procedure will be used to compare the group mean values. The meanbody weight values at initiation and at end of the first year of the control groups will also be compared to those of the treated groups by a multiple pairwise comparison procedure (Games and Howell, 1976). Survival through Week 130 will be analyzed by a life table technique (Sachs, 1959). The incidence of tumors will be analyzed by Fisher's (1954) exact test for proportions and the time to tumor data (if appropriate) will be analyzed by Cox's (1958) exact test for trends in proportions to determine whether a linear trend exists in the tumor incidence rates which would be dose-related. The time to tumor data will be analyzed by the methods of Cox (1972) and Breslow (1970); and for each method Tarone's (1975) test for trend will be used to test for a linear dose-response relationship. T

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All high-dose animals were observed to have compound-colored or silver - colored feces.
During Week 42, clinical signs indicative of a Sialodacryoadenitis viral infection (i.e., periorbital edema, keratoconjunctivitis, and weight loss) were noted for animals at all dose levels. Other clinical signs were considered incidental and not related to compound administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One mid-dose female was found dead shortly following orbital sinus bleeding during Week 27. All remaining'animals survived until scheduled sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights at initiation and Weeks 26 and 50, as well as the growth rates, generally were comparable between the control and the respective compound-treated groups of both sexes, and no dose-related trends were apparent. The apparent viral infection detected in the animals during Week 42 was reflected in slightly decreased mean body weights at Week 42; however, mean weights returned to previous ranges by Week 46.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of mean total food consumption revealed significantly greater food consumption values for the low-dose males when compared to the control males at both the twenty-six- and fifty-week intervals. The mean food consumption values at Week 42 showed slight increases, apparently in response to the viral infection detected at this time. Mean food consumption-values returned to previous ranges at Week 46.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of the hematology data revealed significantly decreased platelet counts for the high-dose females at Week 27 and for the low- and high-dose females at Week 53, however, no biological significance was attributed to these findings. At Week 27, the mean hemoglobin value of the mid-dose males was significantly higher than the control value, and the mean leukocyte count values of the low-dose and mid-dose females were significantly lower than the control values.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of the clinical chemistry data revealed significantly higher mean glucose values in the male compound-treated groups at Week 53. However, all mean values were within the range of historical values for this strain of rat at this laboratory. The remaining significantly altered findings (significantly lower mean albumin/globulin ratios for the low- and high-dose females at Week 27, and significantly lower mean total bilirubin values for the high-dose females at both Weeks 27 and 53) were not considered to be compound-related. All remaining blood chemistry parameters were comparable between the control and compound-treated groups, and no dose-related trends were noted.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Results of the urinalyses were unremarkable.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of the organ weight data revealed generally lower mean absolute and relative thyroid weights for the mid- and high-dose groups. Significantly lower mean absolute and relative thyroid weights were noted for the high-dose (Group 8) males and the mid-dose females when compared to the respective control values. Mean absolute heart weights were noted to be lower in the low-, mid-, and high-dose groups of both sexes. This trend appeared to be dose related for the male rats. Dose-related increases in mean absolute and relative spleen weights also were noted for the male rats. However, no significant differences from controls were detected for any of these values and histopathological examination of these tissues did not reveal possible causes for these organ weight changes. Single occurrences of significant differences in mean absolute and/or relative liver, brain, and pituitary weights were noted between the compound-treated and control animals; however, no dose-related trends involving these organs were apparent.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Observations for the sacrificed animals were incidental and no trends attributed to compound administration were noted. In addition, no unusual lesions were noted for the mid-dose female which was found dead during Week 27.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No histomorphologic lesions were detected which could be attributed to the test materials. A variety of spontaneous disease lesions and incidental findings were noted with similar frequency in control and compound-treated rats. These included perivascular/ peribronchial lymphoid hyperplasia in the lung, nonsuppurative pericholangitis and biliary hyperplasia in the liver, and unilateral ocular and Harderian gland lesions commonly associated with orbital sinus bleeding. In conclusion, no treatment-related histopathologic changes were induced following fifty-two weeks of compound administration.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Effect level:

2 412 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOAEL

Effect level:

3 217 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Conclusions:

Based on these findings, administration of Titanium Dioxide coated mica in the diet of rats at concentrations of 10000, 20000 and 50000 ppm for 52 weeks, did not significantly alter survival nor demonstrate toxic effects.

Executive summary:

The purpose of this study was to provide evidence of the nontoxic nature of Titanium Dioxide coated mica in rats when administered continuously in the diet at levels of 10000, 20000 and 50000 ppm for a period of 52 weeks. Criteria used to evaluate compound effects in the animals were survival, clinical signs, body weights and food consumption, clinical pathology (hematology, clinical chemistry and urinalysis), ophthalmologic findings, organ weight data, and gross and microscopic pathology. One mid-dose female was found dead during Week 27 shortly following orbital sinus bleeding. All remaining animals survived until scheduled sacrifice. All high-dose rats were observed to have compounder silver-colored feces throughout the study. Other clinical signs were considered incidental and common to rats of this age and strain except those indicative of a Sialodacryoadenitis viral infection noted during Week 42 for animals in all groups.

Mean body weights at initiation and Weeks 26 and 50, as well as the growth rates during these same intervals were comparable among all male groups and among all female groups. Analysis of total food consumption revealed significantly greater values for the low-dose males at both the twenty-six and fifty-week intervals; however, this increase in consumption was not considered to be compound related. Significantly decreased platelet counts were noted for the high-dose females at Week 27 and for the low- and high-dose females at Week 53; however, no dose response was apparent. The hemoglobin value of the mid-dose males was significantly higher and the leukocyte count values of the low- and mid-dose females were significantly lower than the respective control group values at Week 27. These hematology changes were considered incidental in nature. Significantly higher glucose values were noted for the compound-treated male groups at Week 53. Other significant changes in the clinical chemistry data were noted infrequently and no dose-related trends were observed. Results of the urinalyses were unremarkable. No compound-related ophthalmologic findings were observed at Week 52. Gross necropsy findings generally were comparable in nature and frequency between the control and compound-treated groups, and no trends attributable to compound administration were noted. Comparisons of the organ weight data revealed dose-related decreases in absolute heart weights and increases in absolute and relative spleen weights for the compound-treated male rats; however, no statistically significant differences from control values were detected. Lower than control absolute and relative thyroid weights were noted for the mid- and high-dose rats, being significantly lower for the high-dose males and the mid-dose females. Histopathological examination did not reveal the cause(s) for these organ weight changes. A variety of spontaneous disease lesions and incidental histopathological findings were noted with similar frequency in control and compound-treated rats. However, no histomorphologic lesions were detected which could be attributed to the test materials.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

550 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Concentrations tested exceeded the maximum tolerated dose (MTD)

Qualifier:

no guideline followed

Principles of method if other than guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Remarks:

CDF (F344)/CrlBR

Sex:

female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.44 µm

Geometric standard deviation (GSD):

1.71

Details on inhalation exposure:

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

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

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

50 mg/m³ air (nominal)

Dose / conc.:

250 mg/m³ air (nominal)

No. of animals per sex per dose:

65 (only females)

Control animals:

yes

Positive control:

none

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes

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

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

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

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

Sacrifice and pathology:

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

Statistics:

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

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEC

Effect level:

10 mg/m³ air (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Lung burden, BALF, inflammation

Key result

Critical effects observed:

not specified

Conclusions:

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

Executive summary:

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

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Concentrations tested exceeded the maximum tolerated dose (MTD)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male rats were exposed to TiO2 particles 6 hours/day, 5 days/week for 4 weeks at concentrations of 5, 50, and 250 mg/m³ and evaluated at selected intervals through 6 months postexposure (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.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 7-8 weeks

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.4 - <= 1.9 µm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Overall mean concentrations were very close to the nominal concentrations (5,4 ± 6, 51.9 ± 16, 252.2 ± 86 mg/m3, respectively)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

5 mg/m³ air (nominal)

Dose / conc.:

50 mg/m³ air (nominal)

Dose / conc.:

250 mg/m³ air (nominal)

No. of animals per sex per dose:

not specified

Control animals:

yes, sham-exposed

Details on study design:

The methods utilized for aerosol generation of titanium dioxide particles have previously been reported (Warheit et al., 1991, Development of a short-term inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107, 350 - 368)

Positive control:

none

Observations and examinations performed and frequency:

- Following exposures, the lungs of exposed animals and aged-matched sham controls were subsequently evaluated by bronchoalveolar lavage fluid analysis, BrdU cell labeling, lung clearance analysis, in vitro macrophage function, and histopathology at hr, 1 week, and 1, 3, and 6 months postexposure
- Bronchoalveolar lavage procedures and biochemical assays on lavaged fluids were conducted
- Phagocytosis: For the phagocytic assay for TiO2-exposed macrophages, a suspension of carbonyl iron particles was incubated with normal rat serum for 1 hour at 40°C and sonicatecl to reduce aggregations of particles. A final concentration of 1.75 mg/mL was added to monolayers.

- Chemotaxis:
Alveolar macrophages were collected from TiO2- or sham-expmed rats by lavage as described above. The chemotaxis assay was carried out as described previously using three concentrations (1, 5, and 10%) of zymosan-activated sera as the chemotactic stimulus

- Pulmonary cell proliferation studies:
Pulmonary cell proliferation experiments were conducted according to methods previously described (Warheit et al., 1992 Pulmonary cellular effects in rats following aerosol exposures to ultrafine Kevlar aramid fibrils: Evidence for biodegradability of inhaled fibrils. Toxicol. Appl. Pharmacol. 116, 225-239.)

Sacrifice and pathology:

GROSS PATHOLOGY: No data

HISTOPATHOLOGY: Yes
The lungs of rats exposed to TiO2 for 4 weeks were prepared for light microscopy by airway infusion. Analyses of lung and lymph node burdens were conducted by digesting tissue specimens in hydrofluoric acid and analyzing for titanium using the method of inductively coupled plasma (ICP-AES) spectroscopy.

Statistics:

Statistics were carried out using a two-tailed Student t-test

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

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:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- lesions in the respiratory system varied with exposure concentration and duration of post-exposure recovery

- 5 mg/m3: concentration produced only minimal effects. Particle-laden macrophages and a minimal diffuse increase in alveolar macrophages (histiocytosis) were evident at 0 days of recovery. The histiocytosis was no longer evident at 1 week postexposure. Individual particle-laden macrophages could be found in very low numbers within air spaces and lymphoid tissue throughout the entire 6-month postexposure period.
- 50 and 250 mg/m³: concentrations produced a wide spectrum of effects within the lung. Free granular pigment of TiO2 was present on the mucosal surfaces of bronchioles and bronchi at 0 days of recovery. Particle-laden macrophages, found individually, were numerous throughout the air spaces at this same time period. Beginning at 1 week post-exposure and persisting thereafter, many dense aggregates of particle-laden macrophages were within alveoli and alveolar ducts. Cellular hypertrophy and hyperplasia were evident at alveolar wall and duct bifurcations that were adjacent to macrophage aggregates. Mucosal hypertrophy and hyperplasia were also observed within the bronchi and bronchioles.
- number of pigment-laden macrophages found individually and in aggregates was much greater in animals exposed to the highest concentration, and occupied a greater portion of the lung.
- severity of cellular hypertrophy and hyperplasia at alveoli and alveolar duct bifurcations was significantly greater in animals exposed to the highest concentration of TiO2 particles.
- severity and character of the lesions changed with time. Free granular pigment was no longer apparent at 1 week post-exposure in any concentration group. Particle-laden macrophages, found individually, decreased in number with time, but were evident in small numbers within the pulmonary air spaces throughout the entire 6-month recovery period. The numbers and size of the dense aggregates of macrophages within alveoli and alveolar ducts, increased during the first month post-exposure but did not expand throughout the remaining 5-month post-exposure period. Minimal mucosal hypertrophy and hyperplasia in bronchi and bronchioles were evident at 1 month postexposure in the two highest concentration groups. Focal cellular hypertrophy and hyperplasia were associated with aggregates of pigmented macrophages, and were evident at alveoli and alveolar duct bifurcations for the entire 6-month postexposure period in the two higher concentration groups. Pigmented macrophages could also be observed within pulmonary lymphoid tissue throughout this time period.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BALF data:
- sustained pulmonary inflammatory responses in animals exposed to 250 mg/m3 and small but sustained inflammatory responses at 50 mg/m3
- elevated alveolar macrophages and neutrophils at high dose
- LDH and protein values in BALF fluid elevated only in high-dose group

PULMONARY CELL PROLIFERATION:
- increase on pulmonary cell proliferation indices measured on lung parenchymal and terminal bronchiolar surfaces at high dose level (effects sustained through 3-6 month post-exposure)

LUNG CLEARANCE:
- clearance of particles was evident during the time interval of 1 week through 1 month postexposure but appeared to be substantially diminished thereafter
- the deposited TiO2 was found to clear with half-times of approximately 68, 110, and 330 days for the 5, 50, and 250 mg/m3 groups, respectively

PARTICLE TRANSLOCATION TO TRACHEOBRONCHIAL LYMPH NODES:
- lymph node burdens of rats exposed to 250 mg/m3 TiO2 demonstrated that substantial amounts of particles had translocated to tracheobronchial lymph nodes, particularly during the 1- to 3- and 3-to 6-month intervals
- no appreciable translocation of TiO2 to lymph nodes ot mid and low dose group

CHEMOTAXIS:
- alveola macrophages exposed to 250 mg/m3 were impaired in their phagocytic responses relative to controls and this effect was sustained through 1 month postexposure

Key result

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Key result

Critical effects observed:

not specified

Conclusions:

A 4-week inhalation exposure high concentrations produced persistent pulmonary effects, many lasting throughout a 6-month post-exposure period.

Executive summary:

Male rats were exposed to TiO2 particles 6 hr/day, 5 days/week, for 4 weeks at aerosol concentrations of 5, 50 and 250 mg/m3 and evaluated at selected intervals through 6 months post-exposure. Indices of pulmonary inflammation as well as alveolar macrophage clearance functions, cell proliferation, and histopathology endpoints were measured at several post-exposure time periods through 6 months.

4-week inhalation exposures to high dust concentrations produced persistent pulmonary effects, many lasting throughout a 6-month post-exposure period. These included pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance mechanisms, deficits in macrophage function, and morphological evidence of macrophage aggregation. However,Titanium dioxide showed adverse pulmonary effects only at concentrations above the maximum tolerated dose (MTD).

Reference 3

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Concentrations tested exceeded the maximum tolerated dose (MTD)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 100 male and 100 female 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, gross pathology, and histopathology.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Diet: ad libitum, Purina Rodent Chow
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.5 - <= 1.7 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Atmospheres of TiO2 were generated by metering the dust into an apparatus containing a vertical elutriator (an inverted Plexiglas U-shaped tube) connected in series to a settling chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: chamber concentrations were determined gravimetrically.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

24 months

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

50 mg/m³ air (nominal)

Dose / conc.:

250 mg/m³ air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Not specified

CLINICAL CHEMISTRY: Not specified

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Not specified

LUNG BURDEN: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Dunnett’s test

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 specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

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):

The lung weights at 10 mg/m3 were comparable to those of the control group., but at 50 mg/m3, an increase in lung weights was statistically significant. The lung weights at 250 mg/m3 were more than twice the weight of control lungs.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In lungs of rats exposed to 10 mg/m3, white foci were scattered sparsely throughout the pleural surface while a more dense accumulation of white faci was ssen in the periphery of the lobes. These faci were increased significantly in number and size at 50 mg/m3. At 250 mg/m3, the lungs were voluminous, showed a white "paint-brushed" appearance, contained rough pleural surfaces, and failed to collapse.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A significant increase in the incidence of rhinitis, tracheitis, and pneumonia was found in all groups of exposed rats. The severity of lesions was dose-dependent and was minimal at 10 mg/m3. The anterior nasal cavity often revealed acute and chronic inflammation with squamous cell metaplasia while the incidence of inflammation in the posterior nasal cavity was comparable to control groups. The epithelium of nasal cavity and trachea was covered with mucus containing dust-laden macrophages (dust cells) and extracellular dust particulates, but the epithelium showed no tissue reaction.
In the lungs of rats exposed to 10 mg/m3, the inhaled particulates were mostly phagocytosed by intraalveolar macrophages (dust cells) in a few alveolar ducts and adjoining the alveoli in an acinus. The alveoli showed slight hyperplasia of the cuboidal
lining cells.
At 50 mg/m3, dust cell aggregates were increased and were sharply confined to alveolar ducts and adjacent alveoli. The alveolar air spaces were obliterated by dust cells, foamy macrophages and hyperplastic lining cells. Some alveoli were
filled with PAS-positive granular or amorphous material, and microscopic findings were compatible with alveolar proteinosis.
At 250 mg/m3, dust cells and foamy macrophages increased markedly in numbers and were distributed throughout the alveoli, with the heaviest concentration in the alveolar duct region. Alveolar cell hyperplasia, cholesterol granulomas, alveolar proteinosis, bronchiolarization, and focal pleurisy were markedly pronounced. A significant collagen fiber deposition occurred often within these cholesterol granulomas, while the alveolar walls enclosing dust cells revealed only minute, linear, collagenized fibrosis. The alveolar cell hyperplasia and bronchiolarization were so marked that the areas were adenoma-lime in appearance.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Bronchioloalveolar adenomas were found in 12 of 77 males and 3 of 74 females, while the incidence of squamous cell carcinomas was sex dominant, 1 in males and 13 in females. Of 13 females, 3 had both types of lung tumors. The bronchioloalveolar adenomas occurred in the alveoli that chowed marked hyperplasia of the cuboidal or columnar alveolar lining cells. There was no tumor metastasis to regional lymph nodes or other organs.

Key result

Dose descriptor:

NOAEC

Effect level:

10 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

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

Executive summary:

Male and female CD-rats each were exposed to titanium dioxide (10, 50, and 250 mg/m3). The test item was administerated 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, gross pathology, and histopathology. The pulmonary lesions with massive dust accumulation appeared to be the result of an overwhelmed lung clearance mechanism at 250 mg/m3 exposure. Bronchioloalveolar adenomas and cystic keratinizing squamous cell carcinomas occured at 250 mg/m3, while no compound-related lung tumors were found in mid or low dose. In addition to excessive dust loading at 250 mg/m3, the lung tumors were different from common human lung cancers in terms of tumor type, anatomic location, tumorigenesis, and were devoid of tumor metastasis. Therefore, the biological relevance of these lung tumors for man is negligible. 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.

Reference 4

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

only one concentration tested, TiO2 was used as negative control in this study

Principles of method if other than guideline:

A chronic inhalation study of a test toner was conducted by exposure of groups of F-344 rats for 6 hr/day, 5 days/week for 24 months. The target test aerosol exposure concentrations were 0, 1.0 (low), 4.0 (medium), and 16.0 (high) mg/m3. Titanium dioxide (5 mg/m3) and crystalline silicon dioxide (1 mg/m3), used as negative and positive controls for fibrogenicity, were also evaluated.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 4 weeks
- Housing: individually housed in metal wire mesh cages
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 40-60
- Air flow: 3.8 m3/min
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.1 µm

Geometric standard deviation (GSD):

1.6

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
- Temperature, humidity, pressure in air chamber: 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

Duration of treatment / exposure:

24 months

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

analytical concentration: 3.87 ± 0.28

No. of animals per sex per dose:

144

Control animals:

yes, concurrent no treatment

Positive control:

yes, SiO2 (1 mg/m3)

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

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes

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

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
The method of Henderson et al. (1987) was used with minor modifications. Following preparation of the lungs, they were lavaged with 2 X 4 mL of saline without massage. The cell concentration was determined using a counting chamber. The lavagate was centrifuged at 160g and the supernatant used for the determination of the biochemical parameters [lactic dehydrogenase (LDH), /3-glucuronidase, total protein]. Cytoslides were prepared for differential cell count.

LUNG BURDEN: Yes

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.

Statistics:

Parametric data were examined by analysis of variance (ANOVA) followed by Dunnett test to compare various treatment groups with controls. Survival data were analyzed by the Kaplan-Meier method using the lifetest program of SAS. For necropsy and tumor occurrence data, simple tests for homogeneity of contingency tables (using X2 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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- 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.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The incidence of primary lung tumors in the basic study was comparable among the three toner exposure groups and the TiO2 and the air-only controls, as well as consistent with historical background values.
In contrast to the above, an increased incidence of lung tumors was observed in the SiO2 exposed group. A total of 20 primary lung tumors were found in 19 animals. Two tumors, an adenoma and an adenocarcinoma, were observed in separate areas of the lung of one male silica-exposed rat.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

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.

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

Conclusions:

The available results support the view that the adverse effects noted are associated with chronic lung overloading.

Executive summary:

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.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Diiron titanium pentaoxide as a single component has not been tested for systemic toxicity after repeated dosing. However, diiron titanium pentaoxide is a constituent of several pigments and it is therefore considered toxicologically more relevant to administer it as a constituent of a typical pigment mixture with a relatively high content. Therefore, a WoE approach was used to investigate the repeated-dose toxicity of diiron titanium pentaoxide using data from pigments which contain diiron titanium pentaoxide and data from the oxides titanium dioxide (TiO2) and iron oxide (Fe2O3) since Diiron titanium pentaoxide is a mixed oxide of titanium dioxide and iron oxide.

Oral

A subchronic feeding toxicity study equal to OECD TG 408 was performed to investigate the toxicity of different mica-based pearlescent pigments. Mica is the core raw material of all substances tested. The pigments tested have been composed of mica, TiO2, Fe2O3 and diiron titanium pentaoxide.

Groups of 40 rats (20 males and 20 females) received the pigments (one composition included 13.5% diiron titanium pentaoxide) incorporated into feed pellets at the highest internationally recommended concentration of 50000 ppm, for the males and females, respectively. One placebo group (20 males and 20 females) received feed pellets containing mica only at 50000 ppm. In addition, a control group consisting of 80 rats (40 males and 40 females) were fed exclusively with standard commercial feed pellets. Treatment was for 3 months followed by a 2-months recovery period. Hematology, clinical chemistry and urinalysis were performed on half of the animals in each group, 4 and 12 weeks after start of the study and once during the follow-up phase (20 weeks after start of the study. Half of the animals were killed at the end of the 2-months recovery phase. All animals were subjected to gross pathological and histopathological examinations.

No treatment related mortalities occurred during the study. A slight increase of body weights was observed in females receiving the one pigment compared to the feed control group 1. Food intake was temporarily or permanently increased in males and females of all groups treated with mica or mica-based pigments due to the 5% mineralic content in the diet. No treatment-related effects were observed in haematology or clinical chemistry. Gross pathology revealed a discoloration of the gut content in the animals of the treatment groups which was not observed in the recovery animals and which is not considered to be an adverse effect. All histopathological findings encountered were considered to have arisen spontaneously. In conclusion, no adverse effects were observed in rats treated with mica pigments at 50000 ppm.

Based on the concentration of Diiron titanium pentaoxide in this pigment (13.5%) and the food consumption during the subchronic toxicity study the mean daily intake of Diiron titanium pentaoxide can be calculated as 534 mg/kg bw/day and 603 mg/kg bw/day for male and female rats, respectively. As these mean daily intakes of diiron titanium pentaoxide by rats over a period of 90 days did not induce any adverse effects, a NOAEL of 550 mg/kg bw/day can be deduced for Diiron titanium pentaoxide on the basis of this study.

In a further study the possible toxicity of Titanium Dioxide Coated Mica (contained 2-7% Diiron titanium pentraoxide) and its oncogenic potential was evaluated using a standardized and accepted test regimen by administration in the diet of Fischer 344 rats (50 m and f per dose group) at levels of 0, 10000, 20000 and 50000 ppm for 104 weeks. No dose-related differences in survival were noted. Compound-colored or silver-colored feces were noted for the high-dose animals from Week 2 throughout the remainder of the study. Growth rates through Week 26 were significantly lower for the mid- and high-dose females compared to controls. Mean absolute body weights at Week 26 were significantly lower for the high-dose males and females compared to controls. Although not statistically significant, mean body weights for the compound-treated groups were lower than controls throughout the remainder of the study until Week 126 when the weights were more comparable among groups. Mean total food consumption through Weeks 26 and 50 was significantly greater for the high-dose males and females, respectively, compared to controls.

Mean food consumption became more comparable among groups during the second year of the study. At Week 104, ophthalmoscopic examinations revealed a dose-related increased incidence of cataracts in the male rats. Except for one male, all cataracts were unilateral. Statistical analysis revealed a significant linear trend in the incidence of cataracts and a significantly higher incidence of cataracts in the high-dose compared to controls. However, histopathological examination did not support this trend as there was no correlation between dose level and cataract incidence. Compound-colored tint on the walls of the stomach and/or cecum, and silver-colored fecal material in the cecum and/or intestines were observed grossly at necropsy in the mid- and high-dose rats. In the animals that died or were sacrificed in extremis, an increased incidence of adrenal medullary hyperplasia was noted microscopically for the high-dose males compared to controls. This increase was not observed for the females, nor was it observed for the males or females that were sacrificed at Week 130. The incidence of mononuclear cell leukemia was unusually high for animals in all groups compared to historical control data. This increased incidence is thought to be due to the length of the study (130 weeks) and the tendency of the Fischer 344 strain to develop this lesion as they age. Based on these findings, administration of Titanium Dioxide coated mica in the diet of rats at concentrations up to 50000 ppm, did not significantly alter survival nor demonstrate toxic effects. Additionally, no indication of oncogenic potential was elicited by the test material at the designated concentration levels.

From the same study reported above satellite groups were administered continuously Titanium Dioxide Coated Mica (contained 2-7% Diiron titanium pentraoxide) in the diet at levels of 10000, 20000 and 50000 ppm for a period of 52 weeks. Criteria used to evaluate compound effects in the animals were survival, clinical signs, body weights and food consumption, clinical pathology (hematology, clinical chemistry and urinalysis), ophthalmologic findings, organ weight data, and gross and microscopic pathology. One mid-dose female was found dead during Week 27 shortly following orbital sinus bleeding. All remaining animals survived until scheduled sacrifice. All high-dose rats were observed to have compounder silver-colored feces throughout the study. Other clinical signs were considered incidental and common to rats of this age and strain except those indicative of a Sialodacryoadenitis viral infection noted during Week 42 for animals in all groups.

Mean body weights at initiation and Weeks 26 and 50, as well as the growth rates during these same intervals were comparable among all male groups and among all female groups. Analysis of total food consumption revealed significantly greater values for the low-dose males at both the twenty-six and fifty-week intervals; however, this increase in consumption was not considered to be compound related. Significantly decreased platelet counts were noted for the high-dose females at Week 27 and for the low- and high-dose females at Week 53; however, no dose response was apparent. The hemoglobin value of the mid-dose males was significantly higher and the leukocyte count values of the low- and mid-dose females were significantly lower than the respective control group values at Week 27. These hematology changes were considered incidental in nature. Significantly higher glucose values were noted for the compound-treated male groups at Week 53. Other significant changes in the clinical chemistry data were noted infrequently and no dose-related trends were observed. Results of the urinalyses were unremarkable. No compound-related ophthalmologic findings were observed at Week 52. Gross necropsy findings generally were comparable in nature and frequency between the control and compound-treated groups, and no trends attributable to compound administration were noted. Comparisons of the organ weight data revealed dose-related decreases in absolute heart weights and increases in absolute and relative spleen weights for the compound-treated male rats; however, no statistically significant differences from control values were detected. Lower than control absolute and relative thyroid weights were noted for the mid- and high-dose rats, being significantly lower for the high-dose males and the mid-dose females. Histopathological examination did not reveal the cause(s) for these organ weight changes. A variety of spontaneous disease lesions and incidental histopathological findings were noted with similar frequency in control and compound-treated rats. However, no histomorphologic lesions were detected which could be attributed to the test materials.

Furthermore, a study according to OECD TG 407 was performed with the read-across substance TiO2. Male Sprague-Dawley rats were exposed by oral gavage for 29 days to 24000 mg/kg bw/day TiO2 particles, or the vehicle water. Under the conditions of this study, the NOEL was 24000 mg/kg bw/day for male rats, based on the lack of any adverse effects at this dose.

Inhalation

Various repeated-dose inhalation studies with Titanium dioxide are available. In these studies, titanium dioxide showed adverse pulmonary effects only at concentrations above the maximum tolerated dose (MTD):

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

In a further study male rats were exposed to TiO2 particles 6 hr/day, 5 days/week, for 4 weeks at aerosol concentrations of 5, 50 and 250 mg/m3 and evaluated at selected intervals through 6 months post-exposure. Indices of pulmonary inflammation as well as alveolar macrophage clearance functions, cell proliferation, and histopathology endpoints were measured at several post-exposure time periods through 6 months.

4-week inhalation exposures to high dust concentrations produced persistent pulmonary effects, many lasting throughout a 6-month post-exposure period. These included pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance mechanisms, deficits in macrophage function, and morphological evidence of macrophage aggregation. However,Titanium dioxide showed adverse pulmonary effects only at concentrations above the maximum tolerated dose (MTD).

In a further study, male and female CD-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, gross pathology, and histopathology. The pulmonary lesions with massive dust accumulation appeared to be the result of an overwhelmed lung clearance mechanism at 250 mg/m3 exposure. Bronchioloalveolar adenomas and cystic keratinizing squamous cell carcinomas occured at 250 mg/m3, while no compound-related lung tumors were found in mid or low dose. In addition to excessive dust loading at 250 mg/m3, the lung tumors were different from common human lung cancers in terms of tumor type, anatomic location, tumorigenesis, and were devoid of tumor metastasis. Therefore, the biological relevance of these lung tumors for man is negligible. 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.

A chronic inhalation study of a test toner was conducted by exposure of groups of F-344 rats for 6 hr/day, 5 days/week for 24 months. The target test aerosol exposure concentrations were 0, 1.0 (low), 4.0 (medium), and 16.0 (high) mg/m3. Titanium dioxide (5 mg/m3) and crystalline silicon dioxide (1 mg/m3), used as negative and positive controls for fibrogenicity, were also evaluated. 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.

In conclusion, in all repeated-dose inhalation studies titanium dioxide showed adverse pulmonary effects in rats, mice and hamsters after repeated inhalation studies only at concentrations above the maximum tolerated dose (MTD).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance is not considered to be classified for repeated dose toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.