Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2005-09-14 to 2006-01-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study. However, only one sex and one dose were used.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
limit test dose is 24 times higher than in guideline, only one sex was used
GLP compliance:
not specified
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Titanium dioxide R-101-A
- Physical state: white solid
- Analytical purity: 100% (see composition of test material)
- Composition of test material, percentage of components: 98.4% Titanium dioxide, 1.2% Aluminum hydroxide and 0.4% Triethanolamine
- Lot/batch No.: H-27201
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

- Name of test material (as cited in study report): Titanium dioxide R-101-B
- Physical state: white solid
- Analytical purity: 100% (see composition of test material)
- Composition of test material, percentage of components: 98.4% Titanium dioxide, 1.2% Aluminum hydroxide and 0.4% Triethanolamine
- Lot/batch No.: H-27203
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

No further details are given.

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: between 6 and 8 weeks of age
- Weight at study initiation: 211 to 214 g
- Housing: All animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Each cage rack contained only animals of one sex.
- Diet: All rats were fed PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum.
- Water: ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in NANOpure water and the dosing suspensions were prepared daily.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to address homogeneity/concentration verification and stability were not done during this study.
Duration of treatment / exposure:
The rats were dosed by intragastric intubation for 29 consecutive days.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
24,000 mg/kg
Basis:
other: nominal
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Under the conditions of a previously conducted study, the no-observed-effect level (NOEL)a for H-27201 and H-27203 was 24,000 mg/kg/day for male rats, based on the lack of any adverse effects at this dose.
- Rationale for animal selection: Rats were selected for use on study on the bases of adequate body weight gain and freedom from clinical signs of disease or injury. They were distributed by computerised, stratified randomisation into study groups as designated in the study design, so that there were no statistically significant differences among group body weight means.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily throughout the study
- Cage side observations checked: Detection of moribund or dead rats and abnormal behaviour and/or appearance among rats.
Each rat was individually handled and examined for abnormal behaviour and appearance (careful clinical observations ) daily approximately 1 -2 hours after receiving the final daily dose. Any abnormal clinical signs were recorded.

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

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

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

FOOD EFFICIENCY: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : No data

OPHTHALMOSCOPIC EXAMINATION: see below (sacrifice and pathology)

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

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

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

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

HISTOPATHOLOGY: Yes
All tissues collected from all rats were processed to slides and evaluated microscopically.
Testes, epididymides, and eyes were fixed in modified Davidson’s solution. All other tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist.
Other examinations:
no
Statistics:
Significance was judged at p < 0.05. Statistical comparisons were made between the control group (group I) and H-27201 (group III) and H-27203 (group V). Statistical comparison was performed between the two treatment groups H-27201 (group III) and H-27203 (group V).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No test substance-related signs were noted on daily or weekly clinical observations during the 28-day oral gavage study. Some non-specific clinical observations were noted in some animals but were not test substance related. These observation which included a neck wound, misshapen ears and hair loss. These types of clinical signs are commonly reported among rats of this sex and age group. One animal, number 305 (H-27201), was found dead at the end of the first week. During the second week, animal number 505 (H-27203) was observed to have breathing noises and was sacrificed. The necropsy report indicated both deaths were attributed to trauma from gavage. The remaining 13 male rats survived until the scheduled sacrifice (test day 29).

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

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

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

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

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

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

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the two rats that died before the terminal sacrifice revealed several lesions associated with esophageal trauma and morbidity (e.g., rupture-associated inflammation, splenic and thymic lymphoid depletion, bone marrow hyperplasia, centrilobular liver necrosis, esophageal reflux and pulmonary aspiration).
There were no test substance-related adverse microscopic findings in the treatment or the control groups.
The observation of gavage material in macrophages within the mesenteric lymph nodes and Peyer’s patches of rats given the test substance was considered to be the expected disposition of a non-toxic particulate. There was no evidence of an adverse cellular response to the presence of the phagocytised particulates. The observation of these particulates was facilitated by their optical birefringence.
Inflammation in the nose of 3/5 Group III rats and 2/5 Group V rats was the result of gastro-esophageal reflux in all but one rat (Group III). This latter rat had minimal focal inflammation in the turbinate mucosa that was interpreted to be incidental. Three of the remaining four rats with evidence of reflux had esophageal perforations identified either by gross or microscopic examination. The remaining rat had minimal gavage material and inflammation and no identified esophageal lesion. All cases of gastro-esophageal reflux and associated inflammation were interpreted to be dosing related and not test substance related.
All other microscopic findings were consistent with either the normal background lesions in rats of this age and strain or were the result of a dosing accident.

Effect levels

Dose descriptor:
NOEL
Effect level:
24 000 mg/kg bw/day (nominal)
Sex:
male

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Male rats were exposed by oral gavage for 29 days to 24,000 mg/kg TiO2 particles H-27201, H-27203, or the vehicle. Under the conditions of this study, the no-observed-effect level (NOEL) for H-27201 and H-27203 was 24,000 mg/kg/day for male rats, based on the lack of any adverse effects at this dose.