Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-11-08 to 2011-08-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011
Reference Type:
publication
Title:
Acute and subchronic oral toxicity studies in rats with nanoscale and pigment grade titanium dioxide particles
Author:
Warheit DB, Brown SC, Donner EM
Year:
2015
Bibliographic source:
Food Chem Toxicol. 84:208-24

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
, adopted 1998-09-21
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Titanium dioxide (pigment grade)
- Physical state: white solid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: approximately 8 weeks
- Housing: animals were housed in pairs in polycarbonate pans that contained bedding with enrichment (i.e., Shepherd’s™ Cob + PLUS™).
- Diet (ad libitum, except when fasted): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26ºC
- Relative humidity: 30-70%
- Photoperiod (hrs dark / hrs light): approximately 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
H-29865 was suspended in 0.5% aqueous methylcellulose. Dose suspensions of the test substance were prepared daily and stored at room temperature until used.

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

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

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

- Rationale for animal assignment: animals of each sex were selected for use on study based on adequate body weight gain and freedom from any ophthalmology abnormalities or clinical signs of disease or injury. The selected animals were distributed by computerized, stratified randomization into study groups as designated in the Study Design so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations: moribund, mortality, abnormal behaviour and/or appearance
An additional cage-site evaluation was conducted daily approximately 2 hours (± 1 hour) postdosing to detect acute clinical signs of systemic toxicity.

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

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

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

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

WATER CONSUMPTION AND COMPOUND INTAKE : No

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

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

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

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

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

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

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- no test substance-related clinical signs were observed.
- no deaths occurred.

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

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

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

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

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

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

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

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

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

GROSS PATHOLOGY
- no test substance-related gross observations.

HISTOPATHOLOGY: NON-NEOPLASTIC
- no test substance-related adverse changes in histopathology were observed

Effect levels

Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (males & females; nominal concentration): >1000 mg/kg/day
NOAEL (males & females; actual concentration): >962 mg/kg/day
The NOAEL is based on a lack of test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameter in rats dosed up to 1000 mg/kg/day.