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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 October 2005 - 19 December 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006
Reference Type:
other: Amendment
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
; adopted on 27July 1995
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation: solid, powder
- Particle size distribution (TEM): 29.1 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: plate
- Surface area of particles: 89 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1566 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable
Specific details on test material used for the study:
- Physical state: Powder / black
- Storage condition of test material: Room temperature
- Analytical purity: 99.9%
- Lot/batch No.: Lot 2005-01
- Expiration date of the lot/batch: unlimited at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 41-43 days old
- Weight at study initiation: 100-112 g
- Housing: up to 5 of one sex to a cage, clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: commercially available laboratory rodent diet (4RF21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 28 October 2005 To: 2 December 2005 (main groups) or 19 December 2005 (recovery groups)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Following addition of the vehicle, the suspension was initially mixed manually with the aid of a spatula, then it was mixed by Silverson for approximately 2-3 minutes and finally stirred for approximately 5-6 minutes. The formulations were prepared daily.

administered dose volume: 10 ml/kg bw

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/ml.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations prepared in weeks 1 and 4 were analysed to check the homogeneity and concentration. Results of the analyses were generally within the limits of acceptance (90-110%). In a single occasion, the measured concentration of group 2 was outside the limits of acceptance and was repeated on Day 2.
Duration of treatment / exposure:
4 consecutive weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (Five additional animals for each sex were included in the high and control groups for recovery assessment - two consecutive weeks)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from preliminary studies.
- Post-exposure recovery period in satellite groups: yes (2 weeks)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Mortality: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Pre- and post-dose observations: All observations were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after and approximately 1 and 2 hours after dosing.
Clinical signs and neurotoxicity assessment:
Once before commencement of treatment and once a week thereafter each animal was subjected to a detailed clinical examination, which included an evaluation of neurotoxicity. Animals were examined in an open arena for a period of a minimum of three minutes. Observed parameters: Removal (from cage), handling reactivity, lachrymation, palpebral closure, salivation, piloerection, rearing, spasms, myoclonia, mobility impairment, arousal (animal activity), vocalisation, stereotypies, unusual respiratory pattern, bizarre behaviour, urination, defecation, tremors, gait

In addition, from Day 6 of treatment, at each change of litter tray paper, an examination of the litter tray was performed and any signs were recorded. Once during week 4 of treatment and once during week 2 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and assessment of grip strength were also performed.

Motor activity assessment (MA)
The motor activity (MA) of all animals was measured once during week 4 of treatment and once during week 2 of recovery by an automated activity recorder.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 4 of treatment, samples of blood were withdrawn from the retro-orbital sinus; prior to necropsy, blood samples were taken from the abdominal vena cava of the main phase rats from each group
- Anaesthetic used for blood collection: Yes (isofluorane anaesthesia)
- Animals fasted: Yes
- How many animals: all surviving male and female rats from each group
- Parameters checked : Haematocrit, haemoglobin, red blood cell count, reticulocyte count (not performed as no signs of anaemia were observed), mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), abnormalities of the blood film, platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 4 of treatment, samples of blood were withdrawn from the retro-orbital sinus; prior to necropsy, blood samples were taken from the abdominal vena cava of the main phase rats from each group
- Animals fasted: Yes
- How many animals: all surviving male and female rats from each group
- Parameters checked: Alkaline phosphatase,alanine aminotransferase, aspartate aminotransferase, gamma -glutamyltransferase, urea, creatinine, glucose, triglycerides, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: During week 4 of treatment, individual overnight (approximately 16 hours) urine samples were collected from all surviving male and female rats from each group
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Appearance, volume, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood; t he sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: Epithelial cells, poly morphonuclear leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes (see: Detailed clinical observations)


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Organ weights
From all animals the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues were fixed and preserved in 10% buffered formol saline (except testes and epididymides which were fixed in Bouin's solution and preserved in 70% ethyl alcohol). One slice from the liver lobes, the lobus dexter lateralis and the lobus sinister lateralis, was fixed in Carnoy's solution. Liver samples fixed in Carnoy's solution from the low, mid and recovery groups were processed onto the paraffin block.

Histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Samples fixed in Carnoy' s solution were cut at 2-3 micrometre thickness. In the first instance the examination was restricted as follows:
a) Tissues from all animals in the control and high dose group killed after 4 weeks of treatment;
b) All abnormalities in all main phase groups.
Pertaining to the liver, only the sections fixed in Carnoy's solution were examined by the pathologist.
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No signs were observed at daily post-dose observations. Detailed clinical signs with neurotoxicity assessment did generally not show any signs which could be correlated to the treatment with the test item. Dark faeces (with a dose-related intensity) were seen in the litter tray of all animals treated with the test item for the duration of the treatment period. This sign was no longer evident by Day 6 of the recovery phase.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weights were noted between treated animals and controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes of toxicological significance were observed in haematological parameters. The slight increments, generally statistically significant, of red blood cells, haemoglobin and the decrement of mean corpuscular volume observed in females treated with 1000 mg/kg/day were considered to be incidental and without toxicological significance, as they were slight, not dose-related and fully within historical control values.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry parameters did not show any changes of toxicological importance. The slight, statistically significant differences observed in some parameters (aspartate aminotransferase, glucose, triglycerides, urea, albumin, sodium and calcium) were not dose related, not consistent between sexes and insufficient in magnitude to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes were observed at analysis of urine parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant differences between treated animals and controls were observed at evaluations of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes, which could be ascribed to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No changes of toxicological significance were observed in the weight of the organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No change of toxicological significance was reported at post mortem examination, in treated animals killed at the end of treatment or at completion of the recovery period, when compared with controls.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histopathological examination did not reveal any evident differences in the incidence of the findings observed in treated and control animals, which could be considered treatment related. The lesions described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results reported, the high-dose of 1000 mg/kg/day, when administered daily for 4 consecutive weeks, was considered the No Observed Adverse Effect Level (NOAEL).
Executive summary:

In a GLP-compliant oral subacute toxicity study following OECD test guideline 407, three groups, each of 5 male and 5 female Sprague Dawley rats received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day for 4 consecutive weeks. A fourth similarly constituted group received the vehicle alone (0.5% CMC) and acted as a control. Five additional animals for each sex were included in the high and control groups for recovery assessment.

No mortality occurred during the study and no clinical signs were observed at daily post-dose observations which could be correlated to the treatment with the test item. No significant differences between treated animals and controls were observed at evaluations of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes, which could be ascribed to treatment. No significant differences in body weights were noted between treated animals and controls. Food consumption was not affected by treatment. No changes of toxicological significance were observed in haematological and clinical parameters. No significant changes were observed at analysis of urine parameters. No changes of toxicological significance were observed in the weight of the organs. No change of toxicological significance was reported at Post mortem examination, in treated animals killed at the end of treatment or at completion of the recovery period, when compared with controls. The histopathological examination did not reveal any evident differences in the incidence of the findings observed in treated and control animals, which could be considered treatment related. The lesions described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

On the basis of these results, no signs of an adverse effect of the test item were seen at any of the dose levels investigated (100, 300 and 1000 mg/kg/day). Therefore, the high-dose of 1000 mg/kg/day, when administered daily for 4 consecutive weeks, was considered the No Observed Adverse Effect Level (NOAEL).