[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 28 August 2001 to 16 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

some animals from all groups decreased or stopped their water intake between days 22 to 29 of the study (due to a problem with the water system). This deviation was not considered to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

753480-32-9

Cas Number:

753480-32-9

IUPAC Name:

753480-32-9

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l’Arbresle, France.
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: mean body weight of 200 g (males) and 169 g (females)
- Fasting period before study: no
- Housing: individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm).
- Diet: free access to A04 C pelleted maintenance diet
- Water: free access to bottles containing tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: approximately 12 cycles/hour
- Photoperiod: 12h dark/12h light

IN-LIFE DATES: from 28 August 2001 (1st day of acclimation) to 18 October 2001 (day of necropsy of the last animal)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of vehicle using an ultraturrax in order to achieve the concentration of 30, 90 or 200 mg/mL, and then homogenized using a magnetic stirrer. The test item dosage forms were made daily and delivered to the animal room within 5 hours (stability period).

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 30, 90 or 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 70K0127

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis were performed using Fluorescence X spectrometry.

Homogeneity:
On day 1, dosage forms were prepared at the lowest and highest concentrations (30 and 200 mg/mL) used in the study. Duplicate samples were immediately taken from three levels (top, middle and bottom), frozen at -20°C and protected from light.
The results of the analysis demonstrated a satisfactory homogeneity and a correspondence between the nominal and the measured concentrations.

Stability:
On day 1, the dosage forms prepared for homogeneity analysis were kept for 5 hours at room temperature and protected from light after preparation and then duplicate samples were frozen at -20°C and protected from light.
The results of the analysis demonstrated a satisfactory stability of the same dosage forms over a 5-hour period at room temperature.

Concentration:
Duplicate samples were taken on each control and test item dosage forms prepared for use on day 1 and day 22. The samples were frozen at -20°C and protected from light.
Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the administered dosage forms since the deviations from nominal concentration were in an acceptable range of +/- 9%.

Duration of treatment / exposure:

29 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

450 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: selected according to the results of a 7-day range-finding toxicity study by oral route performed in the same species, in which no clinical signs or macroscopic findings were noted in any treated group at 150, 450 or 1000 mg/kg/day.
- Rationale for animal assignment: according to a computerized stratification procedure, so that the average body weight of each group was
similar.
- Rationale for selecting satellite groups: at the end of the treatment period, the first five surviving animals per sex and per group were killed and the remaining animals were kept for a 2-week treatment-free period.
- Post-exposure recovery period in satellite groups: yes

Statistical analyses were performed on body weight, food consumption, hematology, blood biochemistry and organ weight data.

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (morbidity and mortality) or once daily (clinical signs)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and once a week thereafter.
Observations included (but were not limited to) changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation of the animals to groups, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The quantity of food consumed by the animals of each cage was recorded once a week, over a 7-day period during study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 4 and for all surviving recovery animals at the end of the treatment-free period (week 6).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight for at least 14 hours)
- How many animals: all animals (5 animals/sex/group at the end of the treatment period and at the end of the free-treatment period)
- Parameters checked: erythrocytes, hemoglobin, Mean Cell Volume, Packed Cell Volume, Mean Cell Hemoglobin Concentration, Mean Cell Hemoglobin, thrombocytes, leucocytes, Differential White Cell count with cell morphology, neutrophils, eosinophils, basophils, lymphocytes, monocytes, Prothrombin Time, Activated Partial Thromboplastin Time, Fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: for all principal animals at the end of week 4.
- Animals fasted: Yes (overnight for at least 14 hours)
- How many animals: for all principal animals
- Parameters checked: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total Bilirubin, total Proteins, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period.
- Dose groups that were examined: all animals
The following parameters were assessed and graded:
. “touch escape” or ease of removal from the cage,
. in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
. in the standard arena (two-minute recording): grooming, palpebral closure, defecation and urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypy, behavior and breathing, ataxia, hypotonia.
Then, the following parameters measurements, reflexes and responses were recorded: touch response, forelimb grip strength, papillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.
Finally, motor activity of all animals were measured by automated infra-red sensor equipment over a 30-minute period once at the end of the treatment period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The organs specified in the table 1 were weighed wet as soon as possible after dissection.
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes (see table 1)
A microscopic examination was performed on:
. all tissues listed in the Table 1 below for principal animals of the control and high dose-level groups (groups 1 and 4) killed at the end of the treatment period and for all animals that died during the study (all groups),
. all macroscopic lesions of all the principal animals of the low and intermediate dose-level groups (groups 2 and 3) killed on completion of the treatment period,
. all macroscopic lesions of all recovery animals at the end of the treatment-free period.

Statistics:

Statistical analyses were performed on body weight, food consumption, hematology, blood biochemistry and organ weight data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the treatment period, the following relevant clinical signs were noted:
- Scabs (forelimbs/back): in males at 0 (2/10), 150 (1/10) and 1000 mg/kg bw/d (1/10)
- Area of hair loss (forelimbs)/hair loss: in males at 150 mg/kg bw/d (2/10) and in females at 0 (1/10), 150 (1/10), 450 (2/10) and 1000 mg/kg bw/d) (1/10)
- Regurgitation: in males at 150 (1/10, day 4) and 450 mg/kg bw/d (1/10, day 4 and 6)
One control female showed round back and thin appearance at the end of the treatment period (from day 29).
During the treatment-free period, areas of hair loss were noted in previously treated females (1/5, 2/5 and 1/5 at 150, 450 and 1000 mg/kg/day, respectively).
In addition, chromodacryorrhea was observed in 1/10 control males during the treatment period (from days 14 to 19) and in 1/5 males given 150 mg/kg/day during the treatment-free period (from day 36).
As all these clinical signs were observed with the same incidence in control and treated animals, and/or without dose-relationship, they were not considered to be related to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male was found dead on day 5 with no clinical sign prior to death. The major factor contributing to death was considered to be accidental as evidenced by the presence of grade 2 alveolar edema in the lungs.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Similar mean body weight gain was noted in treated and control animals, eventhough, during week 4, a reduced mean body weight gain/slight body weight loss was noted with the same incidence in control and treated animals. These changes may be attributed to the reduced food consumption noted in the same animals (due to reduced water consumption).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A slightly lower food consumption was observed in females given 150 mg/kg/day on day 15-21 (-9%) and 36-42 (-10%) and in females given 450 mg/kg/day on day 22-28 (-10%). As it was the contribution of some individuals, these differences were not considered to be of toxicological significance.
Some differences in the food consumption were also observed on day 22-28 between a controls and all treated males, and were due to the contribution of some controls showing a very low food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At the end of treatment period:
. slightly lower packed cell volume was noted in males given 450 and 1000 mg/kg/day (0.43 and 0.42 vs. 0.46 in controls, respectively),
. slightly higher mean cell hemoglobin concentration was noted in males given 150, 450 and 1000 mg/kg/day (34.7, 35.3, 35.2 vs. 34.0 g/dL in controls, respectively),
. slightly lower prothrombin time was noted in females given 1000 mg/kg/day (13.3 vs. 15.1 s in controls) was observed.
Although these differences were statistically significant, they were slight and since all the individual values were within or close to the range of the historical background data. Thus they were considered to be of no toxicological significance.
At the end of treatment-free period, no relevant changes were observed in any parameters when compared to controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No relevant changes were noted in any parameter at the end of the treatment period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no evidence of perturbation of either autonomic or physiological functions at any dose-level. The functional test battery showed no treatment-related changes in any neurotoxicological parameter.
Some animals presented an abnormal fur appearance, but these observations were noted with a similar incidence in control and treated animals, and they were not considered to be related to treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Some differences noted between treated and control animals in organ weights at the end of treatment period:
- Spleen: between +16% and +26% in males and between +10% and +22% in females (organ weight relative to body weight)
- Thymus: between +27% and +55% in males and between +14% and +26% in females (organ weight relative to body weight)
The values at the end of treatment-free period were as follow:
- Spleen: between +0% and +13% in males and between -2% and +1% in females (organ weight relative to body weight)
- Thymus: between -20% and -28% in males and between +7% and +10% in females (organ weight relative to body weight)
As microscopic examination revealed no changes to correspond with these differences in organ weights, they were considered to be of no toxicological importance.
Some other differences were noted between treated and control animals in absolute and relative values. However, as these differences were minimal, and of opposing trend in the two sexes, they were considered to be of no toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related necropsy findings were noted at the end of treatment and treatment-free period.
All the necropsy findings encountered are commonly recorded changes in the rat of this strain and age and were considered to be of no toxicological importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In the control animal that died, death was considered to be accidental as evidenced by the presence of grade 2 alveolar edema in the lungs.
No changes were seen in the other animals in the sections examined that were considered to be related to treatment.
No changes were seen in the sections examined that were considered to be related to treatment.
Those changes that were present were recognized as those that occur commonly in rats of this age maintained in this laboratory. They were present with approximately the same frequency and degree of change in both control and treated animals and were of no toxicological importance.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No relevant effects up to highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 2: Mean body weight changes

 

Mean body weight gain (g)

Males					Females
Dose-level (mg/kg/day)	0	150	450	1000	0	150	450	1000
Days 1 to 15	+125	+126	+127	+119	+52	+50	+50	+53
Days 15 to 29	+41	+64	+60	+68	+20	+22	+16	+20
Days 29 to 43 (treatment-free period)	+68	+56	+76	+53	+15	+19	+28	+20

 

 

Table 3: Mean food comsuption (g/animal/day)

 

Males					Females
Dose-level (mg/kg/day)	0	150	450	1000	0	150	450	1000
Days 1-7	26.6	26.6	27.6	25.6	18.0	17.8	17.1	17.5
- % from controls	-	0	+4	-4	-	-1	-5	-3
Days 15-21	27.3	27.0	27.5	26.2	18.2	16.6	17.9	18.5
- % from controls	-	-1	0	-4	-	-9	-2	+2
Day 22-28	16.7	21.0	22.0	23.1	15.7	14.0	14.1	14.9
- % from controls	-	+26	+32	+38	-	-11	-10	-5
Day 36-42	28.2	28.7	29.0	27.4	19.1	17.2	19.4	20.0
- % from controls	-	+2	+3	-3	-	-10	+2	+5

 

 

Table 4: Major differences (in %) noted between treated and control animals in organ weights

 

End of treatment period:

Males				Females
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	150	450	1000	150	450	1000
. Spleen
- absolute	+15	+20	+25	+23	+17	+5
- relative	+16	+13	+26	+22	+17	+10
. Thymus
- absolute	+26	+40	+53	+17	+24	+21
- relative	+27	+33	+55	+14	+23	+26

 

 

End of treatment-free period:

Males				Females
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	150	450	1000	150	450	1000
. Spleen
- absolute	+10	+17	+2	-6	-8	+1
- relative	+4	+13	+0	+1	-2	+1
. Thymus
- absolute	-14	-26	-20	-13	-15	-10
- relative	-20	-28	-22	-7	-10	-10

Applicant's summary and conclusion

Conclusions:

Due to absence of toxicity in this study, the No Observed Effect Level (NOEL) of the test item is 1000 mg/kg/day for both male and female rats.

Executive summary:

In a subacute toxicity study scored as validity 1 according to Klimisch criteria (OECD guideline 407, GLP, CIT report No. 22314 TSR) Cerium and iron oxide isostearate was administered daily to 10 Sprague-Dawley rats/sex/dose by gavage at dose levels of 0, 150, 450 or 1000 mg/kg/day in corn oil. At the end of the 4-week exposure period, the first five surviving animals per sex and per group were killed and the remaining animals were kept for a 2-week treatment-free period.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded once a week. Detailed clinical observation was performed once a week, and a functional observation battery was performed at the end of the treatment period.

Hematological investigations were performed in the first five males and females per group in week 4 and at the end of the treatment free period. Blood chemistry analyses were performed in the first five males and females per group in week 4. All animals were killed and subjected to a macroscopic post-mortem examination and specified organs were weighed and preserved. Microscopic examination was performed on selected tissues from animals of the control and the 1000 mg/kg/day groups killed at the end of the treatment period

  

No treatment-related death or clinical signs occurred during the study. There were no effects on body weight, body weight gain or food consumption at any dose level. The Functional Observational Battery assessment, haematology and blood chemistry parameters revealed no treatment-related effects. Macroscopic and microscopic examinations at necropsy did not reveal any treatment-related findings and there were no treatment-related changes in organ weights.

 

The No Observed Effect Level (NOEL) was therefore considered to be 1000 mg/kg bw/d for both male and females rats.

  

No classification for repeat-dose toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of UN/EU GHS.

 

This study is classified as acceptable. It satisfies the OECD 407 guideline requirements on repeated dose toxicity testing.