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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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From February 26th to April 18th, 1990 (included recovery period)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: See read-across justification in section 13. Reliability of the original study 1.
Justification for type of information:
See attached document in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Similar substance 1 of Tris(ethyl acetoacetato-O1’,O3)aluminium
IUPAC Name:
Similar substance 1 of Tris(ethyl acetoacetato-O1’,O3)aluminium

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rodent species is acceptable to the regulatory authorities and has documented susceptibility to a wide range of toxic substances. Background data of the strain are available at the testing facility. There are no known contra-indications to its use.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO. BP 0109, 69592 L'Arbresle. Cedex, FRANCE.
- Age at study initiation: 6 weeks.
- Weight at study initiation: males 172.0 - 197.9 g; females: 141.5 - 174.2 g
- Housing: animals were housed in groups of 5 of the same sex and dose group in stainless steel mesh cages (555 x 350 x 200 mm). The cages were placed in one room for the study (room 5A) in an air-conditioned building (Building K barrier protected unit). The animals were identified by ear tattoo while for the cages group-related coloured card containing study number, group number, sex and animal numbers was used.
- Diet: Rat & Mouse pelleted complete diet ad libitum sterilised by irradiation and analysed for specified contaminants. Animals were fasted overnight (about 16 hours) before blood sampling and before necropsy.
- Water: filtered (0.6 μm) mains water, ad libitum, analysed twice a year for chemical and bacteriological contamination.
- Acclimation period: 7 days between animal arrival and start of treatment.

DETAILS OF FOOD AND WATER QUALITY: the diet and the water were analysed before using it in the study. No known contaminants were present in diet or water at levels which might have interfered with the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 25 to 75 %
- Air changes (per hr): minimum 8 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Potential human exposure is by the oral route.
Vehicle:
water
Remarks:
distilled water and water for injection
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the formulation was prepared dissolving 5, 22.5 and 100 mg/ml of the test item in water and it was prepared daily. A sample from each preparation (including control group) was analysed at weeks 1 and 4. Analysis of the low and high dose concentrations gave results which indicated that they were stable over 24 hours at + 4 °C and at room temperature. Analysis of the solutions prepared in weeks 1 and 4 gave results which were generally in good agreement with nominal dose levels.

VEHICLE
- Concentration in vehicle: 5, 22.5 and 100 mg/l
- Amount of vehicle (if gavage): 10 ml/kg/day (individual dose volumes were calculated weekly using the most recent body weight).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical verification of dose and concentration was performed by gas chromatography. The dose were diluted in acetone before the analysis.

INSTRUMENT
- Chromatograph : Varian 3400 with autosampler.
- Detector FID.
- Column OV101 15 m long. x 0.53mm J.W. Scientific
- Injection : 1 μl
- Gas : Nitrogen 10 ml/min.
- Temperature : injector : 210 °C, detector : 260 °C, column oven : 70°C.

LINEARITY: the linearity of the response was checked in the range 50 - 500 mg/I. Correlation was 1.0000 and 0.9997.

LOD: 22 mg/l

REPEATABILITY: The accuracy of the method is the relative standard deviation (RSD) of the 5 concentrations calculate. RSD was 8.2 % at 5 g/l and 1.9 % at 100 g/l.
Duration of treatment / exposure:
28 days (4 weeks) of administration followed only for control and high dosage level by 2 weeks treatment free period.
Frequency of treatment:
Daily for 29 days for males and females killed at week 4 and 28 days for males and females killed at week 6.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
225 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose plus 2 recovery groups (5 males and 5 females for each). Total animals for the study: 60 (30 males and 30 females).
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: the high dose level of 1000 mg/kg/day is the maximal dose recommended by the OECD and EEC guidelines. The low dose level is expected to be a no observable effect level and the intermediate dose level of 225 mg/kg is close to the geometric mean between the low and high dose levels.
- Rationale for animal assignment (if not random): randomly assigned by computer.
- Rationale for selecting satellite groups: satellite groups only for control and high dose to evaluate the possible regression of any toxicological signs during a 2 week treatment-free period.
- Post-exposure recovery period in satellite groups: 2 week treatment-free period.
Positive control:
Not present.

Examinations

Observations and examinations performed and frequency:
MORBIDITY/MORTALITY: all animals were observed twice daily, at the beginning and at the end of the working day to detect any which were dead or moribund.

CLINICAL OBSERVATION: all animals were observed daily before and at least once after dosing to detect any clinical signs or reaction to treatment. A full clinical examination was performed weekly. During the treatment-free period, observations were performed once in the morning.

BODY WEIGHT: individual body weights were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE: food consumption was measured weekly for each cage from the first week of treatment. The ratio of mean food consumption per unit of body weight gain (food conversion ratio) was calculated for each week of the study. The substance was administrated by oral gavage and not in food.

OPHTHALMOSCOPIC EXAMINATION: direct and indirect ophthalmoscopy. A mydriatic agent (tropicamide) was instilled into the eyes before examination. All animals were examined once pretest and at week 4.

HAEMATOLOGY :
- Time schedule for collection of blood: only at week 4. The blood sample was taken from the retro-orbital sinus following light ether anaesthesia.
- Animals fasted: overnight fasted animals (about 16 hours).
- How many animals: 5 animals for each sex/group.
- Parameter checked: haemoglobin, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), packed cell volume, red blood cell count, mean corpuscular volume (MCV), reticulocyte count , platelet count, total white blood cell count , differential white blood cell count.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: only at week 4. The blood sample was taken from the retro-orbital sinus following light ether anaesthesia.
- Animals fasted: overnight fasted animals (about 16 hours).
- How many animals: 5 animals for each sex/group.
- Parameters checked in table: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, blood urea nitrogen, total cholesterol , total bilirubin, total protein, albumin, globulin (calculated) , albumin/globulin ratio, creatinine, Alkaline phosphatase (AP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT)

URINALYSIS:.
- Time schedule for collection of urine: only at week 4.
- Metabolism cages used for collection of urine: urine was collected in metabolism cages (approximately 16 hours).
- Animals fasted: animals were deprived of food and water but receiving 20 ml/kg of mains water by gavage before being placed in the metabolism cages.
Sacrifice and pathology:
GROSS PATHOLOGY:
All animals were submitted to full necropsy procedures including examination of :
- the external surface
-all orifices
- the cranial cavity
- the carcass
- the external surface of the brain and of samples of the spinal cord
- the thoracic, abdominal and pelvic cavities and viscera
- the cervical tissues and organs.

The following organs were weighed at scheduled necropsy for all animals :
- Adrenals
- Testes
- Liver
- Spleen
- Kidneys
- Heart
Paired organs were weighed together. Organs were weighed after dissection of fat and other contiguous tissue

HISTOPATHOLOGY:
The following organs/tissues were sampled for all animals :
- Adrenals (2)
- Bone marrow smears
- Heart
- Kidneys (2)
- Liver (2)
- Lungs (2)
- Spleen
- All gross lesions
All organs/tissues sampled were fixed and preserved in 10 % neutral formalin with the following exception : bone marrow smears fixed in methanol.
Histopathological examinations were performed on all organs/tissues : for all animals in groups 1 (control) and 4 (high dose) at week 4. In each group, histopathological examination was performed for all gross lesions except those for which the diagnosis was judged unnecessary for the outcome of the study by the pathologist.
All sections were stained with haematoxylin and eosin.
Statistics:
The data from concurrent controls and historical data were used to evaluate the effects of the test article.
For food consumption, for body weight, for haematology and blood biochemistry parameters, the arithmetic mean was calculated and only for the last three descriptors also the standard deviation was calculated for each group and sex. Body weight data were analysed using analysis of variance : where the result was significant or close to significance Dunnett’s t-test was used to compare each treated group with the control group. Any intergroup differences which attained statistical significance are indicated.
For organ weights, the number of data, the arithmetic mean, the standard deviation and the difference between the mean of the treated group and that of the control group were reported. For the treated groups, these results are followed by the results of Dunnett's t-test and analysis of variance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In week 2, approximately half the high dose animals (group 4) were salivating immediately after dosing. In weeks 3 and 4, this observation was present in all high dose animals. Salivation is seen occasionally in studies of this type (gavage) and probably represents a local reaction to the test article. It is considered not to represent systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
There was no deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no biologically significant differences between the groups in the amount of body weight gained.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The differences in food consumption between the groups were small and not considered to be treatment-related. Food conversion ratios were similar in all groups. The substance was administrated by oral gavage and not in food.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no eye lesions during the study.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no differences between the groups to indicate an effect of treatment on the haematological parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The clinical chemistry profiles of the treated animals were similar to the controls.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The few microscopic findings were considered to be of agonal or incidental origin (i.e pulmonary haemorrhage). There were no treatment-related microscopic findings.

Effect levels

Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects treatment releated

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOEL of similar substance 1 (repeated oral toxicity rat 28 days) = 1000 mg/kg bw /day equivalent to NOAEL Tris(ethyl acetoacetato-O1',O3)aluminium (repeated oral toxicity) = 1062 mg/kg bw/day.
Executive summary:

The objective of the study was to determine the toxicity and a no-effect level of the test item following daily oral (gavage) administration to the Sprague-Dawley rat for 4 consecutive weeks and to evaluate the possible regression of any toxicological signs during a 2 week treatment-free period.

STUDY DESIGN

The doses used during the study were 0, 50, 225 and 1000 mg/kg/day and each group was composed by 10 animals (5 males+5 females) and a recovery group (10 animals: 5 males+5 females) was included only for control and for highest dosage. Clinical examinations were performed daily. Food consumption was measured weekly for each cage. Individual body weights were recorded weekly. Ophthalmological examination was performed pretest and at week 4. Clinical pathology investigations were performed at week 4. All surviving animals were killed and necropsied at weeks 4 or 6 ; selected organs were weighed at necropsy. Selected tissues were examined histopathologically in group 1 and 4 animals at week 4 kill. Analysis of the solutions gave results which indicated that they were stable under the conditions of administration and that they had been accurately formulated in weeks 1 and 4.

RESULTS

During the study there were no deaths. There were no toxicologically significant clinical observations. Treated animals gained similar amounts of weight to controls. There were no differences in the amount of food consumed across the groups. The efficiency of food conversion was similar in all groups. There were no eye lesions, no treatment-related differences in the haematological profiles of the animals, in the clinical chemistry parameters or in the organ weights indicating an effect of treatment.

CONCLUSION

Oral administration of the test item to the rat for 4 weeks at dose levels up to 1000 mg/kg/day was not associated with any toxicological changes. The dose level of 1000 mg/kg was a no-observable-effect-level under the experimental conditions employed.

NOEL of similar substance 1 (repeated oral toxicity rat 28 days) = 1000 mg/kg bw /day and the recalculated NOAEL for Tris(ethyl acetoacetato-O1',O3)aluminium is 1062 mg/kg bw/day.