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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(92/69/EEC)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-240-7
EC Name:
-
Molecular formula:
C28H42O8Cl2Co2N12Zn4
IUPAC Name:
Zinc hexacyanocobaltate(III), tertiary butyl alcohol/polypropylene glycol complex

Test animals

Species:
rat
Strain:
other: Crl:CDBR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd, Margate, UK
- Age at study initiation: less than 7 weeks
- Weight at study initiation: males 165.8-202.5 g; females 147.0-174.6 g
- Housing: in groups of 5 rats in stainless steel mesh cages (55x34x20 cm)
- Diet and water: ad libitum
- Acclimation period: approx. 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% methyl cellulose in purified water
Details on oral exposure:
Administration volume: 10 ml/kg bw

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily, each dose concentration being individually formulated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared on Day 1 for all dose groups were taken for homogeneity analysis. Further samples were taken following 24 hours of storage at room temperature to assess stability.
Samples of formulations prepared on Day 1 for all dose groups were taken to check for achieved concentration. The concentrations of the test substance in formulated doses were calculated from the measurements of the divalent metals contained in the test substance samples. The method used was the Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES).
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 150 and 1000 mg/kg bw
Basis:
other: nominal
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level was assessed in a 14 days preliminary study (Cheffings, Covance Laboratories, Harrogate. North Yorkshire, UK, report number 1068/24-1050, 1997). Three male and three female rats were treated in a 14-days oral gavage study with 0 and 1000 mg/kg and day. No deaths and no treatment-related clinical signs of toxicity were observed in this preliminary study.
The low and intermediate dose levels of the main study were selected on the basis of the key dose levels relating to the risk phrase R48 as stated in Commission Directive 93/21/EEC.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: daily for signs of ill health, behavioural changes or overt toxicity
Additionally all animals were examined at the beginning and end of each working day to detect any which were dead or moribund.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: before treatment on the first day of dosing, at weekly intervals thereafter and before necropsy.

FOOD CONSUMPTION: Yes, per each cage of animals
- Food consumption calculated as g per animal and week

HAEMATOLOGY: Yes, blood samples obtained for all animals in Week 4 (orbital sinus puncture and samples from abdominal aorta at necropsy)
- Anaesthetic used for blood collection: Yes, light halothane anaestesia prior to orbital sinus puncture
- Animals fasted: Yes, overnight period without food prior to orbital sinus puncture
- Parameters examined: hemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total and differential white blood cell count, platelet count, prothrombin time, activated partial thromboplastin time.
Blood films for reticulocyte count and bone marrow smears prepared, but not examined.

CLINICAL CHEMISTRY: Yes, blood samples obtained for all animals in Week 4 (orbital sinus puncture and samples from abdominal aorta at necropsy)
- Animals fasted: Yes, overnight period without food prior to orbital sinus puncture
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, potassium, sodium, calcium, inorganic phosphorus, chloride, total protein, albumin, albumin/globulin ratio, total cholesterol, glucose, urea, total bilirubin, creatinine.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
The following organs were dissected free from fat and other contiguous tissue and weighed before fixation: adrenals, brain, heart, kidneys, liver, spleen, ovaries, testes and epididymides.

HISTOPATHOLOGY: Yes
The following organs were fixed: adrenals, aorta, brain, cecum, colon, duodenum, eyes, femur with marrow and articular surface, gross lesions, Harderian glands, head, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs (with mainstem bronchi), mammary, mandibular lymph nodes, mesenteric lymph nodes, muscle quadriceps, nasal turbinates, nasopharynx, oesophagus, optic nerves, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerves, seminal vesicles, skin, spinal cord cervical, spinal cord lumbar, spinal cord thoracic, spleen, sternum with bone marrow, stomach, testes and epididymides, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus, vagina, Zymbal glands.

The following organs were evaluated: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes and epididymides from control and high dose animals, gross lesions from all animals.
Statistics:
2-way analysis of variance (ANOVA) for body weight gains, necropsy weight gains, haematology and clinical chemistry variables. Dunnett's test for paiwise comparisons, each sex separately. Regression test for each variable measured and for each sex separately. Levene's test for equality of variances across groups, between sexes and for any interaction. Analysis of Covariance (ANCOVA) for organ weights. Any statistically significant result observed only in the ANOVA or ANCOVA have not been reported.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities. There were no treatment related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
Slight reduction in group mean body weight of the high dose group, influenced by a single high dose male. The toxicological significance was regarded to be equivocal.

FOOD CONSUMPTION
In general, the food consumption of treated animals was comparable with the controls.

HAEMATOLOGY
Minor haematological disturbances generally within 10% of control levels were seen in rats dosed at 1000 mg/kg (haemolglobin levels and clotting times in males, lymphocyte and neutrophil counts in females) were of uncertain toxicological significance.

CLINICAL CHEMISTRY
Small decreases to 75-85% of control levels in plasma alanine and aspartate aminotransferase also seen at 1000 mg/kg were not considered to be indicative of toxic effect.

ORGAN WEIGHTS
An increase in testes and epididymides was noted in the males treated at 15, 150 and 1000 mg/kg, but none dose-related. There was no apparent effect of treatment on other organ weights.

GROSS PATHOLOGY
No treatment-related findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related findings.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: No treatment-related effects up to highest dose (1000 mg/kg).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

An increase in testes and epididymides weight in males of all dose groups did not follow a dose-relationship and was furthermore without a histopathological correlate. Therefore the toxicological significance of this observation was regarded as uncertain.

Without further corroborating findings statistically significant decreased or increased values for hematology or clinical biochemistry were considered to be of uncertain toxicological significance.

Analysis of the divalent metal contents of the formulation samples indicated that measured concentrations were significantly lower than the nominal concentrations. None of the data generated during the preparation of the formulations provided an explanation of the low measured levels of metals. Losses of settlements prior to final analysis may have occurred, alternatively, weakness of the ICP-AES technique was indicated. The author concluded, that the dose formulation records provide a more reliable indicator of doses to which the test animals were exposed; nominal dosage were therefore believed correct, and only theses were discussed elsewhere in the report.

Applicant's summary and conclusion

Executive summary:

A subacute (28 days) toxicity study according to EU Method B.7 was conducted with the test substance administered once daily in nominal doses of 0 (vehicle control), 15, 150 and 1000 mg/kg bw to 5 male and 5 female animals per dose-group.

There were no deaths and no clinical signs observed during the study. The body weight and food consumption of the treated animals was generally comparable to the controls. Minor haematological disturbances seen in rats dosed at 1000 mg/kg (haemolglobin levels and clotting times in males, lymphocyte and neutrophil counts in females) were considered to be of uncertain toxicological significance. Small decreases in plasma alanine and aspartate aminotransferase also seen at this test dosage were also not indicative of toxic effect. There were no changes in organ weights, macroscopic and microscopic examinations that were considered to be an effect of treatment. Overall, no clear indication for systemic availability could be concluded from the study. The NOAEL of the study was set 1000 mg/kg bw.