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Repeated dose toxicity: oral

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Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
according to guideline
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
, see any other information on materials and methods
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
hydrate iron potassium hydride
Constituent 2
Reference substance name:
Potassium ferrite
Potassium ferrite
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Potassium Ferrite, (also known as Mapico CG-20X)
- Substance type: reddish, brown powder
- Composition of test material, percentage of components: K2Fe22O34: 83.8 %, K2Fe10O16: 6.2 %, K2CO3 1.5 H2O: 10.0 %
- Lot/batch No.: 23388-17 CG20X 407448A
- Expiration date of the lot/batch: 6 August 1997
- Storage condition of test material: at ambient temperature, protected from light and desiccated.

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 35 - 42 days
- Weight at study initiation: males 150-200 g, females 100-150 g
- Housing: Five animals per sex per cage. The cages used were Type TR18 (Modular Systems and Developments Company Limited, Hereford, England), which were made of a stainless steel body measuring 54 x 38 x 20 cm with a stainless steel mesh lid and floor. The cages were suspended above absorbent crepe paper which was changed at appropriate intervals.
- Diet: commercially available pelleted rodent diet RM1(E) SQC (Special Diets Services Ltd., Witham, Essex, England) ad libitum, except overnight before routine blood sampling.
- Water: water from the public supply (Essex and Suffalk Water plc, Chelmsford, Essex, England); ad libitum
- Acclimation period: 14 days

- Temperature (°C): Target values within the study room were 21 °C (acceptable range 19-25°C) for temperature
- Humidity (%): 55% (acceptable range 40-70%) for relative humidity
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
maize oil
Details on oral exposure:
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The homogeneity of distribution of Potassium Ferrite in maize oil was assessed analytically in a trial preparation at concentrations of 3 and 100 mg/mL prepared before the commencement of treatment. Six unit dose samples taken from positions evenly spaced throughout the bulk preparation were assayed to determine the homogeneity of Potassium Ferrite in the formulation. The concentrations of test substance were also determined in formulations prepared for one occasion of dosing during Weeks 1 and 4 of treatment. In view of the results obtained in Week 1, an additional analysis was performed on test formulations prepared on one day in Week 2. Acceptable homogeneity of the formulation was demonstrated for Potassium Ferrite at both concentrations, though Group 4 achieved concentration was slightly higher than intended (117%). The concentrations of the analysed preparations during the study were generally satisfactory. Values for Groups 2 and 4 in Week 1 were higher than intended (125 and 112% of intended, respectively). An additional analyses in Week 2 indicated satisfactory achieved concentrations. The variations in achieved concentration in Week 1 were not considered to have affected the integrity of the study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 7 days per week
Doses / concentrations
Doses / Concentrations:
0, 15, 150 and 500 mg/kg bw
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle


Observations and examinations performed and frequency:
- Signs: Animals and the cage-trays were inspected at least twice daily for evidence of reaction to treatment or ill-health. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. Individual observations of all animals were also recorded before and after dosing on each day of treatment. The timing of these observations was performed to establish and confirm any pattern to the signs. The schedule was: Week 1: daily, Weeks 2-4: twice weekly (at the middle and at the end of each week). The detailed Observations were performed at the following times during the specified days: Pre-dose observation; as each animal was returned to its home cage; at the end of each dosing group; between 1 and 2 hours after completion of dosing of all groups; as late as possible in the working day.
- Body weight: The bodyweight of each animal was recorded during the acclimatisation period, on the day that treatment commenced, at weekly intervals throughout the treatment period and before necropsy.
- Food consumption: The weight of food supplied to each cage, that remaining and an estimate of the amount spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.
- Food conversion efficiency: Group mean food conversion efficiencies were calculated for each week of treatment.
- Functional observation battery: Each animal was subjected to the procedures detailed below on the specified occasions. The functional observation battery was performed at the same time of day on each occasion. The procedures were performed by an observer who was unaware of the treatment group to which each animal belonged. Before commencement of each set of observations, the cage labels were covered so that the only information visible to the observer were the animal and cage numbers. Open field observations: Before commencement of treatment and during each week of treatment, at approximately the same time of day, the appearance and behaviour of each animal was assessed. The animal was placed on a clean sheet of absorbent paper in an observation arena (an open topped cage with floor dimensions of approximately 50 x 30 cm). The behaviour during a two minute period was observed and recorded in an area of the test room where visual and auditory stimuli were minimised. The following parameters were assessed and graded: activity, alertness, behaviour, convulsion, defaecation, exophthalmos, fur appearance, gait, grooming frequency, lachrimation, palpebral closure, piloerection, posture, pupil size, removal from cage, respiration rate, salivation, tremor, urination. Sensory reactivity test: Before commencement and during Week 4 of treatment the functional integrity of each animal was assessed using a series of manipulation observations. The observations comprised: auditory pinna reflex, auditory startle reflex, body temperature, flexor (withdrawal) responses, landing footsplay, pain (tail pinch) response, pupil closure response, reaction to handling, righting reflex. Grip strength and motor activity: Before commencement and during Week 4 of treatment, immediately following the sensory reactivity tests, grip strength and motor activity were assessed.
- Haematology, Peripheral blood: During Week 4 of treatment, after completion of the functional observation battery tests, blood samples were obtained from all animals, after overnight starvation (before dosing). Blood samples were withdrawn from the retro-orbital sinus, with the animals held under halothane/nitrous oxide anaesthesia, and collected into EDTA as anticoagulant. All samples were examined for the following characteristics: Haemoglobin concentration, erythrocyte count, mean cell haemoglobin concentration, mean cell haemoglobin, mean cell volume, total and differential: leucocyte count, platelet count. The equipment distinguishes neutrophils, lymphocytes, eosinophils, basophils, monocytes and a small proportion of large unstained cells. Blood film - Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types, including normoblasts. Additional samples were taken into citrate anticoagulant and examined in respect of: prothrombin time. Since two samples collected into citrate anticoagulant in the control male group were found to be clotted, repeat samples were taken from all male animals prior to necropsy. The animals were unstarved prior to this bleed.
- Haematology, bone marrow: bone marrow samples were obtained from the femur at the necropsy of all animals. Smears from these samples were air-dried, fixed in methanol and stained using a May-Grünwald-Giemsa procedure. The smears from all animals of groups 1 and 4 sacrificed on completion of the scheduled treatment period, were examined by counting 100 nucleated cells. The cellularity and composition of the marrow were also assessed.
- Blood chemistry: at the same time as far peripheral haematology further blood samples were taken and collected into lithium heparin as anticoagulant. Samples were taken and analysed in the same sequence as for peripheral haematology. After separation the plasma was examined in respect of: Alkaline phosphatase activity, Alanine amino-transferase activity, Aspartate amino-transferase activity, Gamma-glutamyl transpeptidase activity, Urea concentration, Glucose concentration, Total cholesterol concentration, Creatinine concentration, Total protein concentration, Albumin concentration, Albumin/globulin ratio, sodium and potassium.
Sacrifice and pathology:
Animals sacrificed after 28 days of treatment.
- Euthanasia, all animals were killed by carbon dioxide inhalation.
- Macroscopic pathology: The necropsy included a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. Abnormalities and interactions were noted and the required tissue samples preserved in fixative. The adrenals, brain, epididymides, heart, liver, kidneys, spleen, thymus and testes were dissected free of adjacent fat and other contiguous tissue and were weighted.
- Tissues preserved for histopathology: Tissues of the adrenals, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum, liver, kidney, lungs, lymph nodes (mandibular, mesenteric), ovaries, prostate, sciatic nerve (only one), spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder and uterus with cervix were fixed in 4% neutral buffered formaldehyde (except the testes and epididymides which were initially placed in Bouins fluid and subsequently retained in 70 % industrial methylated spirit) and processed for subsequent light microscopic examination. Femoral bone marrow smears were taken from all animals killed. Samples of any other abnormal tissues were also retained for histopathological examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.
-Tissues preserved, but not examined: The other sciatic nerve was not processed histologically but is held in 4% neutral buffered formaldehyde pending possible future requirement for microscopic examination.
-Histology: Tissue samples from the animals specified below were dehydrated, embedded in paraffin wax, sectioned at approximately five micron thickness and stained with haematoxylin and eosin. Those tissues subjected to histological processing included the following regions: Adrenals (cortex and medulla), Brain (cerebellum, cerebral cortex and medulla), Heart (auricular and ventricular), Kidneys (cortex, medulla and papilla), Liver (section from each of the left and median lobes), Lungs (section from each of the left and right caudal lobes), Spinal cord (transverse section at the cervical, thoracic and lumbar Ievels), Stomach (keratinised and glandular), Thyroid (included parathyroids in section), Uterus (uterus section separate from cervix section). For bilateral organs, sections of both were examined. A single section was prepared from each of the remaining tissues required for microscopic pathology.
-Microscopy: Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used minimal, slight, moderate, marked or severe. The tissues specified above were examined for all animals of Groups 1 and 4. The kidneys, liver and lungs were examined for all animals of groups 2 and 3.
For body temperature, landing foot splay and grip strength (fore and hind), homogeneity of variance was tested using Bartlett's test at the 1% level. Dunnett's test was then used to compare each treated group with the control. For motor activity scores (high and low beam) non-parametric tests were used because the data was not distributed normally. Wilcoxon test (exact Bonferroni-adjusted) was applied to compare each treated group with the control. The significance of inter-group differences in haematology and blood chemistry was assessed by student's t-test using a pooled error variance. Statistical significancesfor eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed. For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fiher test was used to perform pairwise comparisons, otherwise a Dunnett´s test was used. Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.

Results and discussion

Results of examinations

Details on results:
-Signs and mortality: No animals died during the study and no signs of a reaction to treatment were seen.
-Bodyweight: Bodyweights were not adversely affected by treatment. The slightly lower gain in males receiving 15 mg/kg/day and slightly higher gain in females receiving 150 mg/kg/day, in comparison with the controls, were, in the absence of similar findings at 500 mg/kg/day, considered to represent normal biological variation.
-Food consumption: Food consumption was not affected by treatment. The slightly lower food intake by males receiving 15 mg/kg/day and the slightly greater food intake by females receiving 150 mg/kg/day were not attributed to treatment since there was no affect upon food intake for animals receiving 500 mg/kg/day.
-Food conversion efficiency: The efficiency of food conversion was unaffected by treatment.
-Open field observations: Open field observations during the four weeks of treatment showed relatively minor inter-group differences which were considered to be due mainly to natural variation. Two females receiving 500 mg/kg/day, however, showed consistently slight or moderate gait abnormalities during Weeks 2, 3 and 4 of treatment. Gait in these individuals was described as unsteady or slow and/or deliberate on each occasion. During Week 1 of treatment, slight body tremor was noticed during handling in two males receiving 500 mg/kg/day but no tremor was observed in the open field or at any other occasion. Females receiving 500 mg/kg/day appeared to show increased reactivity during removal from the cage during Week 2 of treatment, with three individuals being classified as difficult, but normal reactivity was recorded at subsequent assessments. These findings were not attributed to treatment. One male receiving 500 mg/kg/day showed very dark faeces during both Week 2 and Week 3. One male receiving 15 mg/kg/day showed blood stained faeces during both Week 3 and Week 4. However, it was different individuals within each group on each occasion. These findings were considered to relate to the dark colour and irritant nature of the test material.
-Sensory reactivity: Landing footsplay and body temperature measurements during Week 4 of treatment showed some inter-group variation but there were no consistent treatment or dosage-related trends to suggest an involvement with Potassium Ferrite. Landing footsplay at 500 mg/kg/day was higher than controls for males but lower than controls for females and these differences were not ascribed to treatment. Furthermore, the trend in males was apparent before treatment commenced. All other sensory reactivity responses were similar in all groups.
-Grip strength: Forelimb grip strength values in males receiving 500 mg/kg/day were lower than controls. However, this difference was also apparent pre-treatment and so was not considered to be related to treatment. Hindlimb grip strength values for females in all treated groups were higher than in control animals during Week 4 but, because of the natural variation often seen in this parameter, the lack of a similar effect in males and the lack of dosage relationship, an involvement with treatment was considered unlikely.
-Motor activity: Motor activity scores showed an unusually high degree of inter-group variation when recorded at both pre-treatment and Week 4 of treatment. High beam and low beam scores (rearing and general locomotor activity respectively) for both males and females in all treated groups were lower than those of controls during Week 4. However no dosage-relationship was evident. In addition, scores for males in all treated groups were similarly decreased before treatment began. lt was therefore concluded that motor activity was unaffected by treatment with Potassium Ferrite.
-Haematology. peripheral blood: There were no haematological findings in Week 4 that were considered to be due to treatment. Some minor differences between control and treated animals occurred, none of which was considered to be of any toxicological significance. These included slightly high mean cell haemoglobin and mean cell volume in females receiving 500 mg/kg/day, slightly high lymphocyte count in males and females receiving 500 mg/kg/day and high platelet count in females receiving 150 or 500 mg/kg/day. Other inter-group differences that attained statistical significances were either minor, lacked dosage-relationship or were confined to one sex and were therefore not attributed to treatment.
- Haematology. bone marrow: Treatment with Potassium Ferrite did not affect the cellularity and composition of the bone marrow.
- Blood chemistry: Blood chemistry investigations during Week 4 of treatment revealed, compared with the controls, marginally high total protein concentrations in males receiving 500 mg/kg/day. The difference from controls was small and not considered to be toxicologically significant. Other inter-group differences that attained statistical significances were either minor, lacked dosage-relationship or were confined to one sex and were not attributed to treatment.
- Organ weights: Organ weights were not affected after four weeks of treatment with Potassium Ferrite.
- Macroscopic pathology: Macroscopic examination after four weeks of treatment revealed depressed areas in the stomach of two males that received 500 mg/kg/day and abnormal contents of the gastro-intestinal tract in two females that received 500 mg/kg/day, comprising dark contents of the caecum in one animal and dark contents in the ileum, caecum and colon of the other animal. There were no other findings that were attributed to treatment with Potassium Ferrite.
- Microscopic pathology: All findings were consistent with those expected in a study of this duration in this species. A number of findings were seen in the stomach of animals receiving 500 mg/kg/day that could relate to the irritant nature of the test material. The incidence was, however, low.

Effect levels

Dose descriptor:
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion