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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
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other: GLP compliant guideline study, available as unpublished report, fully adequate for assessment.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
hydrate iron potassium hydride
Details on test material:
- Name of test material (as cited in study report): Potassium ferrite
- Product name: Mapico CG-20X
- Physical state: Reddish brown powder
- Composition of test material, percentage of components: K2Fe22O34; 83,8%, K2Fe10O16; 6.2%, K2CO3 1.5H2O; 10.0%
- Lot/batch No.: 23388-17 CG20X 407448A
- Expiration date of the lot/batch: 6 August 1997
- Storage condition of test material: Room temperature, dark, desiccated
- Stability under test conditions: not determined

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River UK Limited, Margate, Kent, England
- Weight at delivery: Males: 22-24g; females: 28-30g
- Housing: Plastic disposable cages
- Diet: RM1(E)SQC diet (Special Diet Services), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

- Temperature (°C): 22 (19-23)
- Humidity (%): 50 (42-60)
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
- Vehicle used:1 % aqueous methylcellulose
Details on exposure:
- Preliminary toxicity test: The purpose of this test was to determine a suitable dose level for use in the micronucleus test. This part of the study was carried out in two phases. The dosages employed in Phase I were used to give an approximate indication of the maximum tolerated dose, ie the highest dosage which would not induce excessive lethality. The dosages in Phase II were based on the outcome of Phase I and were used to refine the results obtained. In Phase I the following doses were tested: 250, 500, 1000 and 2000 mg/kg bw. In Phase II the dose range was refined as follows: 378, 504, 672, 896 mg/kg bw. During both phases, 2 male and 2 female rats were assigned per treatment group. Following dosing the animals were observed regularly during the working day for a period of 48 hours and any mortalities or clinical signs of reaction during the experiment were recorded. At the end of this period, surviving animals were killed and discarded.
- Micronucleus test: From the results obtained in the preliminary test, dose levels of 126, 252 and 504 mg/kg bodyweight were chosen for the micronucleus test.
Duration of treatment / exposure:
24 (all dose levels) and 48 hours (control and highest dose level)
Frequency of treatment:
Post exposure period:
not applicable
Doses / concentrations
Doses / Concentrations:
0, 126, 252 and 504 mg/kg bw
nominal conc.
No. of animals per sex per dose:
10 for the vehicle control
5 for potassium ferrite low- and mid-dose as well as for the positive control
10+2* for the high dose (* Additional animals, dosed concurrently, to replace any that might die)
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: oral: gavage
- Doses / concentrations: 12 mg/kg bw mitomycin C; 0,6 mg/mL in purified water


Tissues and cell types examined:
Bone marrow was taken from the femora and slides prepared using standard procedures. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCEs) per animal. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.
Details of tissue and slide preparation:
Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Five males and five females from all dose groups were sacrificed 24 hours after dosing. In addition five male and five female animals from the negative control and the highest level treatment group were sacrificed after 48 hours. The animals were killed by cervical dislocation. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 mL of pre-filtered foetal calf serum using a 2 mL disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). At least three smears were made from each animal. The prepared smears were fixed in methanol (>10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1:9 dilution of Gurr's improved R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.
Evaluation criteria:
- A finding is considered positive if the following criteria are met: statistically significant and dose related increase in the incidence of micronucleated immature erythrocytes compared to the current control group (p<0.01). However, individual and/or group mean values should exceed the laboratory historical control range.
- Bone marrow cell toxicity is normally indicated by a substancial and statistically significant dose-related decrease in the proportion of immature erythrocytes.
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided p-values were calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels were carried out with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance was detected (Agresri et al. 1990); for individual intergroup comparisons (i.e. the positive control group) this procedure simplified to a Straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores had been used, i.e. exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
Preliminary toxicity test:
results show that a dose level of 504 mg/kg was approximately the maximum tolerated since administration of slightly higher doses resulted in mortalities. This dose level was considered to be an appropriate maximum for use in the micronucleus test. Dose levels of 126, 252 and 504 mg/kg bodyweight were chosen for use in the main test.
Micronucleus test:
No mortalities were obtained in the micronucleus test. Clinical signs for the high level group were consistent with the MTD having been effectively achieved. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test. The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time [P>0.01]. Mitomycin C caused large, highly significant increases [P<0.001] in the frequency of micronucleated immature erythrocytes. The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time. A statistically significant decrease in the proportion of immature erythrocytes was obtained at the 48 hour sampling time after treatment of the animals with the test substance [P<0.01 using Wilcoxon's test]. This decrease could be indicative of slight bone marrow depression but, since the decrease was only slight and the values obtained were not unusually low (eg they were comparable to those obtained for the vehicle control at the 24 hour sampling time), the apparent decrease is considered more likely to have been the result of chance variation. Mitomycin C did not cause any statistically significant decreases in the proportion [P>0.01] - it should be noted that even very cytotoxic compounds such as Mitomycin C do not always produce a substantial decrease in this proportion as early as the 24 hour sampling time because of the lag caused by erythrocyte maturation.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
Potassium ferrite did not induce micronucleus formation in bone marrow erythrocytes of mice following treatment up to the maximum tolerated concentration (504 mg/kg bw).
Executive summary: