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Toxicological information

Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
not reported.
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Guideline study (OECD 474) with acceptable restrictions. Justification of read-across: Dioctyltin bis (IOMA) and dioctyltinnbis (2-EHMA) are isomers of the same compound and are expected to be chemically and toxicologically equivalent. Restrictions : less than the recommended number of dose levels of test substance were used. Purity and source of test material are not reported and a non-standard positive control substance was used. Test material differs to that being registered.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
less than the recommended number of dose levels of test substance were used.
Principles of method if other than guideline:
In this assessment of the effect of ZK 30 434 on the incidence of micronucleated polychromatic erythrocytes in mice, a total dosage of 4500 mg/kg bodyweight was administered by oral gavage, in two equal dosages, separated by an interval of 24 hours.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Details on test material:
Test article: ZK30434, Dioctyltin bis(IOMA) [CAS No. 26401-97-8]:Octyltin tris [CAS No.26401-86-5] (80:20% mixture)
Lot no.: RU 20/77
Physical properties: colourless liquid

Test animals

Details on test animals and environmental conditions:
Breeder: Charles River Laboratory, Kent
Initial body weights: 23-25 g
Housed in plastic disposable cage (5/cage)
Acclimitisation: one week
Maintained at room temperature 22 ± 2 ºC and 50% relative humidity with 20 changes of air/hour
Photoperiod: illuminated by artifical light for 12 hours/day
Diet: Spratts aLab Diet No. 1 and tap water ad libitum
Fasted overnight before treatment
Mice (CD1) were pretested to determine target dose for micronucleus test.

Administration / exposure

Route of administration:
oral: gavage
1% methylcellulose
Details on exposure:
Total dose (in 2 administrations) were 2250, 4500, 1900 mg/kg; positive control mitomycin C = 14 mg/kg
Information on experimental design
Group Material Conc mg/mL Dose mL/10g Dose mg/kg Total (2 doses) M F
1a control (veh) - 0.1 - - 5 5
5 ZK 30434 112.5 0.1 1125 2250 5 5
6 ZK 30434 225 0.1 2250 4500 5 5
7 ZK 30434 450 0.1 4500 9000 5 5
8 control (pos) 0.7 0.1 7 14 5 5
Duration of treatment / exposure:
72 hours
Frequency of treatment:
Two dosages; twenty-four interval
Post exposure period:
12, 24, 36 and 48 hours post exposure groups
Doses / concentrations
Doses / Concentrations:
4500 mg/kg bw
actual ingested
No. of animals per sex per dose:
20/sex (40) for vehicle and postive controls
5/sex (10) for each time point of ZK 30434 treated animals
Control animals:
yes, concurrent vehicle
Positive control(s):
40 mg/kg body weight methotrexate in physiological saline (two administrations by gavage 24 hours apart.).


Tissues and cell types examined:
bone marrow smear examined for the presenc of micronuclei in 2000 polychromatic and 2000 normochromatic erythrocytes per mouse.
Details of tissue and slide preparation:
A direct bone marrow smear (from epiphysis from each femur) was made and placed on a methanol-cleaned slide. Smears were air dried and fixed in methanol overnight. After fixation the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor, 1 part Giemsa:9 parts buffered distilled water) for 10 minutes. After rinsing in buffered water, slides were air-dried, then defatted in xylene for 10 minutes and mounting in DPX. Stained smears were examined by light microscopy.
Evaluation criteria:
Incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic ertyhrocytes per animal was determined for each animal.
Ratios were analyzed by Barlett's test < 0.001 using non-paraetric methods based on Kruskal-Wallis mean ranks. These method are more than robust agains inequality of variance than classical variance methods.

Postive control group dosed with methotrexate and the groups dosed with ZK 30 434 were compared to vehicle control using the non-parametric equivalent of the least significant differences.

Results and discussion

Test results
However, bone marrow depression observed
at 48 hours hypopnoea and tremors observed; 4 mortalities
Vehicle controls validity:
Negative controls validity:
not specified
Positive controls validity:
Additional information on results:
Bone marrow depression is observed, which is an indication that the test item reached the bone marrow..

Any other information on results incl. tables

Normochromatic to polychromatic ratios: vehicle control gave group mean ratios of 0.97, 1.0, 1.01, and 1.06 ratios at the 12, 24, 36 and 48 hour time points. ZK 30 434 gave group mean ratios higher that those obtained with the concurrent controls: 1.80, 2.09, 3.29, and 11.99. The ratio increased with time. Positive control gave mean ratios of 2.07, 10.76, 19.18 ratios at the 12, 24 and 36 hour time points. After 48 hours too few cells available to count. Micronucleated normochromatic and polychromatic erythrocyte counts: ZK 30 434 normchromatic counts were not different from controls at 12, 24 or 36 hour time points. At 48 hours the micronucleated polychromatic cell counts were significantly different from control. Positve normochromatic and polychromatic cell counts at the 12, 24 and 36 hour time points. At 48 hours the micronucleated normochromatic cell count was not different from control.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
Under the conditions studied, ZK 30 434 failed to show any evidence of mutagenic potential when administered orally in this test procedure. However, evidence of bone marrow depression was observed.
Executive summary:

The effect of ZK 30 434 on the incidence of micronucleated polychromatic erythroctyes in mice was assessed; a total doseage of 4500 mg/kg bodyweight was administered by oral gavage in 2 equal doses, separated by 24 hours. A negative control group (1% methylcellulose) and positive control group (methotrexate, 40 mg/kg body weight) were run concurrently. Mice were sacrificed 12, 24, 36, and 48 hours after the second dose and bone marrow smears were examined for the presence of microncueli in 2000 polychromatic and 200 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also determined in each mouse.

The group mean micronucleated cell counts in polychromatic and normochromatic erythrocytes obtained after ZK 30 434 treatment were comparable with the concurrent control after 12, 24 and 36 hours. After 48 hours the micronucleated normochromatic cell count was slightly higher than that of the concurrent control (least significant difference method). At 48 hours the micronucleated polychromatic erythrocyte count was comparable to control.

The normochromatic to polychromatic ratio at each period was higher than that for the concurrent control value. The positive control produced the expected increase in both the group mean micronucleated cell counts and also in the normochromatic to polychromatic erythrocyte ratio at 12, 24, 36 hours. After 48 hours the micronucleated normochromatic erythrocyte count was comparable with the concurrent control. There were too few polychromatic cells to count and was not possible to use this parameter in analysis.

It was concluded that ZK 30 434 did not show mtuagenic potential when administered orally in the test procedure. However, evidence of bone marrow depression was observed at each kill. The small increase in micronucleated normochromatic cells at the 48 hour timepoint, though significantly different from the concurrent control, cannot be considered evidence of mutagenic potential since a coreesponding rise in micronucelated polychromatic cells had not been observed in an earlier time point.