Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Micronucleus tests in m9ice on 39 food additives and eight miscellaneous chemicals
Author:
Hayashi M. et al.
Year:
1988
Bibliographic source:
Fd Chem. Toxic. 26(6), pp. 487-500

Materials and methods

Principles of method if other than guideline:
In vivo micronucleus assay in mouse by intraperitoneal route by single or repeated (4 times at 24 h interval) exposure.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-week old
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Saline
Details on exposure:
Intaperitoneal route:
- single injection
- 4 injections at 24-hour interval between injections
Frequency of treatment:
Single dose or 4 doses at 24-hour interval.
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
no. of dose: 1
sampling time: 24 h
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
no. of dose: 1
sampling time: 24 h
Dose / conc.:
1 200 mg/kg bw/day (actual dose received)
Remarks:
no. of dose: 1
sampling time: 24 h
Dose / conc.:
2 400 mg/kg bw/day (actual dose received)
Remarks:
no. of dose: 1
sampling time: 24 h
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
no. of dose: 4
time between doses: 24 h
sampling time: 24 h
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
- Substance: mitomycin C
- Route of administration: intraperitoneal
- Doses / concentrations: 2 mg/kg

Examinations

Tissues and cell types examined:
Femoral marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: maximum dose levels set at the supposed maximum tolerated dose referring to LD50

DETAILS OF SLIDE PREPARATION: Femoral marrow cells were flushed out with foetal bovine serum and smeared on clean glass slides. Cells were mixed with methanol for 5 min. and stained with Giemsa.

METHOD OF ANALYSIS: preparations were coded and analysed without any knowledge of the treatment. 1000 polychromatic erythrocytes per mouse were scored using a light microscope, with a high power objective (× 100) and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The portion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Statistics:
Before evaluating the test data statistically, the frequencies of MNPCEs in concurrent negative and positive control groups were compared with the control charts of our historical data to confirm the technical validity of the experiment. To estimate the true spontaneous level, it should be more reliable to use historical control data instead of the results from concurrent controls which could fluctuate.
According to the historical negative control data, the number of MNPCEs per mouse followed binomial distributions with P = 0.00209 and n = 1000 (laboratory
1) and P = 0.00200 and n = 1000 (laboratory 2).
A two-stage statistical procedure was used. In the first step of the procedure, the frequency of MNPCEs in each treatment group was compared with the binomial distribution specified by historical control data from the laboratory (1 or 2).
In the second step, the dose-response relationship was tested by the Cochran-Armitage trend test. A positive result was recorded only when one or more treatment group(s) showed a statistically significant difference (P < 0.01) from the spontaneous level of MNPCEs and the trend test indicated a positive dose response (P < 0.05). This procedure was not applied to tests with multiple treatments which, in most cases, contained only one dose group. Such data were evaluated statistically using the first step only.
A Monte-Carlo simulation study showed that the probability of a type I error (the probability of a false positive), in this two-step procedure was, in general, closer to the nominal significance level (P = 0.01) than that of the usual conditional binomial test which has been widely used to evaluate micronucleus test data.
Similarly, this method was a more powerful method of analysis than the conditional binomial test. A detailed description of this statistical procedure will be reported separately.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Test compound was found to be negative after single treatment, thus it was assayed by the multiple treatement at the maximum tolerated dose expected from the single treatment test. None of the treatments (4 doses) showed a significant induction of micronuclei when compared with historical control data.

dose mg/kg MNPCE (%) PCE (%) mortality trend test
test substance (single dose) 0 0.27 ± 0.08 62.8 ± 6.7 0/6 not significant
300 0.13 ± 0.08 59.6 ± 3.4 0/6
600 0.26 ± 0.23 62.4 ± 8.6 1/6
1200 0.18 ± 0.16 54.2 ± 10.8 0/6
2400 0.1 43.4 5/6
mitomycin C 2 6.15 ± 1.70* 43.5 ± 5.2 0/6
test substance (4 doses at 24 h interval) 300 0.18 ± 0.15 54.3 ± 7.8 0/6

* significantly different from historical control

Applicant's summary and conclusion

Conclusions:
Non mutagenic.
Executive summary:

Method

In vivo micronucleus test in mouse. Test substance in saline vehicle was given to groups of 6 male mice per dose by intraperitoneal route. Single injection at doses of 0 (negative control), 300, 600, 1200 and 2400 mg/kg was done, followed by sampling after 24 hours. Four injections at 24 -hour interval were done at dose of 300 mg/kg. Mitomycin C 2 mg/kg was used as positive control. Mortality, percentage of micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes (PCE) were recorded.

Results

Mortality was recorded starting at dose of 600 mg/kg. MNPCE (%) and PCE (%) did not significantly differ from controls. No significant induction of micronuclei was noted either upon single or repeated treatment.