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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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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
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Secondary literature source

Data source

Reference Type:
Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.
Witt, K.L., Knapton, A., Wehr, C.M., Hook, G.J., Mirsalis J., Shelby M.D., MacGregor J.T.
Bibliographic source:
Environ Mol Mutagen. 2000;36(3):163-94.

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Mouse peripheral blood micronucleus test was performed on samples collected from a 90 day subchronic toxicity study.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
No information provided

Test animals

Details on species / strain selection:
Mice provide a convenient model for the study because unlike rats, mice do not selectively remove micronucleated cells from the peripheral circulation. Therefore, the frequency of micronucleated cells in the circulating blood of mice would be expected to closely reflect the frequency in bone marrow, and unlike bone marrow sampling, repeated peripheral blood measurements can be made, without disturbing the physiology of the animals, by sampling a few microliters of blood at a time.
Details on test animals or test system and environmental conditions:
No information provided

Administration / exposure

Route of administration:
oral: gavage
Corn oil
Details on exposure:
No information provided
Duration of treatment / exposure:
90 days
Frequency of treatment:
No information provided
Post exposure period:
No information provided
Doses / concentrationsopen allclose all
Dose / conc.:
0.075 other: g/kg
Dose / conc.:
0.15 other: g/kg
Dose / conc.:
0.3 other: g/kg
Dose / conc.:
0.6 other: g/kg
Dose / conc.:
1.2 other: g/kg
No. of animals per sex per dose:
Number o animals that were analysed in the end of the study:

Control: 10
0.075 g/kg: 9
0.15 g/kg: 10
0.3 g/kg: 10
0.6 g/kg: 10
1.2 g/kg: 5

Control: 10
0.075 g/kg: 9
0.15 g/kg: 7
0.3 g/kg: 9
0.6 g/kg: 10
1.2 g/kg: 4
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control group was not included in the test. This is because the toxicity bioassay from which the test animals were obtained did not include a positive control group. However, positive control slides were included in the experiment to control for staining and scoring procedures.


Tissues and cell types examined:
Details of tissue and slide preparation:
Blood was obtained immediately before or at the time of sacrifice (90 days).

Drops of blood were spread on precleaned standard glass microscope slides, air dried, and fixed in absolute methanol for 5 min. All slides were coded prior to scoring by a person not involved in reading the slides.
Evaluation criteria:
Identification of micronucleus (MN) was based on fluorescence emission characteristic of the fluorescent stain used (blue with UV excitation and orange with green [540nm] excitation with Hoechst/pyronin stain, or yellow to greenish yellow with acridine orange stain).

Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated method, in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 or 5,000 erythrocytes.
The micronucleus results were tabulated as the mean frequency of micronucleated erythrocytes per 1000 cells per animal, plus or minus the standard error of the mean among animals within a treatment group.

The frequency of micronucleated cells among normochromatic erythrocytes or polychromatic erythrocytes was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group.

The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Results and discussion

Test results
Key result
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

The authors highlight that the chemicals that produced equivocal or negative results in the mouse peripheral blood MN test, predictivity for noncarcinogenic activity in rodents was poor.

Any other information on results incl. tables

Table 1: Micronucleated Erythrocyte Frequencies in Peripheral Blood of B6C3F1 Mice from NTP Subchronic Toxicity Study, males

Dose (g/kg)NMN-NCE/1000 NCEP value% PCE
Control101.85 ± 0.1803.17 ± 0.21
0.07591.94 ± 0.180.23603.44 ± 0.25
0.15101.57 ± 0.100.93423.42 ± 0.31
0.3101.77 ± 0.100.65373.17 ± 0.21
0.6101.50 ± 0.130.97253.29 ± 0.18
1.252.05 ± 0.070.16013.49 ± 0.45
Trend test  p=0.424  
ANOVA   p=0.873


Table 2: Micronucleated Erythrocyte Frequencies in Peripheral Blood of B6C3F1 Mice from NTP Subchronic Toxicity Study, females

Dose (g/kg)NMN-NCE/1000 NCEP value% PCE
Control101.35 ± 0.1303.09 ± 0.23
0.07591.20 ± 0.140.76673.26 ± 0.30
0.1571.38 ± 0.210.43723.14 ± 0.40
0.391.23 ± 0.190.70533.54 ± 0.21
0.6101.27 ± 0.050.61023.27 ± 0.26
1.241.48 ± 0.190.25864.18 ± 0.16
Trend test  p=0.236  
ANOVA   p=0.195




Applicant's summary and conclusion

In this assay the test substance showed a negative result after 90 days exposure via oral gavage.
Executive summary:

In a study conducted by Witt et al (2000) the mouse peripheral blood micronucleus (MN) test was performed on samples collected from a sub-chronic toxicity study conducted by the National Toxicology Program (NTP). The incidence on polychromatic erythrocytes (PCE) provided an index of damage induced within 72 hours of sampling whereas the incidence of MN in the normocromatic erythrocytes (NCE) population at steady state provided an index of the average damage during the 30 -day period preceding sampling.


In a B6C3F1 mouse peripheral blood micronucleus assay, mice were treated by oral gavage with Butyraldehyde at doses of 0, 0.075, 0.15, 0.3, 0.6 and 1.2  g/kg bw. The vehicle was corn oil. Blood samples were taken at the day of termination.  


There was not a significant increase in the frequency of micronucleated NCE and PCE in the mouse peripheral blood micronucleus test.