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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:
supporting study
Study period:
12 JUN 1989 to 29 AUG 1989
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study has been performed according to OECD TG 474 following GLP standards.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
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:
Betaine monohydrate, >98% purity

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Ltd, Maidstone, Kent (UK)
- Age at study initiation: 8-9 weeks
- Weight at study initiation: males: 25.9-35.1g and females 23.6-32.0g
- Assigned to test groups randomly: yes
- Fasting period before study: 4 hours before dosing with cavage
- Housing: solid bottomed polypropylene cages, stainless steel mesh lids, bedding: graded softwood sawdust
- Diet (e.g. ad libitum): R&M expanded diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no information
- Temperature (°C): 21 (+-4)
- Humidity (%): 50 (+-20)
- Air changes (per hr): --
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: betaine is water soluble and saline optimized for mammalian physiology
- Concentration of test material in vehicle: range finding 0.5, 1, 1.5, 2 g/kg (50, 100, 150 and 200 mg/ml) and 0.5, 1, 2 g/kg in the micronuclues study
- Amount of vehicle (if gavage or dermal): 10ml/kg
Details on exposure:
An oral dose rangefinding study in mice was carried out using betaine monohydrate at 500mg, 1, 1.5 and 2 g/kg. From the results obtained no toxicity was observed. The dose levels to be used in the micronucleus test were set at 500mg, 1 and 2g/kg. Groups of 30 animals (15 males and 15 females) were given single oral dose of betaine monohydrate at the above dose levels. A solvent control group of 30 animals received doses of 0.9% saline. 5 male and 5 female animals from the solvent control and each treatment group were sacrificed at 24, 48 and 72 hours after dosing. One group of 10 animals was dosed with cyclophosphamide (CPA) as a positive control and sacrificed 24 hours after dosing. A minimum of 1000 polychromatic erythrocytes (PCE) were counted for each animal for coded slides. Vehicle control groups had micronucleus frequencies which were all within historical limits for control values for study laboratory previous studies. Positive control (CPA) animals, sacrificed at 24 hours, had significantly increased micronucleus frequencies compared to the relevant controls, showing the animals used to be sensitive to the effects of a known clastogen.
Micronucleus study:
10 ml/kg 0.9% saline (negative control)
0.5 g/kg, betaine
1 g/kg, betaine
2 g/kg, betaine
40 mg/kg, cyclophosphamide (positive control)
Duration of treatment / exposure:
single oral dose (gavage); 24, 48 and 72 hours.
Frequency of treatment:
single oral dose (gavage)
No. of animals per sex per dose:
Positive control(s):
- Justification for choice of positive control(s):
- Route of administration: oral cavage
- Doses / concentrations: 40 mg/kg


Tissues and cell types examined:
Bone marrow cells, polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
The femurs were exposed by dissectiing out the surrounding muscles and connective tissues and the shank of the bones removed from the ends. The bone marrow cells were aspirated by a 1ml syringe containing fetal calf serum into labelled centrifuge tubes with caps. Both femurs of each animal were aspirated into one centrifuge tube. The bone marrow cells were centrifuged (800rpm, 5 min) the supernatant removed and the cells were resuspended in a minimal volume of foetal calf serum by mixing on a rotary mixer (Whirlimixer). One drop of cell suspension was placed on each of 2 slides and spread by drawing the edge of a clean microscope slide along from the drop to tge end of the slide. All the slides were left to air dry and age for 24 hours before staining by the method of Gollapudi and Kamra (Mut.Res., 64 (1979) 45-46)
Cells were fixed for 5 minutes in methanol, rinsed twice in deionised water and stained for 10 minutes in Giemsa (1:6 Gurrs Giemsa R66 in deionised water) rinsed several times in tap water and finally in deionised water. After air drying, slides were cleaned in xylene for 1 minute, dried and mounted in Gurrs neutral mounting medium.
The mounted, dried slides were coded by a responsible person not connected with the scoring of the slides. This person devised a unique, unambiguous code for each animal at each time point (including positive controls) and used adhesive labels to cover existing information, so that cytogeneticists could only see the time of sacrifice (24, 48 and 72 hours), the sex of the animal and the new code.
Evaluation criteria:
Scoring of micronuclei. A minmum of 1000 polychromatic erythrocytes (PCE) including micronucleated PCE (MN-PCE) was counted for each animal, the numbers of normochromatic erythrocytes (normocytes, NCE) and micronucleated NCE (MN-NCE) were also recorded. Only areas of slides of good technical quality and appropriate staining charasteristics were scored. The purpose of scoring micronucleated NCE is an internal check on accuracy of scoring. All the available data shows that one would not expect to see more than 5 MN-NCE in each animal. Consistent scoring of more than this number is an indication of other artefacts being counted as micronuclei.
Assessment of whether numbers of micronuclei scored differed significantly between groups and treated animals was by the Mann-Whitney U -test. In this test, individual values are ranked in numerical order for a joint group of control and treated animals. These values are assigned ranks and rank sums (R) calculated for control (Rc) and treated (Rt) animals.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Betaine is not genotoxic in vivo in mouse micronucleus test up to 2 grams per kilogram dose.
Executive summary:

Also, whilst conducting micronucleus test it is common to perform a maximum tolerated dose (80% of LD50) or a dose showing a toxic effect to the bone marrow by alteration of the normal PCE/NCE ratio. In this study, no decrease in PCE/NCE ratio was not found. In the dose range study no toxicity was found. In the actual micronuclei study no indication of genotoxic properties were found with the highest dose 2 g/kg.