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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Betaine has been tested for genetic toxicity in vitro and in vivo, and no toxicity was found. In vitro-testing include EEC Method B14 of Annex V, which was performed in accordance with GLP, EU B.10 standardized method also performed following GLP, and OECD TF 476 performed following GLP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus of L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C, Each batch of frozen cells was tested and found to be free of mycoplasma contamination. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F-68 (F0P). All media contained antibiotics.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Assay - 4.61 to 1180 μg/mL
Mutagenicity Assay - 148, 295, 590, 885 and 1180 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: based on the solubility of the test article and compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (presence and absence of S9), or 24 hours (absence of S9 only)
- Expression time (cells in growth medium): For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment.
Cell population adjustments to 3x10e5 cells/mL were made as follows:
• 4-hour treatment – 1 and 2 days after treatment.
• 24-hour treatment – immediately after test article removal, and 2 and 3 days after treatment.

SELECTION
- Cells from selected dose levels were cultured in triplicate with 2-4 μg TFT/mL at a density of 1x10e6 cells/100-mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100-mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 10 or 11 days.
- The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10-4), and was expressed as TFT-resistant mutants/10e6 surviving cells. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the vehicle control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥90 mutants/10e6 clonable cells at the Aberdeen meeting in 2003

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition relative to the vehicle control
Evaluation criteria:
* A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibit induced mutant frequencies of ≥90 mutants/10e6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/10e6 clonable cells, a doubling of mutant frequency over the vehicle would also be required.
* A result was considered negative if the treated cultures exhibit induced mutant frequencies of less than 90 mutants/10e6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
Statistics:
Cultures treated with a minimum of four concentrations of test article must be evaluated and their mutant frequencies reported. Results may be accepted, with justification, when only three concentrations of test article are evaluated and otherwise meet the other criteria for a valid test. The highest test article concentration must produce 80 to 90% toxicity unless limited by solubility or the maximum required concentration. In the case of a test article with a steep toxicity curve (no concentrations with 10 to 20% survival), the results may be considered acceptable if a concentration spacing of ≤2-fold is used and the highest concentration tested showed <20% survival or total kill
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of precipitation/pH: No visible precipitate was observed at the beginning or end of treatment, and the test article had no adverse impact on the pH of the cultures.
- Water solubility: The test article formed a clear solution in sterile water at a maximum concentration of ~50 mg/mL in the solubility test conducted at BioReliance.

RANGE-FINDING/SCREENING STUDIES:
The test item was prepared in sterile distilled water and evaluated in a preliminary toxicity assay at concentrations from 4.61 to 1180 μg/mL [the highest concentration evaluated approximated the 10 mM limit dose for this assay; dose formulations were adjusted for purity (97%) using a correction factor of 1.03]. Relative suspension growth (RSG) was 110, 105 and 101% at a concentration of 1180 μg/mL (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively).




The treated cultures exhibited 86 to 99%, 92 to 101% and 96 to 115% RSG (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively), and were cloned. Relative total growth of the cloned cultures ranged from 75 to 113% (4-hour treatment with S9), 77 to 98% (4-hour treatment without S9) and 105 to 128% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants per 10e6 clonable cells were observed under any treatment condition.

The trifluorothymidine-resistant colonies for the positive and vehicle control cultures were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Conclusions:
The results indicate the test item was negative in the L5178Y/TK+/- Mouse Lymphoma Assay, in the presence and absence of metabolic activation.
Executive summary:

The test substance was evaluated to determine its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of Aroclor-induced rat liver S9. The test substance was prepared in sterile distilled water and evaluated in a preliminary toxicity assay at concentrations from 4.61 to 1180 μg/mL [the highest concentration evaluated approximated the 10 mM limit dose for this assay; dose formulations were adjusted for purity (97%) using a correction factor of 1.03]. No visible precipitate was observed at the beginning or end of treatment, and the test article had no adverse impact on the pH or osmolality of the cultures. Relative suspension growth (RSG) was 110, 105 and 101% at a concentration of 1180 μg/mL (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively).

Based on the results of the preliminary toxicity assay, cultures were treated at concentrations of 148, 295, 590, 885 and 1180 μg/mL under all three treatment conditions in the mutagenicity assay. No visible precipitate was observed at the beginning or end of treatment, and the test article again had no adverse impact on the pH of the cultures. The treated cultures exhibited 86 to 99%, 92 to 101% and 96 to 115% RSG (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively), and were cloned. Relative total growth of the cloned cultures ranged from 75 to 113% (4-hour treatment with S9), 77 to 98% (4-hour treatment without S9) and 105 to 128% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants per 106 clonable cells were observed under any treatment condition. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. These results indicate the test substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay, in the presence and absence of metabolic activation, under the conditions and according to the criteria of the test protocol.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 APR 1989 to 24 APR 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
EEC Method Method B14 of Annex V study performed in accordance with GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Betaine does not exhibit any genotoxic properties up to 5000 micrograms, when tested according to Method B14 (EEC, Annex V) and following GLP.
Executive summary:

The test substance was evaluated to determine its genotoxic potential to bacterial cells (S. typhimurium TA1535, TA1537, TA98 and TA100) in the presence and absence of metabolic activation according to EU Method B.13/14. The test substance did not exhibit any genotoxic properties up to 5000 µg, the highest dose tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Betaine has been tested for genetic toxicity in vitro and in vivo, and no toxicity was found. For in vivo-testing OECD TG 474 study in accordance with GLP was performed.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 JUN 1989 to 29 AUG 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study has been performed according to OECD TG 474 following GLP standards.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (+-4)
- Humidity (%): 50 (+-20)
- Air changes (per hr): --
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
- 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:
30
Positive control(s):
cyclophosphamide
- 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.
Statistics:
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.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Betaine has been tested in various in vitro and in vivo settings against genetic toxicity. Betaine has not indicated genetic or clastogenic toxicity even with the relatively high doses used in all of the testing.

Justification for classification or non-classification

Not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 based on negative results from Ames, chromosome aberration, and mouse lymphoma tests in vitro, as well as mouse micronucleus in vivo.