Betaine

Endpoint summary

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 OECD TG 471, which was performed in accordance with GLP, EU B.10 standardized method also performed following GLP, and OECD TG 476 performed following GLP.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 MAR 1989 to 01 OCT 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: primary cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9

Vehicle / solvent:

Betaine was completely solved in culture medium: bicarbonate buffered RPMI 1640 medium containing 15% foetal bovine serum (both Gibco Europe Ltd.), 1% phytohaemagglutinin (PHA, Burroughs Wellcome) and 100 units/ml penicillin and streptomycin.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in the presence of S-9 metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

in the absence of S-9 metabolic activation

Evaluation criteria:

Chromatid and chromosome gaps and breaks as well as fragments and re-arrangements were all recorded on raw data sheets with the stage coordinates (vernier readings) of every cell containing aberrations.

Statistics:

Chi squared -test

Key result

Species / strain:

lymphocytes: human

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

Rangefinder Mitotic Index (MI) and Relative Mitotic Index
Point		MI	Relative MI	Point		MI	Relative MI
Control	-S-9	855	100	Control	+S-9	13.45	100
1.6 µg/mL	-S-9	10.17	118.9	1.6 µg/mL	+S-9	11.11	82.6
8 µg/mL	-S-9	9.06	106.0	8 µg/mL	+S-9	8.45	62.8
40 µg/mL	-S-9	7.03	82.5	40 µg/mL	+S-9	7.24	53.8
200 µg/mL	-S-9	8.18	95.7	200 µg/mL	+S-9	7.14	53.1
1000 µg/mL	-S-9	10.51	112.9	1000 µg/mL	+S-9	4.90	36.4
2000 µg/mL	-S-9	9.96	116.5	2000 µg/mL	+S-9	5.97	44.4
4000 µg/mL	-S-9	11.13	130.5	4000 µg/mL	+S-9	3.95	29.4
7000 µg/mL	-S-9	13.17	154.0	7000 µg/mL	+S-9	5.74	42.7
10000 µg/mL	-S-9	14.07	164.6	10000 µg/mL	+S-9	5.16	38.4

 

Metaphase Analysis Without Activation (-S9)
Treatment	Replicate No.	No. cells scored	Gaps	Aberrations per 100 cells				Iso-locus Del.	Total Aberrations		% cells with aberrations		Others*	Mitotic index
				Chromosome		Chromatid
				Del.	Exchange	Del.	Exchange		+gaps	-gaps	+gaps	-gaps
Control	A	100	6	0	0	0	0	0	6	0	6	0	0	9.70
	B	100	6	0	0	0	0	0	6	0	5	0	0	6.06
	Mean/100 cells based on 200 cells scored		6	0	0	0	0	0	6	0	5.5	0	0	7.88
1000 µg/mL	A	100	1	0	0	0	0	0	1	0	1	0	0	13.56
	B	100	1	0	0	2	0	0	3	2	3	2	0	9.93
	Mean/100 cells based on 200 cells scored		1	0	0	1	0	0	2	1	2	1	0	11.75
3333 µg/mL	A	100	5	0	0	0	0	0	5	0	5	0	0	4.64
	B	100	1	1	0	1	0	0	3	2	3	2	0	7.65
	Mean/100 cells based on 200 cells scored		3	0.5	0	0.5	0	0	4	1	4	1	0	6.15
10000 µg/mL	A	100	4	0	0	0	0	0	4	0	4	0	0	7.66
	B	100	2	0	0	0	0	0	2	0	2	0	0	3.77
	Mean/100 cells based on 200 cells scored		3	0	0	0	0	0	3	0	3	0	0	5.72
*Cells which showed fragmentation were excluded from the calculation of aberrations per 100 cells but are included in cells with aberrations.

 

Metaphase Analysis With Activation (+S9)
Treatment	Replicate No.	No. cells scored	Gaps	Aberrations per 100 cells				Iso-locus Del.	Total Aberrations		% cells with aberrations		Others*	Mitotic index
				Chromosome		Chromatid
				Del.	Exchange	Del.	Exchange		+gaps	-gaps	+gaps	-gaps
Control	A	100	7	0	0	0	0	0	7	0	7	0	3	8.57
	B	100	0	0	0	1	0	0	1	1	1	1	0	4.61
	Mean/100 cells based on 200 cells scored		3.5	0	0	0.5	0	0	4	0.5	4	0.5	1.5	6.59
1000 µg/mL	A	100	11	0	0	4	0	0	15	4	14	4	0	5.22
	B	100	6	0	0	2	0	0	8	2	8	2	0	7.88
	Mean/100 cells based on 200 cells scored		8.5	0	0	3	0	0	11.5	3	11	3	0	6.55
3333 µg/mL	A	100	11	0	0	1	0	0	12	1	12	1	1	8.53
	B	100	1	0	0	1	0	0	2	1	2	1	0	7.69
	Mean/100 cells based on 200 cells scored		6	0	0	1	0	0	7	1	7	1	0.5	8.11
10000 µg/mL	A	100	12	0	0	0	0	0	12	0	11	0	1	5.78
	B	100	4	0	0	1	0	0	5	1	5	1	1	3.54
	Mean/100 cells based on 200 cells scored		8	0	0	0.5	0	0	8.5	0.5	8	0.5	1	4.66
*Cells which showed fragmentation were excluded from the calculation of aberrations per 100 cells but are included in cells with aberrations.

 

Positive Controls
Treatment	Replicate No.	No. cells scored	Gaps	Aberrations per 100 cells				Iso-locus Del.	Total Aberrations		% cells with aberrations		Others*
				Chromosome		Chromatid
				Del.	Exchange	Del.	Exchange		+gaps	-gaps	+gaps	-gaps
MMC (-S9)	A	25	12	0	4	20	12	8	56	44	40	36	0
	B	25	12	4	0	12	4	8	40	28	44	36	8
	Mean/100 cells based on 50 cells scored		12	2	2	16	8	8	48	36	42	36	4
CPA (+S9)	A	25	12	8	0	16	0	8	44	32	36	32	0
	B	25	8	0	0	12	0	12	32	24	32	28	4
	Mean/100 cells based on 50 cells scored		10	4	0	14	0	10	38	28	34	30	2
*Cells which showed fragmentation were excluded from the calculation of aberrations per 100 cells but are included in cells with aberrations.

 

Conclusions:

Based on this study betaine does not have clastogenic properties, when tested in human lymphocytes at concentrations up to a limit of 10000 microgram per millilitre.

Executive summary:

An in vitro mammalian chromosome aberration test with primary human lymphocytes was performed with the test substance according to EU Method B.10. No genotoxicity or cytotoxicity was observed during the study. The test substance does not have clastogenic properties, when tested in human lymphocytes at concentrations up to a limit of 10000 µg/mL.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2022 to 07 December 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

- S. typhimurium: Histidine locus
- E. coli: Tryptophan locus

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Preliminary test: 50, 100, 200, 400, 800, 1600, 3200 and 5000 g/plate
- Based on the results of the preliminary test the following doses were selected for the main test: 50, 158, 500, 1581 and 5000 μg/plate

Vehicle / solvent:

Sterile water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other:

Details on test system and experimental conditions:

Preliminary Toxicity Test:
- Tester Strain Culture: A loopful of the tester strain, TA 100 was inoculated into a tube containing Oxoid Nutrient broth No. 2 and the tube was incubated at 37 ± 1 ºC for 17 hours.
- Plating Procedure: One hundred microlitres (100 μL) each of the respective dilutions and the stock solution equivalent to test doses (50, 100, 200, 400, 800, 1600, 3200 and 5000 g/plate) was mixed with 2 mL of soft agar containing histidine and biotin, 500 μL of S9 mix (for the test in the presence of metabolic activation) or 500 μL of PBS (for the test in the absence of metabolic activation), 100 μL of overnight TA100 culture and overlaid onto pre-labeled VB agar plates in duplicate. A sterile water control, similarly treated, was maintained. After the agar had set, these plates were incubated at 37 ± 1 °C for 68 hours. The number of revertant colonies on the VB agar plates was counted and the bacterial background lawn was evaluated for the evidence.

Mutation Assay:
- Test Strain Cultures: Each of the tester strains from the master plates were inoculated into the respective tubes containing Oxoid Nutrient broth No. 2 and the tubes were incubated at 37 ± 1 °C for approximately 17 hours.
- Performance of the Assay:
No. of Replicates: Three replicate plates were maintained for the initial as well as the confirmatory mutation assays.
Plating Procedure: Initial mutation assay was conducted using the direct plate incorporation method. The test constituents were transferred into sterile test tubes, mixed and overlaid onto VB agar plates and allowed to set. The plates were then incubated at 37 ± 1 °C for 68 hours. Revertant colonies were counted manually and the plates were examined for bacterial background lawn. The confirmatory mutation assay was conducted using the pre-incubation method. The test constituents were transferred into sterile test tubes and kept in an incubator shaker for 20 minutes at 37ºC. After this period, 2 mL soft agar containing histidine-biotin / tryptophan were added to each of the tubes. The tube contents were mixed and overlaid onto VB agar plates and allowed to set. The plates were then incubated at 37 ± 1 °C for 68 hours. Revertant colonies were counted manually and the plates were examined for bacterial background lawn.
Viable Counts: The bacterial suspension of each tester strain was diluted up to 10-6 dilution in 9 ml PBS. One hundred microlitres from the 10-6 dilution of each tester strain was mixed with 2 mL of soft agar and plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1 °C for 68 hours for the initial as well as the confirmatory mutation assays. After incubation, the number of colonies in each plate were manually counted and expressed as the number of colony forming units per mL of the bacterial suspension.

CRITERIA FOR ACCEPTABILITY OF THE TEST:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• Tester strain integrity
• All Salmonella typhimurium tester strains and the Escherichia coli tester strain used in this assay must demonstrate their growth potential in the
• presence of histidine and tryptophan, respectively.
• All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and to ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation, respectively.
• The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of uvrA mutation.
• Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.
• The spontaneous reversion rates in the vehicle control must be in the range of in-house historical data.
• There must be at least five non-toxic dose levels.
• The top dose selected should demonstrate toxicity. In case of non-toxic test items, the top dose tested should be 5000 μg/plate.
• All tester strain culture titers must be in the range of 1-2x109 cells/mL to ensure that appropriate numbers of bacteria are used for plating.
• The positive control substances should produce a more than 3-fold increase in mutant colony frequencies when compared to the respective vehicle control plates.

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two
increasing concentrations of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle
control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and
TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility Test: The test item was soluble in Sterile water at 50 mg/mL.
Preliminary Toxicity Test and Justification for the Selection of Target Top Dose: The test item did not cause precipitation on the basal agar plates at any of the tested doses. In a preliminary toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the OECD 471-recommended top dose of 5000 g/plate was tested in the mutation assay.
Viable Counts of the Overnight Tester Strains: Viable counts of all the tester strains were within the required range of 1-2 x 109 CFU/mL for the initial toxicity-mutation as well as the confirmatory mutation assay.

Conclusions:

Under the study conditions, Betaine is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The study was conducted to determine the mutagenic potential of Betaine in Bacterial Reverse Mutation Test in accordance with the OECD Guideline 471 in compliance with GLP. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay both in the presence and absence of metabolic activation system.

The test item was soluble in Sterile Water at 50 mg/mL. In a preliminary toxicity test, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the OECD 471-recommended top dose of 5000 g/plate was tested in the mutation assay.

The bacterial tester strains were exposed to the test item in triplicate at 50, 158, 500, 1581 and 5000 μg/plate using the direct plate incorporation mode of exposure in the initial mutation assay and using the preincubation mode of exposure in the confirmatory mutation assay. The vehicle control (Sterile water) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results of the study from both the initial and confirmatory mutation assay showed that the test item did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

Under the study conditions, Betaine is considered to be non-mutagenic in the bacterial reverse mutation assay.

Reference 3

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

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 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

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system 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.

Key result

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.

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.