Betaine

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Betaine

EC Number:

203-490-6

EC Name:

Betaine

Cas Number:

107-43-7

Molecular formula:

C5H11NO2

IUPAC Name:

(trimethylammonio)acetate

Details on test material:

Betaine monohydrate, >95% purity

Method

Species / strain

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.

Controls

Controls 1

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

Controls 2

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

Results and discussion

Test results

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

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.