Choline hydrogen carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: chromosome aberration and DNA damage and/or repair

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The present study is combined study on the effects of the read-across substance choline chloride on both the induction of sister chromatid exchanges and chromosomal aberrations in CHO cells and was performed in two independent laboratories similar to OECD Test Guidelines 473 & 479. No deviations were identified, and the documentation is sufficient to assess the reliability of the study results. The only reason leading to the classification of this study as Klimisch 2 but one is that the study was performed on a read-across substance and not Choline bicarbonate itself.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test and sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Choline chloride

EC Number:

200-655-4

EC Name:

Choline chloride

Cas Number:

67-48-1

IUPAC Name:

2-hydroxy-N,N,N-trimethylethanaminium chloride

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Choline chloride (ChCl; CAS No. 67-48-11)
- Other: supplied by the National Toxicology Program chemical repository (Radian Corp., Austin, TX)

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5A medium supplemented with 10 % fetal calf serum (FCS), and L-glutamine (2 mM). Penicillin (100 units/mL) and streptomycin (100 µg/mL) were used at Litton Bionetics, Inc. (LBI) but not routinely at Columbia University (CU)
- Properly maintained: yes: To maintain karyotypic stability, the cells were used at no more than 15 passages after cloning; they were recovered routinely from stocks stored in liquid nitrogen, and maintained by transferring twice a week

Additional strain / cell type characteristics:

other: obtained from Dr. Sheldon Wolff, cloned, and designated CHO-W-B 1

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction prepared from male Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0, 0.005, 0.05, 0.5, 50, 500 µg/mL (SCE & CA CU, ±S9)
0, 0.005, 0.05, 0.5, 5, 50, 500 µg/mL (SCE, LBI, ±S9)
0, 0.05, 0.5, 5, 50, 500, 5000 µg/mL (CA, LBI, ±S9)
0, 1000, 2000, 3000, 4000, 5000 µg/mL (CA, LBI, -S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Controls

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

triethylenemelamine

benzo(a)pyrene

cyclophosphamide

mitomycin C

other: N-Methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 2 h
- Exposure duration: total 26 h
- Expression time (cells in growth medium): 26 h
- Fixation time (start of exposure up to fixation or harvest of cells): 28-29 h (SCE), 34-38 h (CA)

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays):
- SCE: fluorescence plus Giemsa (FPG) technique: BrdUrd, Hoechst 33258, Giemsa
- CA: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: SCE: 50 cells /dose level; CA: 100 / dose level

DETERMINATION OF CYTOTOXICITY
- Method: other: Cultures were exposed to a five-log range of concentrations of test compound in a half-log series of doses. Immediately before fixation, the cultures were examined under the inverted microscope. The degree of confluence of the monolayer and the occurrence of large, round healthy cells (mitotic cells) on the surface of the cell sheet or floating in the medium was noted. If there was no evidence of toxicity, cells from only the top five or six dose levels were fixed. For toxic compounds, cells from the highest dose likely to yield analyzable metaphases were fixed, together with cells from five successively lower dose levels.

Evaluation criteria:

As described more precisely in 'Any other information on materials and methods incl. tables', the evaluation reflects a blend of statistical analysis and biological intuition. The former is influenced by the evidence for dose response, determined by tests for trend, whereas the latter takes into account reproducibility, as judged by the number of doses at which the results are elevated compared with the appropriate control. Analyses for SCE and CA assess the evidence both for a dose response and for an absolute increase at any dose.

Statistics:

As described more precisely in 'Any other information on materials and methods incl. tables', because the capacity of this free-text field is limited, the statistical analysis of the test results regarding SCEs was performed according:
The statistical analysis of SCE data was based on an assumption of random sampling from a Poisson density for the number of SCEs scored in cells under common experimental conditions. Extensive analyses of counts of SCEs per cell from a control flask or from replicate control flasks within the same trial strongly support the Poisson model.
The statistical analysis involved regressing linearly the average number of SCEs / chromosome on the logarithm of the test compound dose [ArmitageP(1955): Tests for linear trends in proportions and frequencies. Biometrics 11:375-386].
Since logarithm of zero is undefined, the control was incorporated into this analysis as if, on a logarithmic scale, it were spaced below the lowest log-dose an amount equal to the average spacing of consecutive log-doses. The log-dose was used as the independent variable in the regression because to use the doses themselves when they are in a log or half-log series would give extreme weight to the response at the highest dose. The resulting trend test statistic was referred to a table of normal probabilities from which a P-value or observed level of significance was read.
Based on experience with the SCE test, they decided to examine the number of doses at which the average SCE/cell was elevated 20% over the concurrent control. Statistically this translates into a conservative criterion: when 50 cells are scored for each of three doses, and the solvent control data are in the range of 7.5-9.5 SCE/cell, the probability under Poisson sampling that the mean for a single dose will be elevated 20% by chance (“false positive”) is<0.01: the probability of observing by chance two or three of the three doses with means at least 20% above the control mean is approximately 0.001.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

for both SCE and CA induction

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

other: positive, but lacking in dose-response (LBI) / only one dose positive (CU), trend P<0.005, for SCE induction; confirmation required

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

other: negative (LBI) / only one dose positive (CU, contributed by simple breaks), for CA induction

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2A: Induction of SCEs by Choline chloride, CU, -S9, Trial decision: ?+

Dose / µg/mL	Total chromosomes	Total SCE	SCE per cell
0.00	1046	353	7.09
0.50 E-02	1045	337	6.77
0.50 E-01	1046	328	6.59
0.50	1047	388	7.78
0.50 E+02	1047	415	8.32
0.50 E+03	1044	434	8.73*

Trend statistic: 0.44 E+01; Trend probability: 0.55 E-05

 

Table 2B: Induction of SCEs by Choline chloride, CU, +S9, Trial decision: ?w

Dose / µg/mL	Total chromosomes	Total SCE	SCE per cell
0.00	1048	361	7.23
0.50 E-02	1047	385	7.72
0.50 E-01	1047	382	7.66
0.50	1046	410	8.23
0.50 E+02	1047	408	8.18
0.50 E+03	1048	431	8.64

Trend statistic: 0.26 E+01; Trend probability: 0.42 E-02

 

Table 2C: Induction of SCEs by Choline chloride, LBI, -S9, Trial decision: NA

Dose / µg/mL	Total chromosomes	Total SCE	SCE per cell
0.00	526	145	5.79
0.50 E-01	1051	315	6.29
0.50	1111	362	6.84
0.50 E+01	1067	362	7.12*
0.50 E+02	1046	339	6.81
0.50 E+03	1032	396	8.06*
0.50 E+04	1067	323	6.36

Trend statistic: 0.20 E+01; Trend probability: 0.22 E-01

 

Table 2D: Induction of SCEs by Choline chloride, LBI, +S9, Trial decision: +*

Dose / µg/mL	Total chromosomes	Total SCE	SCE per cell
0.00	1051	354	7.07
0.50 E-01	1058	441	8.75*
0.50	1048	414	8.30
0.50 E+01	1062	474	9.37*
0.50 E+02	1056	404	8.03
0.50 E+03	1048	390	7.81
0.50 E+04	1040	391	7.90

Trend statistic: 0.15; Trend probability: 0.44

 

 

Table 3A: Induction of Chromosome aberrations by Choline chloride, CU, -S9, Trial decision: ?+

Dose / µg/mL	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.00	100	1	1	0
0.50 E-02	100	0	0	0
0.50 E-01	100	0	0	0
0.50	100	0	0	0
0.50 E+02	100	3	3	0
0.50 E+03	100	10*	7	0
Trend statistic		0.44 E+01	0.38E+01	0.00
Trend probability		0.45E-05	0.13E-03	0.50
Endpoint summary		?+	?w	-

 

Table 3B: Induction of Chromosome aberrations by Choline chloride, CU, +S9, Trial decision: -

Dose / µg/mL	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.00	100	2	2	0
0.50 E-02	100	3	3	0
0.50 E-01	100	3	3	0
0.50	100	5	3	2
0.50 E+02	100	4	4	1
0.50 E+03	100	4	3	1
Trend statistic		0.91	0.58	0.13 E+01
Trend probability		0.17	0.28	0.89 E-01
Endpoint summary		-	-	-

 

Table 3C: Induction of Chromosome aberrations by Choline chloride, LBI, -S9, Trial decision: -

Dose / µg/mL	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.00	100	0	0	0
0.50 E-01	74	3	0	1
0.50	100	0	0	0
0.50 E+01	100	1	0	1
0.50 E+02	100	2	0	2
0.50 E+03	100	2	0	2
0.50 E+04	100	1	1	0
Trend statistic		0.78	0.15 E+01	0.71
Trend probability		0.22	0.72 E-01	0.24
Endpoint summary		-	-	-

 

Table 3D: Induction of Chromosome aberrations by Choline chloride, LBI, -S9, Trial decision: -

Dose / µg/mL	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.00	100	0	0	0
0.10 E+04	100	2	2	0
0.20 E+04	100	1	0	1
0.30 E+04	100	0	0	0
0.40 E+04	100	6*	2	4
0.50 E+04	100	1	1	0
Trend statistic		0.15 E+01	0.45	0.16 E+01
Trend probability		0.73 E-01	0.33	0.55 E-01
Endpoint summary		-	-	-

 

Table 3E: Induction of Chromosome aberrations by Choline chloride, LBI, +S9, Trial decision: -

Dose / µg/mL	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.00	100	0	0	0
0.50 E-01	100	1	0	1
0.50	100	2	0	2
0.50 E+01	100	1	0	1
0.50 E+02	100	0	0	0
0.50 E+03	100	0	0	0
0.50 E+04	100	3	2	1
Trend statistic		0.95	0.21 E+01	-0.22
Trend probability		0.17	0.17 E-01	0.28
Endpoint summary		-	-	-

 

 

CU: Columbia University

LBI: Litton Bionetics, Inc.

*: Significantly increased values are marked with an asterisk.

?+, ?w: see Table 1 (Summary Judgment of SCE and CA)

NA: analyzable owing to lack of an adequate number of cells in dose or control

-: negative

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation partly ambigous, responses are not attributed to the compound in dose-dependent manner

The present study is a combined study on the effects of the read-across substance choline chloride on both the induction of sister chromatid exchanges and chromosomal aberrations in CHO cells and was performed in two independent laboratories similar to OECD Test Guidelines 473 & 479. No deviations from the guideline were identified, and the documentation is sufficient to assess the reliability of the study, which was assessed as reliable with restrictions only due to the fact that it was performed on a read-across substance. Consequently, the results are also reliable and sufficient to cover the endpoint “Genetic toxicity in vitro – chromosome aberrations”.
Also, choline chloride serves as a read-across substance for choline bicarbonate without relevant restrictions: When dissolved in water, both choline salts dissociate readily in the organic choline cation and the corresponding anion, i.e. chloride or bicarbonate. So, the results obtained from choline chloride in the available in vitro genotoxicity tests can be transferred without any modification to choline bicarbonate.
The results on both SCE and CA induction with metabolic activation are negative and are considered to be the more reliable ones because they mimic an in vivo situation more precisely. Choline as a dietary supplement is excessively metabolized to the methyl donor betaine and other substances in the liver of e.g. rats (Finkelstein et al.(1982), Arch. Biochem. Biophys. Vol. 218, No. 1, October 1, pp. 169-173). Without metabolic activation, one lab (LBI) observed positive results for SCE induction, but lacking in dose-response, and the other lab (CU) observed a positive result for only one dose. The authors concluded that these results need a confirmation to be considered as positive. Additionally, the latter positive result was obtained at a very high dose (0.5 mg/mL) and may therefore be due to osmolality. Regarding the induction of Chromosome aberrations without metabolic activation, choline chloride was tested negative by LBI and only one positive result for one dose was obtained by CU, which was mainly contributed by simple breaks and may be considered therefore not as specific for the test item.
In summary, the negative results obtained from testing choline chloride with metabolic activation and partly ambiguous results obtained from testing without S9 mix for the induction of both SCE and CA allow the conclusion to consider choline chloride and hence choline bicarbonate as negative in both assays. Hence, choline chloride and so choline bicarbonate does not need to be classified as mutagen, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

Executive summary:

In two combined mammalian cell cytogenetic assays (Chromosome aberrations (CA) and sister chromatid exchange (SCE), performed in two laboratories on the read-across substance choline chloride similar to OECD Test Guidelines 473 & 479), CHO cell cultures were exposed to choline chloride in culture medium at concentrations of 0, 0.005, 0.05, 0.5, 50, 500 µg/mL (SCE & CA CU, ±S9), 0, 0.005, 0.05, 0.5, 5, 50, 500 µg/mL (SCE, LBI, ±S9), 0, 0.05, 0.5, 5, 50, 500, 5000 µg/mL (CA, LBI, ±S9) and 0, 1000, 2000, 3000, 4000, 5000 µg/mL (CA, LBI, -S9) with and without metabolic activation (Liver S9 fraction prepared from male Sprague-Dawley rats induced with Aroclor 1254). Choline chloride was tested up to concentrations of 5 mg/mL, which corresponds to 5.9 mg/mL choline bicarbonate.

Choline chloride was tested negative for both CA and SCE induction with metabolic activation. Without metabolic activation, there were partly ambiguous results for the induction of SCEs (lacking dose response (LBI) and only one positive dose (CU)) and mainly negantive results for the induction of CA (except only one dose positive (CU, contributed by simple breaks)). Hence, these results are not attributed to the compound in dose-dependent manner and can therefore be neglected.

Positive controls induced the appropriate response. In summary, there was no evidence or a concentration related clear positive response of chromosome aberrations or SCEs induced over background with or without metabolic activation.

This study is classified as acceptable with restrictions, i.e. reliability Klimisch 2. It satisfies the requirements for both OECD Test Guidelines 473 (In Vitro Mammalian Chromosome Aberration Test) and 479 (Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells) for in vitro mutagenicity data.