Choline hydrogen carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: - Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium strains TA1535, TA1537, TA98, TA100 : negative with and without metabolic activation (similar to OECD Guideline 471 on read-across substance Choline chloride) - Chromosome aberration (in vitro mammalian chromosome aberration test) and DNA damage and/or repair (sister chromatid exchange assays in mammalian cells): CHO cells, negative with and without metabolic activation (similar to OECD Test Guidelines 473 and 479 on read-across substance Choline chloride) - Chromosome aberration (in vitro mammalian chromosome aberration test) and DNA damage and/or repair (sister chromatid exchange assays in mammalian cells): CHO cells, negative with and without metabolic activation (no guideline available / information not sufficient, on read-across substance Choline chloride)

Link to relevant study records

Reference

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.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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

Target gene:

not applicable

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

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.

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

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

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'.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In general, all available results were derived from Choline chloride, which serves as a read-across substance for Choline hydrogen carbonate 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 organic choline moiety is the toxicologically more relevant part here. The bicarbonate anion, or the carbonate anion in general, which is in equilibrium with its decomposition products water and carbon dioxide, are ubiquitous present in the body or the cell in general. Also, in bacteria or cell culture experiments, possible changes in the pH value are neutralized by the buffer-containing media. Furthermore, the atmosphere in incubation chambers usually contain approx. 5 % CO2 and so carbon dioxide or the related carbonates would be present in the cell culture medium anyhow. So, the outcome of mutagenicity tests can be reasonably expected to be identical to the ones with Choline chloride, which indicate no mutagenic potential of choline at all.

In addition, there are several literature data on bicarbonates, i.e. Sodium bicarbonate, available, which state the bicarbonate also not to have any mutagenic activity:

Sodium hydrogen carbonate was tested negative in the Ames test (plate incorporation, ± S9 mix) using S. typhimurium strains TA1535, TA 100, TA1538, TA 98 and TA 1537, leading to less than 0.0002 revertants per nmole. It was tested also negative in the E.coli DNA repair test (rec-assay) in the E.coli strains WP2, WP69 and CM871 ± S9 mix (De Flora S et al. (1984), Mutation Research, 133 (1984) 161-198). Also, sodium bicarbonate was tested negative in both Ames test (S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94, TA98, 10 mg/plate, ± S9 mix) and chromosome aberration test (Chinese hamster fibroblast cell line, CHL, without S9 mix, 48 h incubation of 1 mg/ml) (Ishidate M et al. (1984), Fd Chem. Toxic. Vol. 22, No. 8, pp. 623-636).

 

So, regarding the available studies evaluating the mutagenic potential of choline, the following observations and conclusion were made:

 

The available study on Gene mutation in bacteria (Key study, Bacterial reverse mutation assay / Ames test, Haworth, 1983) was performed independently in three laboratories revealing consistent negative results with and without metabolic activation. This study was performed similar to OECD Guideline 471 and assessed as reliable with minor restrictions due to the fact that it was performed on the read-across substance Choline chloride and only S. typhimurium strains TA1535, TA1537, TA98 and TA100 were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of Choline chloride or choline bicarbonate, this restriction is considered to be negligible. As a consequence, the results on Choline chloride obtained in these studies can be considered to be reliable due to their consistency and the good performance of the studies, and are sufficient to cover the endpoint ‘Gene mutation in bacteria’ with negative results also for choline bicarbonate.

 

The stipulated endpoint ‘Mammalian chromosome aberration’ was covered with two studies on chromosome aberrations, one reliable with minor restrictions (due to read-across from Choline chloride; key study, Galloway 1985) and a non-assignable supporting study with consistent results (Bloom, 1982). Both studies do not only contain results on chromosome aberrations but also on the induction of sister chromatid exchanges. The key study was performed in two independent laboratories similar to OECD Test Guidelines 473 & 479 and no deviations were identified. 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, partly ambiguous results were obtained from for the induction of both SCE and CA. However, e.g. the lacking in dose-response or the fact that a positive result was obtained only for one dose, allow to draw the conclusion that these partly ambiguous results may be considered therefore not as specific for the test item and hence, Choline chloride and so Choline bicarbonate can be considered as negative in both SCE and CA assays in both studies and in all testing laboratories. Hence also based on these results, Choline chloride and so Choline bicarbonate does not need to be classified as mutagen.

 

A study explicitly performed to cover the endpoint ‘Gene mutation in mammalian cells’, e.g. according OECD 476, is not available. However, mutations are hereditary modifications of the DNA base pair sequence. The transversion from modifications to mutations can occur due to mispairing of the complementary strain during DNA replication because the modified base was not recognized properly. Also, which is the relevant part here, it can occur due to defective DNA repair.

Normally, when the modified base, which could lead to possibly lethal mutations, was recognized by the nucleotide excision repair, helicases are recruited and cut out an oligonucleotide which includes the damaged nucleotide. Afterwards, the resulting gap is filled again by repair synthesis with the complementary strain as model. In case the complementary strain is not available, the sister chromatid, if available, serves as matrix and the gap is filled by homologous recombination. Therefore, so-called Holliday junctions are formed between the chromosomes. If this bond between the two sister chromatids is not cleaved properly after finishing the repair process, parts of the sister chromatids are exchanged which would lead to a positive result in a sister chromatid exchange assay.

A positive result in a mammalian cell mutation assay is based on a forward mutation of the gene of interest in the recent assay. Also, this occurs when a base modification is not recognized and repaired properly during cell repair and protection mechanisms.

So, since both sister chromatid exchanges and mutations and hence the either positive or negative results detected in the relevant assays are mainly due to defective DNA repair. So, the results obtained in a SCE assay, although it is assigned to cytogenicity assays, are strongly indicative for the results which can be obtained by a forward mutation assay, and can consequently serve as a surrogate for an e.g. OECD 476 study.

Since the key study on sister chromatide exchanges was assessed as reliable with minor restrictions only due to read-across, revealed negative results with and without metabolic activation and serves as a surrogate for a forward mutation assay, the endpoint ‘Gene mutation in mammalian cells’ can be considered to be covered with negative results, too.

 

According to REACH Regulation 1907/2006/EC Annex VIII column 2 “Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.” and Annex IX column 2 states that “If there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant.“

Since the reliable results gained in the three in vitro endpoints ‘Gene mutation in bacteria’, ‘Mammalian chromosome aberration’ and ‘Gene mutation in mammalian cells’ (from deduction) are negative and Choline chloride as well as choline bicarbonate is not identified as a mutagen, an in vivo study does not need to be performed, neither out of regulatory nor scientific reasons: All in vitro assays were additionally performed in the presence metabolic activation, and therefore the in vivo situation (i.e. in humans) is mimicked precisely enough to allow to draw justified conclusions of the possible outcome of an in vivo study.

 

Consequently, no data gaps were identified as the available data are sufficient to cover this endpoint, and Choline hydrogen carbonate does not need to be classified as mutagenic, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.

Justification for selection of genetic toxicity endpoint
Most reliable key study (Klimisch 1) covering two endpoints: in vitro mammalian chromosome aberration assay (chromosome aberration) and sister chromatid exchange assay in mammalian cells (DNA damage and repair).

Justification for classification or non-classification

All available studies to cover this endpoint (‘Gene mutation in bacteria’, ‘Mammalian chromosome aberration’ and Sister chromatide exchange, strongly indicative for ‘Gene mutation in mammalian cells’), performed on the read-across substance choline chloride, have a negative outcome and consequently choline chloride and so choline bicarbonate does not need to be classified as mutagenic, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.