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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)

- 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)

- 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)

- Gene mutation study in mammalian cells - In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes: negative with and without metabolic activation (according to OECD Guideline 476)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
chromosome aberration and DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The present study is combined study on the effects of 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.
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
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
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

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: negative with metabolic activation and 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 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, which was assessed as reliable without restrictions. Consequently, the results are also reliable and sufficient to cover the endpoint “Genetic toxicity in vitro – chromosome aberrations”. 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 as negative in both assays. Hence, Choline chloride 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 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.

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 negative 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 without restrictions, i.e. reliability Klimisch 1. 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:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.02.2020 (Study plan) - 20.10.2020 (Study initiation: 21.03.2020; Experimental Start: 02.06.2020; Experiment completion: 31.07.2020; Study completion: 10.10.2020)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
adopted by the Council on July 29, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
(EPA 712-C-98-221), August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Exception: Test item charact. (identity/strength/purity/composition/stability/method of synthesis/location of documents for synthesis) is Sponsors responsibility. Dose formulations were used within 2h of formulating, but were not subject to analyt. verif.
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL : confidential

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in original container as supplied by the Sponsor, Room temperature (15 to 30 °C) - at a dry and cool and well-ventilated place

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Test item was diluted with distilled water
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: 185.4 µL/mL
Target gene:
hprt (gene that is responsible for Hypoxanthine-Guanine Phosphoribosyl Transferase)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-K1 cell line obtained from the Janapese Collection of Research Bioresources (JCRB)
- Suitability of cells: The relatively low spontaneous mutation frequency and the stable and easily recognizable karyotype has made the CHO-K1 cell line one of the suitable test models for mutagen-induced cytotoxicity and gene mutation studies. The Chinese Hamster Ovary (CHO-K1) cell line is easy to maintain, culture and it is the recommended test system by the guidelines, to detect the mutation at the hprt locus. The Chinese hamster ovary (CHO) cell line, CHO-K1 is suited for determining forward mutations at the hprt locus because it is a well characterized and validated system (Hsie el ah, 1981).


For cell lines:
- Absence of Mycoplasma contamination: free from mycoplasma contamination
- Number of passages if applicable: passage no. 27 was used in the cytotoxicty test and passage no. 35 was used in the main study
- Methods for maintenance in cell culture: a-MEM (Minimum Essential Medium, Eagle a-Modification with nucleosides) with nucleosides (Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was supplemented with antibiotic and antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 µg/mL and Amphotericin B: 0.25-0.5 µg/mL).
The medium to eliminate the existing mutants in the culture for treatment was prepared by addition of 2 mL of reconstituted HAT supplement to 98 mL of a-MEM w/o NS with 5% fetal bovine serum [50X vial of HAT media supplement was reconstituted using 10 mL of sterile a-MEM w/o NS. The reconstituted supplement contains 5 x 10^-5 Hypoxanthine, 2 x 10^-5 M Aminopterine and 8 x 10^-4 M Thymidine].
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 hours
- Modal number of chromosomes: 22
- Periodically ‘cleansed’ of spontaneous mutants: yes


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: a-MEM (Minimum Essential Medium, Eagle a-Modification with nucleosides) with nucleosides (Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was supplemented with antibiotic and antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 µg/mL and Amphotericin B: 0.25-0.5 µg/mL). At the time of selection Minimum Essential Medium Eagle a-modification without nucleosides (a-MEM w/o NS) with 10% dialyzed fetal bovine serum was used. Cells were incubated in a CO2 incubator at 37 ± 1 °C and 5% CO2
Cytokinesis block (if used):
none
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Moltox (Lot Number 4214)
- method of preparation of S9 mix: The S9 fraction is buffered and supplemented with the essential co-factors beta-NADP, KCL and Glucose-6- phosphate to form the "S9 mix".
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 fraction was used at a concentration of 2 % v/v in the final culture medium for main study.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): confirmed by Moltox
Test concentrations with justification for top dose:
The stock solution of 185.4 µL/mL was serially diluted to obtain stock solutions of 92.7, 46.35, 23.18, 11.59 µL/mL. A volume of 50 µL of relevant stock solutions was added to 4.950 mL of culture medium to obtain the final concentration in the culture medium.
Based on the results of solubility and precipitation tests, cultures were exposed to Choline Chloride at five concentration levels (two cultures/concentration level) from 0.116 to 1.854 µL/mL of culture medium (0.116 µL/mL, 0.232 µL/mL, 0.464 µL/mL, 0.927 µL/mL and 1.854 µL/mL), in the absence and presence of the metabolic activation (2 % v/v S9 mix) in the cytotoxicity test. There was no reduction in the relative survival up to the highest tested concentration, hence, 1.854 µL/mL (10 mM) of culture medium was selected as the highest test concentration for the main study as recommended in the OECD TG.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [aqueous solvents (water or saline or culture medium)] : distilled water
- Justification for choice of solvent/vehicle: the solvent of first choice is water
- Justification for percentage of solvent in the final culture medium: not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2 x 10E6 cells per culture flask
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): At the end of the exposure period, the treatment medium was removed from the flasks and the cell surface washed using Dulbecco's Phosphate Buffered Saline (DPBS). The cells were trypsinized and suspended in complete medium to obtain single cells. The cell concentration was determined using a hemocytometer and adjusted accordingly with complete medium. The cell concentration in the flasks was adjusted to 1 - 2 x 10E5 cells/mL. A sample taken from the cell suspension was serially diluted with complete medium to approximately 1000 - 2000 cells/mL. An aliquot of 100 µL was then dispensed onto the center of 60 mm tissue culture dishes and 5 mL of complete medium added. The plates were incubated for 8 days to determine relative survival and also to demonstrate the cytotoxic effect of selected test concentrations.
An appropriate volume was transferred to fresh twenty-eight tissue culture flasks to receive approximately 2 x 10E5 cells for each treatment, negative and positive control. These flasks were kept as expression flasks for mutation. All plates and flasks were incubated in a CO2 incubator at 37 ± 1 °C and 5 % CO2. At the end of the 8-day incubation period the cytotoxicity plates were removed and the medium decanted. The colonies were fixed, stained and washed. The colonies formed on each plate were counted and recorded for calculation of relative survival/cloning efficiency following treatment.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days (The cultures for mutation expression were subcultured on days 3 and 5, following culture processing on day 1. During the expression period (7 - 9 days) the cell concentration in flasks was adjusted by sub culturing on day 3 and day 5. The cells in each flask were Trypsinized and the cell concentration was adjusted to approximately 2 x 10E6 cells/culture, on day 3 and day 5, respectively and supplemented with fresh complete medium and incubated under standard conditions.)
- Selection time (if incubation with a selective agent): 8-days (An approximate volume of 1-2 mL of suspension (2 x 10E5 cells/mL) (Gupta R.S., 1984) was added into 100 mm culture dish with 10 mL of selective medium (a-MEM without nucleoside - complete medium containing 5 µg/mL of 6-thioguanine). For each treatment 12 dishes were maintained per replicate, i.e. 24 dishes/concentration. The plates were incubated at 37 ± 1 °C and 5% CO2 in humidified air using a CO2 incubator for 8 days.)
- Fixation time (start of exposure up to fixation or harvest of cells): approximately 16 days and 4 hours
- Method used: tissue culture flasks
- Seletive agent used: 2-amino-6-mercaptopurine (6-thioguanine) was used as selective agent at a concentration of 5 µg/mL alpha-MEM without nucleosides.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: An approximate volume of 1-2 mL of suspension (2 x 10E5 cells/mL) (Gupta R.S., 1984) was added into 100 mm culture dish with 10 mL of selective medium (a-MEM without nucleoside - complete medium containing 5 µg/mL of 6-thioguanine). For each treatment 12 dishes were maintained per replicate, i.e. 24 dishes/concentration. The plates were incubated at 37 ± 1 °C and 5% CO2 in humidified air using a CO2 incubator for 8 days.
- Number of cells seeded (2 + 10E5 cells/mL) and method to enumerate numbers of viable and mutants cells: On day 16, the plates were removed from the incubator and the medium decanted, colonies were then fixed using 3.7% formaldehyde for 10 minutes. The fixative was removed, and the colonies stained using 0.4% methylene blue for 10 minutes. The number of colonies formed on each replicate plate was counted and recorded. The number of colonies were used to calculate the absolute cloning efficiency at the time of selection, number of clonable cells and the mutant frequency per 1 x 10E6 clonable cells.
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: percent relative cloning efficiency
- Any supplementary information relevant to cytotoxicity: Precipitation was not observed up to the highest tested concentration of 1.854 µL/mL, in the absence and presence of the metabolic activation (2% v/v S9 mix).
The pH and osmolality at the beginning of the treatment at 1.854 µL/mL was 7.39 and 300 mOsm/kg H2O, respectively (compared to 7.45 and 318 mOsm/kg H2O in the negative control) in the absence of metabolic activation, while pH and osmolality at 1.854 µL/mL was 7.36 and 312 mOsm/kg H2O, respectively (compared to 7.41 and 317 mOsm/kg H2O in the negative control), in the presence of the metabolic activation. Hence, any significant change in the pH or osmolality was not observed up to 1.854 µL/mL in the absence and presence of the metabolic activation.
The percent relative cloning efficiency observed was 89.87, 83.97, 80.84, 78.25, and 63.39 in the absence of metabolic activation and 81.45, 79.56, 70.41, 59.82, and 44.10 at 0.116, 0.232, 0.464, 0.927, and 1.854 µL/mL in the presence of the metabolic activation, respectively.
Since no significant cytotoxicity was observed up to 1.854 µL/mL, 1.854 µL/mL of the culture media was selected as the highest concentration in the absence and presence of the metabolic activation for the main study experiment.
Rationale for test conditions:
according to guideline
Evaluation criteria:
Assay Acceptance criteria
This mutation assay was considered acceptable as it met the following criteria:
a. The concurrent negative control was considered acceptable for addition to the laboratory historical negative control database.
b. Concurrent positive controls induced responses that were compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
c. Two experimental conditions (i.e., with and without metabolic activation) were tested.
d. Five concentrations (not including the solvent and positive controls) with adequate number of cells and appropriate cytotoxicity were evaluated.

Assay Evaluation Criteria
The test item was considered clearly negative as:
a. None of the test concentrations exhibits a statistically significant increase compared with the concuwir hrrent negative control.
b. There is no concentration-related increase when evaluated with an appropriate trend test.
c. All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly negative response.
Statistics:
Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on treatment groups against the negative control group (excluding positive controls).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Any significant change in pH (± 1 unit) or osmolality (> 50 mOsm/kg H2O) was not observed at 0 and 4 h in any tested concentration (0.116, 0.232, 0.464, 0.927, 1.854 µL/mL) of culture medium.
- Data on osmolality: No relevant influence of the test item, either on pH or osmolality, was observed in the absence or presence of the metabolic activation.
- Possibility of evaporation from medium: not specified
- Water solubility: The test item was soluble in distilled water at 185.4 µL/mL
- Precipitation and time of the determination: No precipitation was observed up to 1.854 µL/mL
- Definition of acceptable cells for analysis: not specified

RANGE-FINDING/SCREENING STUDIES (if applicable): firstly a solublity test and additional precipitation, pH and Osmolality Tests were conducted. Moreover Cytotoxicity tests were conducted.

Solubility, Precipitation, pH and Osmolality Tests

Based on its molecular weight (139.6) and purity, the 10 mM concentration was selected as the highest applicable concentration. Hence, solubility of Choline Chloride was performed at 100X (185.4 µL/mL) concentration, in distilled water (DW) and additionally in DMSO for selection of vehicle. The test item was soluble in distilled water, and in dimethyl sulfoxide (DMSO), at 185.4 µL/mL. Hence, aqueous vehicle (distilled water) was selected as the suitable vehicle.

Any significant change in pH (± 1 unit) or osmolality (> 50 mOsm/kg H2O) was not observed at 0 and 4 h in any tested concentration (0.116, 0.232, 0.464, 0.927, 1.854 µL/mL) of culture medium. No precipitation was observed up to 1.854 µL/mL.

Cytotoxicity Test

Based on the results of solubility, precipitation, and osmolality test, cytotoxicity test was conducted at 0.116, 0.232, 0.464, 0.927, 1.854 µL of Choline Chloride/mL. Cytotoxicity due to the test item was assessed by calculating the percent relative cloning efficiency following the treatment. Precipitation was not observed up to the highest tested concentration of 1.854 µL/mL, in the absence and presence of the metabolic activation (2 % v/v S9 mix).

The pH and osmolality at the beginning of the treatment at 1.854 µL/mL was 7.39 and 300 mOsm/kg H2O, respectively (compared to 7.45 and 318 mOsm/kg H2O in the negative control) in the absence of metabolic activation, while pH and osmolality at 1.854 µL/mL was 7.36 and 312 mOsm/kg H2O, respectively (compared to 7.41 and 317 mOsm/kg H2O in the negative control), in the presence of the metabolic activation. Hence, any significant change in the pH or osmolality was not observed up to 1.854 µL/mL in the absence and presence of the metabolic activation.

The percent relative cloning efficiency observed was 89.87, 83.97, 80.84, 78.25, and 63.39 in the absence of metabolic activation and 81.45, 79.56, 70.41, 59.82, and 44.10 at 0.116, 0.232, 0.464, 0.927, and 1.854 µL/mL in the presence of the metabolic activation, respectively.

Since no significant cytotoxicity was observed up to 1.854 µL/mL, 1.854 µL/mL of the culture media was selected as the highest concentration in the absence and presence of the metabolic activation for the main study experiment.

Mutagenicity Test

No relevant influence of the test item on osmolality and pH value was observed in the absence and presence of the metabolic activation during the main study.

Weighted regression analysis was performed to evaluate the significant dose-related effect in the mutation frequency of cultures treated with the negative control group. Statistical analysis was not performed for the positive controls. The mean mutant frequency of the positive control exhibited a clear increase over the mean value of the negative control demonstrating that positive control had potential to induce gene mutations at the hprt locus of CHO-K1 in the absence and presence of the metabolic activation. This demonstrated that the S9 mix was capable of metabolizing a pro-mutagen to its mutagenic form(s), thus, demonstrating integrity of the S9 mix.

The regression equations for the main study are given below:

In the Main study, the regression equation for experiments in the absence of metabolic activation is as follows: Y = 1.1793 X + 15.276

In the Main study, the regression for experiments in the presence of metabolic activation is as follows: Y= 1.191 X + 15.212

The mutant frequency, in the negative control group was less than 20 per 10E6 clonable cells during the main study, validating the acceptability of the test system (Li, A.P. et al., 1987). Any significant dose-related increase in the mutation frequency was not observed in any treated concentration and the mutation frequency was comparable to that with the negative control group. The increased mutant if equency observed in the positive controls in main study demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

CONCLUSION

From results of this study, it is concluded that Choline Chloride does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, either in the absence or presence of the metabolic activation (2 % v/v S9 mix), under these experimental conditions, and it is considered to be negative for mutagenicity.

Conclusions:
Based on the results from this study, it is concluded that Choline Chloride does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells either in the absence or presence of the metabolic activation system (2 % v/v S9 mix) under the present experimental conditions.
Executive summary:

In a mammalian cell gene mutation assay [hprt locus], CHO-K1 cells cultured in vitro were exposed to Choline Chloride at different concentrations, in the absence and presence of the metabolic activation (2 % v/v S9 mix) for a period of 4 hours according to OECD TG 476 and under GLP compliance.

Based on the results of solubility and precipitation tests, cultures were exposed to Choline Chloride at five concentration levels (two cultures/concentration level) from 0.116 to 1.854 µL/mL of culture medium, in the absence and presence of the metabolic activation (2 % v/v S9 mix) in the cytotoxicity test. There was no reduction in the relative survival up to the highest tested concentration, hence, 1.854 µL/mL of culture medium was selected as the highest test concentration for the main study.

In the main study, the cultures were treated at five different concentration levels (two cultures/ concentration level) from 0.116 to 1.854 µL/mL of culture medium, in the absence and presence of the metabolic activation (2 % v/v S9 mix). No relevant influence of the test item, either on pH or osmolality, was observed in the absence or presence of the metabolic activation. No significant concentration related increase in the mutation frequencies were observed in any concentrations tested up to 1.854 µL/mL of culture medium in absence and presence of the metabolic activation (2 % v/v S9 mix). The induced mutation frequencies were comparable to that of the negative control group. All negative controls were within the historical control limit and positive controls showed an increase in the mutation frequency.

All criteria for a valid study were met as described in the study plan. Based on the results from this study, it is concluded that Choline Chloride does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells either in the absence or presence of the metabolic activation system (2 % v/v S9 mix) under the present experimental conditions.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-documented study similar to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, 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 TA1535, TA1537, TA98 and TA100 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, this restriction is considered to be negligible.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four S. typhimurium strains tested (TA1535, TA1537, TA98, TA100)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 fractions from male Sprague-Dawley rats and male Syrian hamsters, injected, ip, with Aroclor 1254 (200 mg/mL in corn oil) at 500 mg/kg
Test concentrations with justification for top dose:
Tests were performed in three laboratories with varying doses:
CWR: 0, 333, 1000, 3333, 10000, 20830 µg/plate
EGG, SRI: 0, 100, 333, 1000, 3333, 10000 µg/plate

Dose-setting experiment: 10 mg/plate or the limit of solubility

CWR: Case Western Reserve University. Dr. William Speck
EGG: Microbiological Associates (formerly EG&G Mason Research Institute), Dr. Steve Haworth
SRI: SRI International, Dr. Kristien Mortelmans
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Remarks:
glycerol, glycine, mannitol, sodium phosphate
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-AA)
Remarks:
tested on all strains in the presence of rat and hamster S-9
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine (NOPD)
Remarks:
tested on TA98 without S-9
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
tested on TA100 and TA1535 without S-9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
tested on TA1537 without S-9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
- Expression time (cells in growth medium): 1-2 × 10 exp8 cells
- Selection time (if incubation with a selection agent): 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: One or more parameters were used as an indication of toxicity: viability on complete medium (EGG) and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn (CWR, EGG, SRI)

CWR: Case Western Reserve University. Dr. William Speck
EGG: Microbiological Associates (formerly EG&G Mason Research Institute), Dr. Steve Haworth
SRI: SRI International, Dr. Kristien Mortelmans
Evaluation criteria:
A positive response was indicated by a reproducible, dose-related increase of revertant colonies, whether it be twofold over background or not.
Statistics:
According to Margolin, B.H., Kaplan, N., Zeiger, E. ( 1981): Statistical analysis of the Ames Salmonella/microsome test. Proc Natl Acad Sci USA 78:3779-3783.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
congruent results from all three testing laboratories
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Table 1: Mutagenic responses on Choline chloride on four S. typhimurium strains, Case Western Reserve University

Dose

µg/plate

TA 100

TA 1535

TA 1537

TA 98

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0

132 ± 12.1

183 ± 12.8

182 ± 25.6

6 ± 1.5

10 ± 1.8

11 ± 2.9

7 ± 1.2

9 ± 2.3

7 ± 0.3

10 ± 3.2

23 ± 1.5

25 ± 2.4

333

170 ± 14.6

203 ± 5.8

195 ±4.1

9 ± 1.7

7 ± 0.6

12 ± 2.4

7 ± 1.2

7 ± 2.0

8 ± 0.6

17 ± 3.2

24 ± 2.2

17 ± 2.1

1000

160 ± 10.7

199 ± 16.2

192 ± 11.5

10 ± 2.1

8 ± 0.9

12 ± 3.5

3 ± 0.3

10 ± 1.5

8 ± 2.7

14 ± 3.5

24 ± 4.0

31 ± 1.7

3333

174 ± 6.4

204 ± 17.1

208 ± 7.9

5 ± 0.9

5 ± 1.5

11 ± 3.5

3 ± 1.2

10 ± 1.2

7 ± 1.0

15 ± 2.6

22 ± 0.6

22 ± 4.0

10000

175 ± 11.0

193 ± 14.6

208 ± 20.6

9 ± 2.8

8 ± 1.9

9 ± 3.2

4 ± 0.3

11 ± 1.9

9 ± 1.5

14 ± 1.5

25 ± 4.4

20 ± 3.4

20830

185 ± 9.5

203 ± 6.7

201 ± 11.8

9 ± 0.6

9 ± 0.9

12 ± 2.6

5 ± 1.9

12 ± 1.5

6 ± 1.8

17 ± 3.2

25 ± 2.7

24 ± 2.7

POS

471 ± 7.8

2551 ± 47.3

2132 ± 23.0

450 ± 7.6

111 ± 6.0

121 ± 6.8

1315 ± 109.8

122 ± 9.9

178 ± 19.0

479 ± 22.8

2089 ± 207.8

2269 ± 106.2

 

Table 2: Mutagenic responses on Choline chloride on four S. typhimurium strains, Microbiological Associates (formerly EG&G Mason Research Institute)

Dose

µg/plate

TA 100

TA 1535

TA 1537

TA 98

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0

192 ± 3.5

160 ± 14.1

141 ± 2.5

20 ± 4.1

14 ± 1.8

13 ± 1.3

8 ± 1.8

10 ± 1.2

8 ± 0.6

20 ± 2.8

31 ± 1.5

31 ± 1.8

100

172 ± 2.3

173 ± 10.0

135 ± 4.3

18 ± 3.0

16 ± 1.7

13 ± 2.0

8 ± 2.1

11 ± 2.3

11 ± 1.7

18 ± 2.4

36 ± 5.4

32 ± 3.5

333

170 ± 7.3

158 ± 10.1

128 ± 6.7

21 ± 2.2

15 ± 2.3

12 ± 0.6

10 ± 2.9

8 ± 0.3

13 ± 1.5

18 ± 2.3

21 ± 1.5

31 ± 3.2

1000

174 ± 2.4

170 ± 4.9

147 ± 6.4

18 ± 2.3

15 ± 0.3

13 ± 3.8

9 ± 1.9

12 ± 4.0

10 ± 4.2

20 ± 3.5

36 ± 3.3

37 ± 2.4

3333

167 ± 13.1

169 ± 12.2

137 ± 6.2

20 ± 3.1

12 ± 2.5

8 ± 0.7

9 ± 1.7

9 ± 2.6

9 ± 3.1

21 ± 1.5

26 ± 3.5

31 ± 4.1

10000

171 ± 8.3

163 ± 7.8

142 ± 2.0

14 ± 0.9

17 ± 3.8

13 ± 1.2

9 ± 1.8

9 ± 2.6

12 ± 1.5

23 ± 2.0

37 ± 3.5

36 ± 1.3

POS

1123 ± 10.7

1155 ± 34.2

1204 ± 38.1

378 ± 25.9

74 ± 4.9

85 ± 2.7

469 ± 36.7

87 ± 2.5

110 ± 11.2

1459 ± 8.6

960 ± 28.6

1115 ± 16.0

 

Table 3: Mutagenic responses on Choline chloride on four S. typhimurium strains, SRI International

Dose

µg/plate

TA 100

TA 1535

TA 1537

TA 98

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0

102 ± 5.3

111 ± 8.1

141 ± 2.5

25 ± 3.1

9 ± 0.3

10 ± 0.7

16 ± 1.5

24 ± 4.3

41 ± 1.2

46 ± 2.4

60 ± 5.0

53 ± 3.4

100

105 ± 6.5

89 ± 6.1

135 ± 4.3

24 ± v2.3

7 ± 0.9

9 ± 1.5

19 ± 1.2

17 ± 2.1

37 ± 2.6

47 ± 3.6

53 ± 0.3

33 ± 3.6

333

118 ± 7.2

90 ± 10.7

124 ± 10.2

23 ± 2.8

9 ± 2.0

12 ± 3.4

25 ± 3.2

11 ± 1.0

41 ± 6.1

46 ± 4.5

59 ± 4.7

34 ± 2.8

1000

110 ± 2.6

86 ± 3.2

113 ± 5.2

26 ± 3.5

6 ± 1.3

11 ± 2.7

26 ± 0.6

15 ± 1.2

34 ± 1.9

49 ± 4.3

54 ± 6.2

36 ± 4.0

3333

111 ± 4.3

85 ± 2.9

120 ± 8.9

20 ± 2.5

12 ± 3.6

11 ± 2.3

24 ± 2.6

12 ± 1.7

40 ± 2.0

44 ± 5.4

58 ± 5.0

24 ± 2.7

10000

113 ± 8.2

85 ± 5.4

120 ± 5.4

23 ± 4.1

4 ± 1.0

12 ± 1.3

18 ± 1.8

6 ± 1.5

31 ± 1.0

47 ± 3.5

47 ± 2.8

28 ± 0.3

POS

526 ± 12.0

627 ± 18.2

1278 ± 34.7

444 ± 17.0

374 ± 12.8

300 ± 6.3

166 ± 5.6

238 ± 45.9

358 ± 6.0

850 ± 18.0

509 ± 20.5

1170 ± 9.5

 

Table 4: Concentrations of Positive Control Chemicals (µg/plate)

 

TA 98

TA 100

TA 1535

TA 1537

-S9

NOPD

+S9

2-AA

-S9

SA

+S9

2-AA

-S9

SA

+S9

2-AA

-S9

9AAD

+S9

2-AA

CWR

3.3

1.0

3.2

1.0

3.3

2.0

33.0

2.0

EGG

12.0

RLI 1.5b

HLI 0.75

2.5

RLI 1.5

HLI 0.75

2.5

RLI 1.5

HLI 0.75

80.0

RLI 1.5

HLI 0.75

SRL

5.0

1.0

1.0

1.0

1.0

2.5

50.0

2.5

 

NA, not activated

RLI, rat liver S-9, Aroclor 1254 induced

HLI, hamster liver S-9, Aroclor 1254 induced

POS, positive control, see Table 4

NOPD, 4-nitro+-phenylenediamine

2-AA, 2-aminoanthracene

SA, sodium azide

9AAD, 9-aminoacridine.

bDifferent concentrations for each S-9 source.

CWR: Case Western Reserve University. Dr. William Speck

EGG: Microbiological Associates (formerly EG&G Mason Research Institute), Dr. Steve Haworth

SRI: SRI International, Dr. Kristien Mortelmans

Conclusions:
Interpretation of results: negative with metabolic activation and negative without metabolic activation

This study was performed similar to OECD guideline 471 and assessed as reliable with minor restrictions due to the fact that 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, this restriction is considered to be negligible. Furthermore, Choline chloride was tested independently in three laboratories revealing consistent negative results ± S9 and the number of revertants per plate are also in the same order of magnitude. The results on 2-Aminoanthracen (positive control) were added exemplarily to this endpoint study record to demonstrate the good intra- and interlaboratory correlation and hence validity of the results.
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 this endpoint.
These three bacterial reverse mutation studies revealed consistent negative results with and without metabolic activation. Consequently, Choline chloride does not need to be classified as mutagenic, neither according Regulation 1272/2008/EC nor Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay in bacteria similar to OECD 471, S. typhimurium strains TA1535, TA1537, TA98 and TA100 were exposed to Choline chloride in water at concentrations of 0, 100, 333, 1000, 3333, 10000 µg/plate (two laboratories) resp. 0, 333, 1000, 3333, 10000, 20830 µg/plate (one laboratory). All experiments were performed in the presence and absence of metabolic activation, i.e. either 0.1 M PO4 buffer or liver Aroclor 1254-induced S-9 fractions derived from male Sprague-Dawley rats and male Syrian hamsters, using the pre-incubation, plate-incorporation method.

Choline chloride was tested up tot 20830 µg/plate in non-cytotoxic ranges as determined in preliminary experiments. The number of revertants was not elevated two-fold over control in any experiment and strain. The positive controls induced the appropriate responses in the corresponding strains.

There was no concentration-related positive response or evidence of induced mutant colonies over background.

This study was classified as acceptable with minor restrictions and satisfied the requirements for OECD Test Guideline 471 for in vitro mutagenicity data (bacterial reverse gene mutation).

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

Additional information

Additional information from genetic toxicity in vitro:

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 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, 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.

 

The stipulated endpoint ‘Mammalian chromosome aberration’ was covered with two studies on chromosome aberrations, one reliable without restrictions (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 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 does not need to be classified as mutagen.

 

A study performed to cover the endpoint ‘Gene mutation in mammalian cells’, e.g. according OECD 476, is now available. In this mammalian cell gene mutation assay [hprt locus], CHO-K1 cells cultured in vitro were exposed to Choline Chloride at different concentrations, in the absence and presence of the metabolic activation (2 % v/v S9 mix) for a period of 4 hours.

Based on the results of solubility and precipitation tests, cultures were exposed to Choline Chloride at five concentration levels (two cultures/concentration level) from 0.116 to 1.854 µL/mL of culture medium, in the absence and presence of the metabolic activation (2 % v/v S9 mix) in the cytotoxicity test. There was no reduction in the relative survival up to the highest tested concentration, hence, 1.854 µL/mL of culture medium was selected as the highest test concentration for the main study.

In the main study, the cultures were treated at five different concentration levels (two cultures/ concentration level) from 0.116 to 1.854 µL/mL of culture medium, in the absence and presence of the metabolic activation (2 % v/v S9 mix). No relevant influence of the test item, either on pH or osmolality, was observed in the absence or presence of the metabolic activation. No significant concentration related increase in the mutation frequencies were observed in any concentrations tested up to 1.854 µL/mL of culture medium in absence and presence of the metabolic activation (2 % v/v S9 mix). The induced mutation frequencies were comparable to that of the negative control group. All negative controls were within the historical control limit and positive controls showed an increase in the mutation frequency.

All criteria for a valid study were met as described in the study plan. Based on the results from this study, it is concluded that Choline Chloride does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells either in the absence or presence of the metabolic activation system (2 % v/v S9 mix) under the present experimental conditions.

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’ are negative and Choline chloride 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 chloride does not need to be classified as mutagenic according Regulation 1272/2008/E.


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

Justification for classification or non-classification

All available studies to cover this endpoint (‘Gene mutation in bacteria’, ‘Mammalian chromosome aberration’ and Sister chromatide exchange and ‘Gene mutation in mammalian cells’) have a negative outcome and consequently choline chloride does not need to be classified as mutagenic according Regulation 1272/2008/EC .