Trimethyl[2-(phosphonooxy)ethyl]ammonium chloride, calcium salt (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria:

The available data refer to the degradation products of calcium phosphorylcholine chloride:

- Key study: Similar to OECD 471. Choline chloride did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation up to 20830 µg/plate.

- Key study: Similar to OECD 471. Choline chloride did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation up to 10000 µg/plate.

- Key study. Similar to OECD 471. Choline chloride did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation up to 10000 µg/plate.

- Supporting study. Similar to OECD 471. Under the experimental conditions used, monocalcium phosphate was found to be non-mutagenic in Salmonella thyphimurium TA1535, TA1537 and TA1538.

- Key study. Similar to OECD 471. Disodium phosphate did not induce any mutagenic change in Salmonella typhimurium TA 92, TA1535, TA 100, TA 1537, TA 94 and TA 98, with and without metabolic activation up to 100 mg/plate.

- Key study. Similar to OECD 471. Phosphoric acid did not induce any mutagenic change in Salmonella typhimurium TA1535, TA 97, TA 98, TA 100 and TA 102, with and without metabolic activation up to 75-85% solution.

- Key study. Similar to OECD 471. Phosphoric acid did not induce any mutagenic change in Salmonella typhimurium TA 97, TA 98, TA 100 and TA 10, with and without metabolic activation up to 2 µg/plate.

In-vitro gene mutation in yeast:

The available data refer to the degradation product of calcium phosphorylcholine chloride which has not mutagenic effects.

- Supporting study. Similar to OECD 480. Under the experimental conditions used, monocalcium phosphate was found to be non-mutagenic in Sacharomyces cerevisae D4.

In-vitro cytogenicity study in mammalian cells:

The available data refer to the degradation products of calcium phosphorylcholine chloride:

- Key study. Similar to OECD 473. Choline chloride does not induce chromosomal aberration in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as not mutagenic.

- Key study. Similar to OECD 473. Choline chloride slightly induces chromosomal aberrations in Chinese Hamster Ovary (CHO) cells without metabolic activation but not with metabolic activation.

- Supporting study. Similar to OECD 473. Choline chloride does not induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells with and without metabolic activation up to 5000 µg/mL.

- Key study. Similar to OECD 473. Disodium phosphate does not induce chromosomal aberration in Chinese Hamster Lung fibroblasts (CHL) without metabolic activation. Therefore, the test item can be considered as not mutagenic.

- Key study. Similar to OECD 479. Choline chloride does not induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as not mutagenic.

- Key study. Similar to OECD 479. Choline chloride does weakly induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as slightly mutagenic.

- Supporting study. Similar to OECD 479. Choline chloride does not induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as not mutagenic.

In-vitro mammalian cell gene mutation test using the thymidine kinase gene:

The available data refer to 2 -trimethylazaniumylethyl phosphate chloride, tetrahydrate:

- Key study: OECD 490. GLP study. Under these experimental conditions, the test item is considered genotoxic with the mouse lymphoma assay. In addition, this genotoxic effect is more mutagenic than clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979 - 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

National Toxicoloxy Program. A standarised protocol was followed.

Deviations:

yes

Remarks:

only 4 strains tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Lot number:022277

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333 and 10000 µg. The maximum concentration used was 10 mg/plate because no apparent toxicity was found in the preliminary test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the chemical was soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene, 4-Nitro-o-phenylenediamine,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation
All chemicals were tested using the preincubation procedure of the Salmonella assay (Ames et al. 1975) as described by Yahagi et al. (1975). 0.5 ml of S9 mix or 0.1 M PO4 buffer was dispensed into an appropiate number of 13x 100 mm culturee tubes maintained at 37ºC in a dry-bath. Then 0.05 ml of cells and 0.05 ml of solvent or chemical solution were added to each tobe. The mixture was vortexed and allowed to incubate standing for 20 minutes at 37 ºC. Following the preincubation period, 2 ml of molten top agar (45ºC) supplemented with 0.5 ml L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar (Vogel and Bonner, 1956) in a 15 x 100 mm plastic petri dish (Falcon Muta-Assay, 1028). After the overlay solidified, the plates were inverted and incubated at 37ºC for 48 h. Each strain was tested in the presence of S9 mix or buffer. The experiment was repeated no less than 1 week after completion of the initial test.

- Cell density at seeding (if applicable): not specified

DURATION
- Preincubation period: 20 minutes at 37ºC
- Exposure duration: 48 h at 37ºC

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S9 mix. One or more parameters were used as an indication of toxicity: reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn.

- OTHER:
Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected ip with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. Five days after injection, the animals were sacrified and the livers were removed aseptically. The animals were fasted for 12-24 h immediately preceding sacrifice. Liver homogenates were prepared aseptically at 0-4ºC. Excised livers were rinsed with 0.15 M KCl, then minced and homogenized. The homogenate was centrifuged for 10 min at 9000 ga t 4ºC. The supernatant (S9) was decanted and distributed into freezing ampules and stored at -70ºC. The microsomal enzyme reaction mix (S9 mix) was prepared immediately prior to each assay. Unused S9 mix was discarded and not refrozen. One milliliter of S9 mix has the following composition: S9, 0.1 ml; 0.04 M MgCl2, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M beta-nicotinamide adenine dinucleotide phosphate (NADP), 0.1 ml; 0.05 M glucose-6-phosphate, 0.1 ml; 1 M NaH2PO4 (pH 7.4), 0.1 ml; and distilled water, 0.56 ml.

Evaluation criteria:

The data were evaluated in an hoc manner by each testing laboratory and by NTP personnel. A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.

Statistics:

The data were evaluated using an analysis based on the models presented by Margolin et al (1981).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test item is soluble in water
- Precipitation: no precipitation was found

RANGE-FINDING/SCREENING STUDIES: a range finding study was performed to set the maximal dose, no toxicity was found. Then, the test item was tested at 10 mg/plate.

Table 2. Mutagenic response of Salmonella tester strains TA100, TA1535, TA1537 and TA98 to choline chloride. SRI International (SRI).

 

TA100

TA1535

TA1537

TA98

Dose (µg/plate)

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

102±5.3

111±8.1

108±9.7

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

100.0

105±6.5

89±6.1

110±9.8

24±2.3

7±0.9

9±1.5

19±1.2

17±2.1

37±2.6

47±3.6

53±0.3

33±3.6

333.0

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

1000.0

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

3333.0

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

10000.0

113±8.2

85±5.4

120±5.4

23±4.1

4±1.0

12±1.3

18±1.0

6±1.5

31±1.0

47±3.5

47±2.8

28±0.3

POS

526±12

627±18.2

1278±34.7

444±17

374±12.8

300±6.3

166±5.6

238±45.9

358±6.0

850±18

509±20.5

1170±9.5

 

Abbreviations:

POS: Positive control

NA: Not activated

RLI: Rat liver S9, Aroclor 1254 induced

HLI: Hamster liver S9, Aroclor 1254 induced

Conclusions:

Choline chloride did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation up to 10000 µg/plate.

Executive summary:

The ability of choline chloride to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to ta National Toxicology Program standardised method, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 20, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 100 well-spread metaphases were observed

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: National Toxicology Program chemical repository

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) cells were obtained from Dr. Sheldon Wolff, cloned, and designated CHO-W-B1. To mantain kariotip stability, the cells were used at no more than 15 passages after cloning.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

500 - 50 - 0.5 - 0.05 - 0.005 µg/ml
The highest concentration tested for cytogenetic activity was the concetration estimated to reduce cell numbers by about 50%; lower, nontoxic doses were also included.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHO cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in air, in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), and L-glutamine (2mM). Cultures were set up the day before treatment at a uniform cell density, chosen to allow treatment of exponentially growing cultures (eg, approximately 1E06 cells per 75 cm2 flask). Duplicate 25 cm2 flasks were used for each experimental point; half the cells scored were from each culture.
In the assay without metabolic activation, one day after culture initiation, the medium was replaced and cells were treated with test of control chemicals for 8 to 10 h. Medium was removed and fresh medium containing colcemid (final concentration 0.1 µg/ml) was added, and incubation continued for 2 to 2.5 h before fixation. In the assay with metabolic activation, cells were treated with test of control chemicals and S9 mix for 2 h. After the incubation with colcemid, cells were collected by trypsinization (CU) and trated for up to 3 min at room temperature with hypotonic KCl (75 mM).

DURATION
- Exposure duration: 8 -10 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3 responses were analyzed for each trial

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were selected for scoring on the basis of good morphology and a chromosome number of 21 ± 2. For control of bias, all slides were coded before scoring. All slides from a test (control and doses) were read by the same reader. Slides were stained in Giemsa.

NUMBER OF CELLS EVALUATED: 100 cells per dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 of metaphase spreads were analysed per dose.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 occurence 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.

OTHER:
- The S9 fraction was prepared from Sprague-Dawley rats induced with Aroclor 1254. The same batch of liver homogenate was used in both labs and was stored at -80ºC. S9 fraction was thawed immediately before use, mixed with an NADPH-generating system, and added to medium to give the following final concentrations in culture: NADP (sodium salt), 2.4 mg/ml; isocitric acid (trisodium salt), 4.5 mg/ml, and S9 fraction, 15 µl/ml.

Evaluation criteria:

The evaluation reflects a blend of statistical analysis and biological intuition.The summary of judgement is shown in Table 1. For each test, an efferot has been made to recognize any response to the chemical under test while guarding against flase-positive evaluations that could arise by chance as a result of the multiple tests performed.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

weakly positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

For chromosome aberration assay without metabolic activation, there was a questionble positive point (?+), that it was contributed by simple breaks (Table 2).

Table 2. Results of the chromosomic aberrations test for Choline chloride without metabolic activation.

		Dose µg/mL	Total Cells examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
				No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs
Vehicle control:	Water	0	100	1	0.010	1.0	0	0.000	0.0	1	0.010	1.0	0	0.000	0.0
Test chemical:	Choline chloride	0.005	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		0.05	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		0.5	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		50	100	3	0.030	3.0	0	0.000	0.0	3	0.030	3.0	0	0.000	0.0
		500	100	10	0.100	10.0	0	0.000	0.0	7	0.070	7.0	3	0.030	3.0
Positive control:	Triethylenemelamine	0.25	100	46	0.460	38.0	16	0.160	15.0	28	0.280	23.0	2	0.020	2.0
Trend:				4.441			0.000			3.649
Probability:				0.000			0.000			0.000

 

Table 3. Results of the chromosomic aberrations test for Choline chloride with metabolic activation (induced rat liver S9).

		Dose µg/mL	Total Cells examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
				No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs
Vehicle control:	Water	0	100	2	0.020	2.0	0	0.000	0.0	2	0.020	2.0	0	0.000	0.0
Test chemical:	Choline chloride	0.005	100	3	0.030	3.0	0	0.000	0.0	3	0.030	3.0	0	0.000	0.0
		0.05	100	3	0.030	3.0	0	0.000	0.0	3	0.030	3.0	0	0.000	0.0
		0.5	100	5	0.050	5.0	2	0.020	2.0	3	0.030	3.0	0	0.000	0.0
		50	100	8	0.080	4.0	1	0.010	1.0	7	0.070	4.0	0	0.000	0.0
		500	100	5	0.050	4.0	1	0.010	1.0	4	0.040	3.0	0	0.000	0.0
Positive control:	Cyclophosphamide	25	100	86	0.860	51.0	38	0.380	30.0	46	0.460	28.0	2	0.020	2.0
Trend:				0.908			1.347			0.579
Probability:				0.182			0.089			0.281

 

Conclusions:

Choline chloride slightly induces chromosomal aberrations in Chinese Hamster Ovary (CHO) cells without metabolic activation but not with metabolic activation.

Executive summary:

In order to determine the ability of the choline chloride to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 473. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (500, 50, 0.5, 0.05 and 0.005 µg/mL) for 8 -10 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation with colcemid (0.1  g/ml), cells were fixed and stained. Negative and positive controls were run in parallel. 100 cells were examined per dose level and the results for chromosome aberration assay without metabolic activation, there was a single questionable positive, that it was contributed by simple breaks. Under these test conditions, the test item was found to slightly induce chromosomal aberrations in CHO cells without metabolic activation but not with metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 16, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: National Toxicology Program chemical repository

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) cells were obtained from Dr. Sheldon Wolff, cloned, and designated CHO-W-B1. To mantain kariotip stability, the cells were used at no more than 15 passages after cloning.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5000 - 500 - 50 - 5 - 0.5 - 0.05 µg/ml
The highest concentration tested for cytogenetic activity was the concentration estimated to reduce cell numbers by about 50%; lower, nontoxic doses were also included.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHO cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in air, in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), and L-glutamine (2mM). Penicillin (100 units/ml) and streptomycin (100 µg/ml) were used. Cultures were set up the day before treatment at a uniform cell density, chosen to allow treatment of exponentially growing cultures (eg, approximately 1E06 cells per 75 cm2 flask). Duplicate 25 cm2 flasks were used for each experimental point; half the cells scored were from each culture.
Assay without metabolic activation: One day after culture initiation, the medium was replaced and cells were treated with test of control chemicals for 2 h to allow interaction with cells before addition of BrdUrd. BrdUrd was added (final concentration 10 µM) and incubation continued for about 24 h. Medium was removed to allow an opportunity to wash off any test chemical precipitate that might interfere with cell fixation and to avoid harvest of cultures containing test chemicals. Fresh medium containing 10 µM BrdUrd and colcemid (final concentration 0.1 µg/ml) was added, and incubation continued for 2 to 3 h.
The total incubation time test chemical was thus about 26 h, and total time with BrdUrd was also about 26 h, beginning about 2 h after addition of test chemical.
Assay with metabolic activation: One day after culture initiation, medium was replaced with S9 mix in medium without fetal calf serum. After exposure for 2 h to test compound, cells were washed at least twice with buffered saline, and culture medium containing 10% FCS and 10 µM BrdUrd was added. Cells were incubated for a further 26 h, with colcemid (0.1 µg/ml) present for the final 2 to 3 h.
Two to three hours after addition of colcemid, cells were collected by mitotic shake-off and treated for up to 3 min at room temperature with hypotonic KCl (75 mM). cell were then washed twice with fixative (3:1, methanol:glacial acetic acid, v/v), dropped onto slides, and air-dried.

DURATION
- Exposure duration: 26 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 28-29 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)

STAIN (for cytogenetic assays): Hoechst 33258 and Giemsa

NUMBER OF REPLICATIONS: 3 responses were analyzed for each trial

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were selected for scoring on the basis of good morphology and a chromosome number of 21 ± 2. For control of bias, all slides were coded before scoring. All slides from a test (control and doses) were read by the same reader. Slides were stained in Giemsa.

NUMBER OF CELLS EVALUATED: 50 cells per dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 of metaphase spreads were analysed per dose.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 occurence 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.

OTHER:
- The S9 fraction was prepared from Sprague-Dawley rats induced with Aroclor 1254. The same batch of liver homogenate was used in both labs and was stored at -80ºC. S9 fraction was thawed immediately before use, mixed with an NADPH-generating system, and added to medium to give the following final concentrations in culture: NADP (sodium salt), 2.4 mg/ml; isocitric acid (trisodium salt), 4.5 mg/ml, and S9 fraction, 15 µl/ml.

Evaluation criteria:

The evaluation reflects a blend of statistical analysis and biological intuition.The summary of judgement is shown in Table 1. For each test, an efferot has been made to recognize any response to the chemical under test while guarding against flase-positive evaluations that could arise by chance as a result of the multiple tests performed.

Statistics:

Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control . An sister chromatid exchange (SCE) frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01, the probability for such a chance occurred at two dese points is less than 0.001. An increase of 20%, or greater, at any single dose, was considered weak evidence of activity (weak positive), increases at two or more doses resulted in a determination that the trial was positive. A trend P-value of less than 0.005, in the absence of any responses reaching 20% above background, led to a call of equivocal for the trial. Positive and weak positive trials were repeated. The overall assay result was based on an evaluation of the responses in all trials within an activation condition.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 2. Results of the sister chromatid exchange test for Choline chloride without metabolic activation.

		Dose µg/mL	Total Cells Examined	Number Chromosomes Examined	Total Number SCEs	SCE / Chromosome	SCE / Cell	Hours in BRDU	% Increase Over Solvent Control
Vehicle control:	Water	0	25	526	145	0.276	5.80	26.0	0.000
Test chemical:	Choline chloride	0.05	50	1051	315	0.300	6.30	26.0	8.724
		0.5	50	1111	362	0.326	7.24	26.0	18.199
		5	50	1067	362	0.339	7.24	26.0	23.073
		50	50	1046	339	0.324	6.78	26.0	17.567
		500	50	1032	396	0.384	7.92	26.0	39.198
		5000	50	1067	323	0.303	6.46	26.0	9.814
Positive control:	Triethylenemelamine	0.015	50	1029	2023	1.966	40.46	26.0	613.179
Trend:					2.008
Probability:					0.022

 

Table 3. Results of the sister chromatid exchange test for Choline chloride with metabolic activation.

		Dose µg/mL	Total Cells Examined	Number Chromosomes Examined	Total Number SCEs	SCE / Chromosome	SCE / Cell	Hours in BRDU	% Increase Over Solvent Control
Vehicle control:	Water	0	50	1051	354	0.337	7.08	26.0	0.000
Test chemical:	Choline chloride	0.05	50	1058	441	0.417	8.82	26.0	23.752
		0.5	50	1048	414	0.395	8.28	26.0	17.284
		5	50	1062	474	0.446	9.48	26.0	32.511
		50	50	1056	404	0.383	8.08	26.0	13.584
		500	50	1048	390	0.372	7.80	26.0	10.485
		5000	50	1040	391	0.376	7.82	26.0	11.620
Positive control:	Cyclophosphamide	1.5	49	1015	2373	2.338	48.43	26.0	594.115
Trend:					0.155
Probability:					0.439

Conclusions:

Choline chloride does not induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as not mutagenic.

Executive summary:

In order to determine the ability of choline chloride to induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells studies iwere performed by a method similar to OECD 479. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (5000, 500, 50, 5, 0.5 and 0.05 µg/mL) for 26 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation, the medium with test chemical was removed and replaced with fresh medium plus BedUrD and colcemid (0.1  g/ml), without test chemical, and incubated for further 2 hours. Then, cells were harvested, fixed and stained with Hoechdt 33258 and Giemsa. Negative and positive controls were run in parallel. 50 cells were examined per dose level and the results for sister chromatid exchange assay were negative.Under these test conditions, the test item was found to be non mutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 20, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: National Toxicology Program chemical repository.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) cells were obtained from Dr. Sheldon Wolff, cloned, and designated CHO-W-B1. To mantain kariotip stability, the cells were used at no more than 15 passages after cloning.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

500 - 50 - 0.5 - 0.05 - 0.005 µg/ml
The highest concentration tested for cytogenetic activity was the concentration estimated to reduce cell numbers by about 50%; lower, nontoxic doses were also included.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHO cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in air, in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), and L-glutamine (2mM). Penicillin (100 units/ml) and streptomycin (100 µg/ml) were used. Cultures were set up the day before treatment at a uniform cell density, chosen to allow treatment of exponentially growing cultures (eg, approximately 1E06 cells per 75 cm2 flask). Duplicate 25 cm2 flasks were used for each experimental point; half the cells scored were from each culture.
Assay without metabolic activation: One day after culture initiation, the medium was replaced and cells were treated with test of control chemicals for 2 h to allow interaction with cells before addition of BrdUrd. BrdUrd was added (final concentration 10 µM) and incubation continued for about 24 h. Medium was removed to allow an opportunity to wash off any test chemical precipitate that might interfere with cell fixation and to avoid harvest of cultures containing test chemicals. Fresh medium containing 10 µM BrdUrd and colcemid (final concentration 0.1 µg/ml) was added, and incubation continued for 2 to 3 h.
The total incubation time test chemical was thus about 26 h, and total time with BrdUrd was also about 26 h, beginning about 2 h after addition of test chemical.
Assay with metabolic activation: One day after culture initiation, medium was replaced with S9 mix in medium without fetal calf serum. After exposure for 2 h to test compound, cells were washed at least twice with buffered saline, and culture medium containing 10% FCS and 10 µM BrdUrd was added. Cells were incubated for a further 26 h, with colcemid (0.1 µg/ml) present for the final 2 to 3 h.
Two to three hours after addition of colcemid, cells were collected by trypsinization and treated for up to 3 min at room temperature with hypotonic KCl (75 mM). cell were then washed twice with fixative (3:1, methanol:glacial acetic acid, v/v), dropped onto slides, and air-dried.

DURATION
- Exposure duration: 26 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 28-29 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)

STAIN (for cytogenetic assays): Hoechst 33258 and Giemsa

NUMBER OF REPLICATIONS: 3 responses were analyzed for each trial

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were selected for scoring on the basis of good morphology and a chromosome number of 21 ± 2. For control of bias, all slides were coded before scoring. All slides from a test (control and doses) were read by the same reader. Slides were stained in Giemsa.

NUMBER OF CELLS EVALUATED: 50 cells per dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 of metaphase spreads were analysed per dose.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 occurence 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.

OTHER:
- The S9 fraction was prepared from Sprague-Dawley rats induced with Aroclor 1254. The same batch of liver homogenate was used in both labs and was stored at -80ºC. S9 fraction was thawed immediately before use, mixed with an NADPH-generating system, and added to medium to give the following final concentrations in culture: NADP (sodium salt), 2.4 mg/ml; isocitric acid (trisodium salt), 4.5 mg/ml, and S9 fraction, 15 µl/ml.

Evaluation criteria:

The evaluation reflects a blend of statistical analysis and biological intuition.The summary of judgement is shown in Table 1. For each test, an efferot has been made to recognize any response to the chemical under test while guarding against flase-positive evaluations that could arise by chance as a result of the multiple tests performed.

Statistics:

Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control . An sister chromatid exchange (SCE) frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01, the probability for such a chance occurred at two dese points is less than 0.001. An increase of 20%, or greater, at any single dose, was considered weak evidence of activity (weak positive), increases at two or more doses resulted in a determination that the trial was positive. A trend P-value of less than 0.005, in the absence of any responses reaching 20% above background, led to a call of equivocal for the trial. Positive and weak positive trials were repeated. The overall assay result was based on an evaluation of the responses in all trials within an activation condition.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

weakly positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 2. Results of the sister chromatid exchange test for Choline chloride without metabolic activation.

		Dose µg/mL	Total Cells Examined	Number Chromosomes Examined	Total Number SCEs	SCE / Chromosome	SCE / Cell	Hours in BRDU	% Increase Over Solvent Control
Vehicle control:	Water	0	50	1046	353	0.337	7.06	26.0	0.000
Test chemical:	Choline chloride	0.005	50	1045	337	0.322	6.74	26.0	-4.441
		0.05	50	1046	328	0.314	6.56	26.0	-7.082
		0.5	50	1047	388	0.371	7.76	26.0	9.810
		50	50	1047	415	0.396	8.30	26.0	17.451
		500	50	1044	434	0.416	8.68	26.0	23.182
Positive control:	Triethylenemelamine	0.025	50	1046	1654	1.581	33.08	26.0	368.555
Trend:					4.397
Probability:					0.000

 

Table 3. Results of the sister chromatid exchange test for Choline chloride with metabolic activation.

		Dose µg/mL	Total Cells Examined	Number Chromosomes Examined	Total Number SCEs	SCE / Chromosome	SCE / Cell	Hours in BRDU	% Increase Over Solvent Control
Vehicle control:	Water	0	50	1048	361	0.344	7.22	26.0	0.000
Test chemical:	Choline chloride	0.005	50	1047	385	0.368	7.70	26.0	6.750
		0.05	50	1047	382	0.365	7.64	26.0	5.918
		0.5	50	1046	410	0.392	8.20	26.0	13.791
		50	50	1047	408	0.390	8.16	26.0	13.127
		500	50	1048	4.31	0.411	8.62	26.0	19.391
Positive control:	Cyclophosphamide	1.5	50	1046	1851	1.770	37.02	26.0	413.723
Trend:					2.634
Probability:					0.004

 

Conclusions:

Choline chloride does weakly induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as slightly mutagenic.

Executive summary:

In order to determine the ability of choline chloride to induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells studies iwere performed by a method similar to OECD 479. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (5000, 500, 50, 5, 0.5 and 0.05 µg/mL) for 26 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation, the medium with test chemical was removed and replaced with fresh medium plus BedUrD and colcemid (0.1  g/ml), without test chemical, and incubated for further 2 hours. Then, cells were harvested, fixed and stained with Hoechdt 33258 and Giemsa. Negative and positive controls were run in parallel. 50 cells were examined per dose level and the results for sister chromatid exchange assay were weakly positive.Under these test conditions, the test item was found to be slightly mutagenic.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 28, 2017 - April 26, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent: Yes

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: JHSF (Japan Health Science Fundation)
- Number of passages if applicable: 27 (Test 1 without metabolic activation); 32 (Test 1 with metabolic activation); 42 (Test 2)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Four concentrations, within the limits of 80 to 90% of cytotoxicity (equivalent to 10 to 20 RSG) were chosen for the tests 1 and 2:
62.5, 125, 250 and 500 µL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: the test item is soluble in water and it is dissoved in culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
TEST 1
Short treatment (4 h) without metabolic activation: Day 0: A series of tubes containing 6x10E6 cells in suspension/tube in R5 medium was seeded. The different concentrations of the test item and positive controls were added directly into the tubes (2 cultures per concentration). The negative control was added to the same volume as for the test item. At the end of the incubation period of 4 hours, the tubes were centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of R10 medium were deposited in each culture. A count of the cells was performed on each tube (N0), then treated cells were transferred into flasks and incubated 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 1: After incubation period, cell counting was performed on each condition (N24h). Cells culture were adjusted if necessary to 2x10E5 cells/ml and incubated for 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 2: After incubation period, cell counting was performed again on each condition (N48h), i.e. 48 hours after the end of the treatment. These counts allowed assessing the cytotoxicity expressed as a percentage of relative cell growth (RSG). Gene mutation test was performed by treated cells cloning on 4 concentrations of the test item as determined by cytoxicity.
The cells were seeded in 96-well plate at 2 cells/200 µl in non-selective culture medium (2 plates/condition) to determine the viability. Plate were incubated at 37ºC (± 0.5ºC), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the survival rate (cloning efficiency, CE) to determine the viability. On the other hand, 96-well plates were seeded at 2x10E3 cells/200 µl in selective culture medium to determine the mutation frequency (4 plates/condition). Plates are incubated at 37ºC (± 0.5ºC), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the mutation frequency.
Short treatment (4 h) with metabolic activation: The test is the same as described in the short treatment without metabolic activation except the culture medium which was replaced by medium containing 20% of S9 mix (2% final).
TEST 2
Long treatment without metabolic activation (24 h): Day 0: A series of flasks containing 1.5x10E6 cells in suspension/tube in R5 medium was seeded. 1mL of the different concentrations of the test item or positive control were added directly into the tubes and then incubated for 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2. The negative control was added to the same volume as for the test item.
Day 1: After incubation period, the cultures were transferred to 50 ml sterile tubes (each identified) and were centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of R10 medium were deposited in each culture. A count of the cells was performed on each tube (N0). The treated cells were placed in new 25 cm2 culture flasks at 2x10E5 cells/ml and incubated for 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 2 and Day 3: The test was carried out as Day 1 and Day 2 in the "Short treatment without metabolic activation" assay.

DURATION
- Exposure duration: Test 1: 4 hours ± 10 min, Test 2: 24 hours ± 1 hour
- Expression time (cells in growth medium): 2 days at 37ºC (± 0.5ºC), 5% CO2.
- Selection time (if incubation with a selection agent): 10 to 12 days, at 37ºC (± 0.5ºC), 5% CO2.

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT), 3 µg/ml in culture medium R20

NUMBER OF REPLICATIONS: 2 replicates per dose (Negative control: 2 replicates; Positive control: 2 replicates)

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and relative cloning efficiency:
Cytotoxicity assessment was integrated in each test. the highest concentration of the test item corresponded to the mother solution diluted 10 times. The concentration tested in the short treatment (4 h) with and without metabolic activation were: 2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.625 µg/ml. In the long treatment (24 h) without metabolic activation the concentrations tested were: 500, 250, 125, 62.5, 31.25, 15.6, 7.8 and 3.9 µg/ml. Cell cultures were treated with the different concentrations of the test item in duplicate. Positive and negative controls were carried out in parallel.
Cytotoxicity was determined by calculating the percentage of relative growth of cells (RSG: Relative Survival Growth) with SG (Suspension Growth)
- Relative Survival Growth (RSG)= SG test item/ SG Neg x D0 factor x 100; where D0 factor=(N0 test item/ N0 neg)
- Suspension Growth (SG)= Number of cells at 24 hours x Number of cells at 48 hours = N24h x N48h
Viability was determined by calculating the percentage of relative total growth of cells (RTG) with Cloning efficiency (CE)
- Relative Total Growth (RTG) = RSG x SR / 100
- Cloning Efficiency (CE) = -Ln(P0) / Number of cells/ well; where P0 = total number of empty wells / total number of seeded (192 for the viability); and, Number of cells/well= 2 in non-selective medium (R20) (CE in non-selctive medium is calculated using a Poisson distribution).
- Relative survival (RS) = CE Test item / CE Neg x 100 (RS was expressed as a percentage and was calculated from the cloning efficiency)
- Mutant Frequency (MF) = CE in the selective medium (mutant) / CE in nonselective medium (viability); with CE = -Ln(P0) / Number of cells/ well; where P0 = total number of empty wells / total number of seeded (384 for the mutation test); and, Number of cells/well= 2000 in selective medium (R20 + TFT)
4 concentrations, within the limits of 80 to 90% of cytotoxicity (equivalent to 10 to 10% of RSG), have been retained for cloning for the evaluation of mutations.

OTHER EXAMINATIONS:
Clastogenicity: The size of the colonies were estimated, a small colony is estimate to less than 25% of the diameter of the well and the size of a large colony greater than 25% of the diameter of the well. The presence of small colonies reflects a clastogenic effect indicating an alteration of chromosomes (chromosome aberrations). Large colonies reflect point mutations or deletions.
In the case of a negative result, the frequency of mutants in small and large colonies will be given for negative and positive controls. In the case of a positive result, the frequency of mutants in small and large colonies will be given for at least one concentration of the test item and negative and positive controls.

Evaluation criteria:

The test item should be considered potentially mutagenic if the following criteria are met: the mutation rate induced is significantly higher than the negative control, the observed effect is dose-dependent, the observed effect is reproducible, the rate of mutation is greater than the value of the GET (126) + MF negative control.

Statistics:

The mutant frequency for each series studied was subjected to a Chi-2 test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: A relative humidity nearly of 95% is maintained in the incubator by evaporation of the water in the water pan. The presence of water in the pan is checked daily and recorded in the FL HSE 09 form.
- Water solubility: the test item is soluble in water
- Precipitation: no precipitation was observed

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined. Cytotoxicity was observed after a short treatment (4 hours) with and without metabolic at concentrations 2000 and 1000 µl. for the long treatment without metabolic activation, the concentrations range was adapted.
Acording to the results obtained, the following four doses were tested for the gene mutation test: 500, 250, 125 and 62.5 µg/ml

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Mutation frequency of Methylmethanesulfonate (MMS) (without metabolic activation) or Cyclophosphamide monohydrate (CP) (with metabolic activation):
Short treatment without S9 (10 µg/ml of MMS): n= 15; 848 ± 258.5 (mean ± SD); 491 / 1368 (min / max);
Short treatment with S9 (1.5 µg/ ml of CP): n= 23; 572 ± 172.3 (mean ± SD); 305 / 1009 (min / max)
Long treatment without S9 (2 µg/ml of MMS): n= 12; 521 ± 206.2 (mean ± SD); 258 / 1059 (min / max)
- Negative (solvent/vehicle) historical control data:
Short treatment without S9: n= 15; 98 ± 38 (mean ± SD); 63 / 167 (min / max);
Short treatment with S9: n= 23; 104 ± 39.8 (mean ± SD); 53 / 198 (min / max)
Long treatment without S9: n= 12; 99 ± 25.7 (mean ± SD); 70 / 152 (min / max)

OTHER EXAMINATIONS:
Clastogenicity: There is no increase in the presence of little colonies. This indicates that the test item shows a mutagenic effect but no clastogenic activity.

Concentration determination for the mutation test (viability determination: % RSG)

Table 1. Test 1, viability without S9 after a short treatment, 4 hours.

4H-S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	60.25	104.75	120.25	31.49	-	-	-
Positive Control MMS 10 µg/ml	61.25	83.25	99.5	20.71	1.02	0.67	67.00
2000 µg/ml	0.25	-	-	-	-	-	-
1000 µg/ml	4.5	-	-	-	-	-	-
500 µg/ml	41.25	103.5	127.25	32.93	0.68	0.71	71.00
250 µg/ml	60.5	109.75	141.5	38.82	1.00	1.23	123.00
125 µg/ml	64	104.5	136	35.53	1.06	1.20	120.00
62.5 µg/ml	57.25	104.5	138.75	36.25	0.95	1.09	109.00
31.25 µg/ml	60.25	101	-	-	-	-	-
15.6 µg/ml	61.5	94.5	-	-	-	-	-

RSG: relative growth suspension

*N₀is the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

Table 2. Test 1, viability with S9 after a short treatment, 4 hours.

4H+S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	51.75	91.25	119.5	27.26	-	-	-
Positive Control CP 1.5 µg/ml	46.75	71.5	107.75	19.26	0.90	0.64	64.00
2000 µg/ml	9	-	-	-	-	-	-
1000 µg/ml	17.25	-	-	-	-	-	-
500 µg/ml	46.25	97.75	123	30.06	0.89	0.98	98.00
250 µg/ml	45.25	91.25	110.5	25.21	0.87	0.80	80.00
125 µg/ml	49.75	85.5	109.25	23.35	0.96	0.82	82.00
62.5 µg/ml	48.75	83.75	102.5	21.46	0.94	0.74	74.00
31.25 µg/ml	52	88.25	-	-	-	-	-
15.6 µg/ml	52.5	91.25	-	-	-	-	-

RSG: relative growth suspension

*N₀is the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

Table 3. Test 2, viability without S9 after a long treatment, 24 hours.

24H-S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	62.25	117.25	126.5	161.3	-	-	-
Positive Control MMS 2 µg/ml	62.25	96.5	125	125.15	0.95	0.74	74.00
500 µg/ml	58.5	112.75	145.25	159.68	0.90	0.89	89.00
250 µg/ml	60	92.75	118.5	109.91	0.92	0.63	63.00
125 µg/ml	63.75	108.75	133	153.68	0.98	0.93	93.00
62.5 µg/ml	58	109.75	128	135.80	0.89	0.75	75.00
31.25 µg/ml	58.75	116	-	-	-	-	-
15.6 µg/ml	70.25	108	-	-	-	-	-
7.8 µg/ml	59	102.25	-	-	-	-	-
3.9 µg/ml	56.5	92.55	-	-	-	-	-

RSG: relative growth suspension

*N₀is the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

Gene mutation test (% RS and MF)

Table 4. Test 1, relative survival without S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	20	192	2	0.76	-	-
	22
	42
Positive Control MMS 10 µg/ml	37	192	2	0.48	63.16	42.32
	37
	74
500 µg/ml	38	192	2	0.56	73.68	52.31
	25
	63
250 µg/ml	20	192	2	0.69	90.79	111.67
	28
	48
125 µg/ml	39	192	2	0.65	85.53	102.64
	13
	52
62.5 µg/ml	25	192	2	0.77	101.32	110.44
	16
	41

RS: Relative survival

CE: Cloning efficacy

 

Table 5. Test 1, relative survival with S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	15	192	2	0.82	-	-
	22
	37
Positive Control CP 1.5 µg/ml	30	192	2	0.61	74.39	47.61
	27
	57
500 µg/ml	21	192	2	0.91	110.98	108.76
	10
	31
250 µg/ml	19	192	2	0.85	103.66	82.93
	16
	35
125 µg/ml	10	192	2	0.96	117.07	96.00
	18
	28
62.5 µg/ml	20	192	2	0.70	85.37	63.17
	27
	47

RS: Relative survival

CE: Cloning efficacy

 

Table 6. Test 2, relative survival without S9 after a long treatment (24 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	13	192	2	1.00	-	-
	13
	26
Positive Control MMS 2 µg/ml	39	192	2	0.45	45.00	33.30
	39
	78
500 µg/ml	63	192	2	0.46	46.00	40.94
	13
	76
250 µg/ml	15	192	2	0.81	81.00	51.03
	23
	38
125 µg/ml	4	192	2	1.35	135.00	125.55
	9
	13
62.5 µg/ml	6	192	2	1.66	166.00	124.50
	1
	7

RS: Relative survival

CE: Cloning efficacy

 

Table 7. Test 1, frequency of mutants without S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	76	384	2000	128.46	169.03	-	-	-
	71
	76
	74
	297
Positive Control MMS 10 µg/ml	53	384	2000	410.37	854.94	88.40	P ≤0.05	Significant
	44
	34
	38
	169
500 µg/ml	59	384	2000	194.98	348.18	13.44	P ≤0.05	Significant
	67
	66
	68
	260
250 µg/ml	58	384	2000	222.66	322.70	10.68	P ≤0.05	Significant
	65
	68
	55
	246
125 µg/ml	48	384	2000	292.33	449.74	31.80	P ≤0.05	Significant
	58
	54
	54
	214
62.5 µg/ml	67	384	2000	204.69	265.83	5.38	P ≤0.05	Significant
	68
	59
	61
	255

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

Table 8. Test 1, frequency of mutants with S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	76	384	2000	130.14	158.71	-	-	-
	72
	76
	72
	296
Positive Control CP 1.5 µg/ml	42	384	2000	373.32	612.00	61.32	P ≤0.05	Significant
	43
	43
	54
	182
500 µg/ml	54	384	2000	267.27	293.70	13.00	P ≤0.05	Significant
	54
	58
	59
	225
250 µg/ml	49	384	2000	278.51	327.66	17.60	P ≤0.05	Significant
	52
	61
	58
	220
125 µg/ml	53	384	2000	278.51	290.11	11.63	P ≤0.05	Significant
	59
	52
	56
	220
62.5 µg/ml	65 66	384	2000	204.69	292.41	9.20	P ≤0.05	Significant
	65
	59
	255

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

Table 9. Test 2, frequency of mutants without S9 after a long treatment (24 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	70	384	2000	157.93	157.93	-	-	-
	72
	68
	70
	280
Positive Control MMS 2 µg/ml	44	384	2000	384.43	854.29	93.47	P ≤0.05	Significant
	47
	50
	37
	178
500 µg/ml	59	384	2000	258.46	561.87	39.56	P ≤0.05	Significant
	55
	59
	56
	229
250 µg/ml	51	384	2000	306.55	378.46	24.67	P ≤0.05	Significant
	46
	55
	56
	208
125 µg/ml	59	384	2000	292.33	216.54	2.19	P> 0.05	Non significant
	54
	53
	48
	214
62.5 µg/ml	50 48	384	2000	316.26	190.52	0.60	P> 0.05	Non significant
	50
	56
	204

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

 

Small and large colonies

Table 10. Distribution of small (SC) and large colonies (LC)

4H-S9		Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
		500	250	125	62.5	vehicle	MMS 10 µg/ml
Test 1	SC	19	13	18	15	17	93
	LC	105	125	152	114	70	122
	% SC	15.32	9.42	10.59	11.63	19.54	43.26

 

4H+S9		Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
		500	250	125	62.5	vehicle	CP 1.5 µg/ml
Test 1	SC	20	24	19	14	12	85
	LC	139	140	144	115	76	117
	% SC	12.58	14.63	11.66	10.85	13.64	42.08

 

24H-S9		Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
		500	250	125	62.5	vehicle	MMS 2 µg/ml
Test 2	SC	20	14	13	19	13	82
	LC	135	162	157	161	91	114
	% SC	12.90	7.95	7.65	10.56	12.50	41.84

 

(viability determination: % RSG)

Table 1. Test 1, viability without S9 after a short treatment, 4 hours.

RSG: relative growth suspension

*N₀is the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

Table 2. Test 1, viability with S9 after a short treatment, 4 hours.

4H+S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	51.75	91.25	119.5	27.26	-	-	-
Positive Control CP 1.5 µg/ml	46.75	71.5	107.75	19.26	0.90	0.64	64.00
2000 µg/ml	9	-	-	-	-	-	-
1000 µg/ml	17.25	-	-	-	-	-	-
500 µg/ml	46.25	97.75	123	30.06	0.89	0.98	98.00
250 µg/ml	45.25	91.25	110.5	25.21	0.87	0.80	80.00
125 µg/ml	49.75	85.5	109.25	23.35	0.96	0.82	82.00
62.5 µg/ml	48.75	83.75	102.5	21.46	0.94	0.74	74.00
31.25 µg/ml	52	88.25	-	-	-	-	-
15.6 µg/ml	52.5	91.25	-	-	-	-	-

RSG: relative growth suspension

*N₀is the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

Table 3. Test 2, viability without S9 after a long treatment, 24 hours.

24H-S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	62.25	117.2	126.5	161.3	-	-	-
Positive Control MMS 2 µg/ml	62.25	96.5	125	125.15	0.95	0.74	74.00
500 µg/ml	58.5	112.75	145.25	159.68	0.90	0.89	89.00
250 µg/ml	60	92.75	118.5	109.91	0.92	0.63	63.00
125 µg/ml	63.75	108.75	133	153.68	0.98	0.93	93.00
62.5 µg/ml	58	109.75	128	135.80	0.89	0.75	75.00
31.25 µg/ml	58.75	116	-	-	-	-	-
15.6 µg/ml	70.25	108	-	-	-	-	-
7.8 µg/ml	59	102.25	-	-	-	-	-
3.9 µg/ml	56.5	92.55	-	-	-	-	-

RSG: relative growth suspension

*N₀is the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

Gene mutation test (% RS and MF)

Table 4. Test 1, relative survival without S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	20	192	2	0.76	-	-
	22
	42
Positive Control MMS 10 µg/ml	37	192	2	0.48	63.16	42.32
	37
	74
500 µg/ml	38	192	2	0.56	73.68	52.31
	25
	63
250 µg/ml	20	192	2	0.69	90.79	111.67
	28
	48
125 µg/ml	39	192	2	0.65	85.53	102.64
	13
	52
62.5 µg/ml	25	192	2	0.77	101.32	110.44
	16
	41

RS: Relative survival

CE: Cloning efficacy

 

Table 5. Test 1, relative survival with S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	15	192	2	0.82	-	-
	22
	37
Positive Control CP 1.5 µg/ml	30	192	2	0.61	74.39	47.61
	27
	57
500 µg/ml	21	192	2	0.91	110.98	108.76
	10
	31
250 µg/ml	19	192	2	0.85	103.66	82.93
	16
	35
125 µg/ml	10	192	2	0.96	117.07	96.00
	18
	28
62.5 µg/ml	20	192	2	0.70	85.37	63.17
	27
	47

RS: Relative survival

CE: Cloning efficacy

 

Table 6. Test 2, relative survival without S9 after a long treatment (24 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	13	192	2	1.00	-	-
	13
	26
Positive Control MMS 2 µg/ml	39	192	2	0.45	45.00	33.30
	39
	78
500 µg/ml	63	192	2	0.46	46.00	40.94
	13
	76
250 µg/ml	15	192	2	0.81	81.00	51.03
	23
	38
125 µg/ml	4	192	2	1.35	135.00	125.55
	9
	13
62.5 µg/ml	6	192	2	1.66	166.00	124.50
	1
	7

RS: Relative survival

CE: Cloning efficacy

 

Table 7. Test 1, frequency of mutants without S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	76	384	2000	128.46	169.03	-	-	-
	71
	76
	74
	297
Positive Control MMS 10 µg/ml	53	384	2000	410.37	854.94	88.40	P ≤0.05	Significant
	44
	34
	38
	169
500 µg/ml	59	384	2000	194.98	348.18	13.44	P ≤0.05	Significant
	67
	66
	68
	260
250 µg/ml	58	384	2000	222.66	322.70	10.68	P ≤0.05	Significant
	65
	68
	55
	246
125 µg/ml	48	384	2000	292.33	449.74	31.80	P ≤0.05	Significant
	58
	54
	54
	214
62.5 µg/ml	67	384	2000	204.69	265.83	5.38	P ≤0.05	Significant
	68
	59
	61
	255

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

Table 8. Test 1, frequency of mutants with S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	76	384	2000	130.14	158.71	-	-	-
	72
	76
	72
	296
Positive Control CP 1.5 µg/ml	42	384	2000	373.32	612.00	61.32	P ≤0.05	Significant
	43
	43
	54
	182
500 µg/ml	54	384	2000	267.27	293.70	13.00	P ≤0.05	Significant
	54
	58
	59
	225
250 µg/ml	49	384	2000	278.51	327.66	17.60	P ≤0.05	Significant
	52
	61
	58
	220
125 µg/ml	53	384	2000	278.51	290.11	11.63	P ≤0.05	Significant
	59
	52
	56
	220
62.5 µg/ml	65 66	384	2000	204.69	292.41	9.20	P ≤0.05	Significant
	65
	59
	255

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

Table 9. Test 2, frequency of mutants without S9 after a long treatment (24 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	70	384	2000	157.93	157.93	-	-	-
	72
	68
	70
	280
Positive Control MMS 2 µg/ml	44	384	2000	384.43	854.29	93.47	P ≤0.05	Significant
	47
	50
	37
	178
500 µg/ml	59	384	2000	258.46	561.87	39.56	P ≤0.05	Significant
	55
	59
	56
	229
250 µg/ml	51	384	2000	306.55	378.46	24.67	P ≤0.05	Significant
	46
	55
	56
	208
125 µg/ml	59	384	2000	292.33	216.54	2.19	P> 0.05	Non significant
	54
	53
	48
	214
62.5 µg/ml	50 48	384	2000	316.26	190.52	0.60	P> 0.05	Non significant
	50
	56
	204

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

 

Small and large colonies

Table 10. Distribution of small (SC) and large colonies (LC)

4H-S9		Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
		500	250	125	62.5	vehicle	MMS 10 µg/ml
Test 1	SC	19	13	18	15	17	93
	LC	105	125	152	114	70	122
	% SC	15.32	9.42	10.59	11.63	19.54	43.26

 

4H+S9		Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
		500	250	125	62.5	vehicle	CP 1.5 µg/ml
Test 1	SC	20	24	19	14	12	85
	LC	139	140	144	115	76	117
	% SC	12.58	14.63	11.66	10.85	13.64	42.08

 

24H-S9		Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
		500	250	125	62.5	vehicle	MMS 2 µg/ml
Test 2	SC	20	14	13	19	13	82
	LC	135	162	157	161	91	114
	% SC	12.90	7.95	7.65	10.56	12.50	41.84

 

Conclusions:

Under these experimental conditions, the test item is considered genotoxic with the mouse lymphoma assay. In addition, this genotoxic effect is more mutagenic than clastogenic.

Executive summary:

The potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus was studied using the cell line L5178Y according to OECD 490, following GLP. A cytotoxicity test included in the gene mutation test was performed on 8 doses. According to the results, four doses were selected, in all treatments with and without metabolic activation the doses tested were 500, 250, 125 and 62.5 µg/ml. Negative and a positive controls were performed in parallel. Positive controls led to an increase in mutation frequency at least 300 x 10E-6 with at least 40% in small colony, which validate the test. The frequencies of mutations observed for the test item were statistically different from those observed for the negative control. Under these experimental conditions, the test item is considered genotoxic with the mouse lymphoma assay. In addition, this genotoxic effect is more mutagenic than clastogenic.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

National Toxicoloxy Program. A standarised protocol was followed.

Deviations:

yes

Remarks:

only 4 strains tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Lot number:022277

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 333, 1000, 3333, 10000 and 20830 µg/plate. The maximum concentration used was the upper limit of solubility.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the chemical was soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene, 4-Nitro-o-phenylenediamine,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation
All chemicals were tested using the preincubation procedure of the Salmonella assay (Ames et al. 1975) as described by Yahagi et al. (1975). 0.5 ml of S9 mix or 0.1 M PO4 buffer was dispensed into an appropiate number of 13x 100 mm culturee tubes maintained at 37ºC in a dry-bath. Then 0.05 ml of cells and 0.05 ml of solvent or chemical solution were added to each tobe. The mixture was vortexed and allowed to incubate with shaking in the early tests for 20 minutes at 37 ºC. Following the preincubation period, 2 ml of molten top agar (45ºC) supplemented with 0.5 ml L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar (Vogel and Bonner, 1956) in Fisher Scientific petri dishes. After the overlay solidified, the plates were inverted and incubated at 37ºC for 48 h. Each strain was tested in the presence of S9 mix or buffer. The experiment was repeated no less than 1 week after completion of the initial test.

- Cell density at seeding (if applicable): not specified

DURATION
- Preincubation period: 20 min at 37ºC
- Exposure duration: 48 h at 37ºC

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S9 mix. One or more parameters were used as an indication of toxicity: reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn.

- OTHER:
Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected ip with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. Five days after injection, the animals were sacrified and the livers were removed aseptically. The animals were fasted for 12-24 h immediately preceding sacrifice. Liver homogenates were prepared aseptically at 0-4ºC. Excised livers were rinsed with 0.15 M KCl, then minced and homogenized. The homogenate was centrifuged for 10 min at 9000 ga t 4ºC. The supernatant (S9) was decanted and distributed into freezing ampules and stored at -70ºC. The microsomal enzyme reaction mix (S9 mix) was prepared immediately prior to each assay. Unused S9 mix was discarded and not refrozen. One milliliter of S9 mix has the following composition: S9, 0.1 ml; 0.04 M MgCl2, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M beta-nicotinamide adenine dinucleotide phosphate (NADP), 0.1 ml; 0.05 M glucose-6-phosphate, 0.1 ml; 1 M NaH2PO4 (pH 7.4), 0.1 ml; and distilled water, 0.56 ml.

Evaluation criteria:

The data were evaluated in an hoc manner by each testing laboratory and by NTP personnel. A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.

Statistics:

The data were evaluated using an analysis based on the models presented by Margolin et al (1981).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test item is soluble in water
- Precipitation: no precipitation was found

RANGE-FINDING/SCREENING STUDIES: a range finding study was performed to set the maximal dose, no toxicity was found. Then, the test item was tested at 20.83 mg/plate.

Table 2. Mutagenic response of Salmonella tester strains TA100, TA1535, TA1537 and TA98 to choline chloride. Case Western Reserve University (CWR).

 

TA100

TA1535

TA1537

TA98

Dose (µg/plate)

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

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

333.0

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±5.2

24±2.2

17±2.1

1000.0

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

3333.0

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

10000.0

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

20830.0

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

 

Abbreviations:

POS: Positive control

NA: Not activated

RLI: Rat liver S9, Aroclor 1254 induced

HLI: Hamster liver S9, Aroclor 1254 induced

Conclusions:

Choline chloride did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation up to 20830 µg/plate.

Executive summary:

The ability of choline chloride to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to a National Toxicology Program standardised method, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to exposed to 0, 333, 1000, 3333, 10000 and 20830 µg/ml of test item in the presence and absence of S9 metabolic activation (Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

National Toxicoloxy Program. A standarised protocol was followed.

Deviations:

yes

Remarks:

only 4 strains tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Lot number:022277

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were prepared for storage as described in Ames et al. 1975. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333 and 10000 µg. The maximum concentration used was 10 mg/plate because no apparent toxicity was found in the preliminary test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the chemical was soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene, 4-Nitro-o-phenylenediamine,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation
All chemicals were tested using the preincubation procedure of the Salmonella assay (Ames et al. 1975) as described by Yahagi et al. (1975). 0.5 ml of S9 mix or 0.1 M PO4 buffer was dispensed into an appropiate number of 13x 100 mm culturee tubes maintained at 37ºC in a dry-bath. Then 0.05 ml of cells and 0.05 ml of solvent or chemical solution were added to each tobe. The mixture was vortexed and allowed to incubate with shaking in the early tests for 20 minutes at 37 ºC. Following the preincubation period, 2.5 ml of molten top agar (45ºC) supplemented with 0.5 ml L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar (Vogel and Bonner, 1956) in a 15 x 100 mm plastic petri dish (Falcon Muta-Assay, 1028) After the overlay solidified, the plates were inverted and incubated at 37ºC for 48 h. Each strain was tested in the presence of S9 mix or buffer. The experiment was repeated no less than 1 week after completion of the initial test.

- Cell density at seeding (if applicable): not specified

DURATION
- Preincubation period: 20 minutes at 37 ºC
- Exposure duration: 48 h at 37 ºC

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: the test chemicals were checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn.

- OTHER:
Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected ip with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. Five days after injection, the animals were sacrified and the livers were removed aseptically. The animals were fasted for 12-24 h immediately preceding sacrifice. Liver homogenates were prepared aseptically at 0-4ºC. Excised livers were rinsed with 0.15 M KCl, then minced and homogenized. The homogenate was centrifuged for 10 min at 9000 ga t 4ºC. The supernatant (S9) was decanted and distributed into freezing ampules and stored at -70ºC. The microsomal enzyme reaction mix (S9 mix) was prepared immediately prior to each assay. Unused S9 mix was discarded and not refrozen. One milliliter of S9 mix has the following composition: S9, 0.1 ml; 0.04 M MgCl2, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M beta-nicotinamide adenine dinucleotide phosphate (NADP), 0.1 ml; 0.05 M glucose-6-phosphate, 0.1 ml; 1 M NaH2PO4 (pH 7.4), 0.1 ml; and distilled water, 0.56 ml.

Evaluation criteria:

The data were evaluated in an hoc manner by each testing laboratory and by NTP personnel. A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.

Statistics:

The data were evaluated using an analysis based on the models presented by Margolin et al (1981).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test item is soluble in water
- Precipitation: no precipitation was found

RANGE-FINDING/SCREENING STUDIES: a range finding study was performed to set the maximal dose, no toxicity was found. Then, the test item was tested at 10 mg/plate.

Table 2. Mutagenic response of Salmonella tester strains TA100, TA1535, TA1537 and TA98 to choline chloride. EG&G Mason Research corporation (EEG).

 

TA100

TA1535

TA1537

TA98

Dose (µg/plate)

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

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

100.0

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

333.0

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

1000.0

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

3333.0

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±3.7

26±3.5

31±4.1

10000.0

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

   

Abbreviations:

POS: Positive control

NA: Not activated

RLI: Rat liver S9, Aroclor 1254 induced

HLI: Hamster liver S9, Aroclor 1254 induced

Conclusions:

Choline chloride did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 97, TA 98 and TA 100 with and without metabolic activation up to 10000 µg/plate.

Executive summary:

The ability of choline chloride to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the a National Toxicology Program standarised method, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 of the 5 recommended strains are tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Source: Japan Food Additives Association, Tokyo
Purity: 98.7 %

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by Ames, University of California, Bekerly, USA

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by Ames, University of California, Bekerly, USA

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by Ames, University of California, Bekerly, USA

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by Ames, University of California, Bekerly, USA

Species / strain / cell type:

S. typhimurium, other: TA 92

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by Ames, University of California, Bekerly, USA

Species / strain / cell type:

S. typhimurium, other: TA 94

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Provided by Ames, University of California, Bekerly, USA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Six different concentrations, maximal dose 100 mg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffer

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): no data
The experiment was carried out according to the method of Ames, McCann and Uamasaki (1975) [see Attached background material].

DURATION
- Preincubation period: 20 minutes at 37ºC
- Exposure duration: 48 hours at 37ºC

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 2

- OTHER:
Preparation of S9 mix:
The liver microsome fraction (S9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S9 mix) contained 5 mM glucose 6-phosphate, 4 mM NADPH, 4 mM NADH, 33 mM KCl, 8 mM MgCl2, 100 mM phosphate buffer (pH 7.4) and 3.75 ml S9 (129 mg protein) in a total volume of 12.5 ml.

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 92

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 94

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Mutagenicity of synthetic food additives in salmonella/microsome tests in vitro.

Ames test Additive CAS no. Purity (%) Max dose (mg/ml) solvent Result Sodium phosphate 7558-79-4 98.7 100 Phosphate buffer Negative

Conclusions:

Disodium phosphate did not induce any mutagenic change in Salmonella typhimurium TA 92, TA1535, TA 100, TA 1537, TA 94 and TA 98, with and without metabolic activation up to 100 mg/plate.

Executive summary:

The ability of disodium phosphate to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames (1975), similar to OECD 471. Six histidine dependent strains of Salmonella typhimurium (TA92, TA1535, TA97, TA98, TA100 and TA94) were exposed to exposed to six concentrations of test item up to a maximal dose of 100 mg/plate of test item in the presence and absence of S9 metabolic activation (Kanchlor KC-400- induced rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one concentration tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

75-85% solution (concentration of solutions not specified)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: MMS; daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
The standard plate incorporation assay was carried out according to the procedure developed by Ames (1975) and Maron and Ames et al. (1983) [see attached background material]. Test compound, the bacterial tester strain and S9 mix are combined in soft agar and poured onto a minimal agar plate. Positive and negative control are also included in the assay. After incubation at 37ºC for 48 h, revertant colonies are counted.
S9 microsomal fraction was prepared from liver homogenates of Sprague-Dawley rats induced with Aroclor 1254.

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Phosphoric acid did not induce any mutagenic change in Salmonella typhimurium TA1535, TA 97, TA 98, TA 100 and TA 102, with and without metabolic activation up to 75-85% solution.

Executive summary:

The ability of phosphoric acid to induce mutation was assessed by a similar protocol to the bacterial reverse mutation test (OECD 471). Five histidine dependent strains of Salmonella typhimurium TA1535, TA97, TA98, TA100 and TA102 were exposed to phosphoric acid 75 -85% solution in the presence and absence of S9 metabolic activation (Aroclor 1254- induced rat liver homogenates), by the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 10

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 22, 1980 - August 28, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 100 well-spread metaphases were observed

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: National Toxicology Program chemical repository

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) cells were obtained from Dr. Sheldon Wolff, cloned, and designated CHO-W-B1. To mantain kariotip stability, the cells were used at no more than 15 passages after cloning.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Assay without metabolic activation: 5000, 4000, 3000, 2000 and 1000 µg/mL
Assay with metabolic activation: 5000 - 500 - 50 - 5 - 0.5 - 0.05 µg/mL
The highest concentration tested for cytogenetic activity was the concentration estimated to reduce cell numbers by about 50%; lower, nontoxic doses were also included.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHO cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in air, in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), and L-glutamine (2mM). Penicillin (100 units/ml) and streptomycin (100 µg/ml) were used. Cultures were set up the day before treatment at a uniform cell density, chosen to allow treatment of exponentially growing cultures (eg, approximately 1E06 cells per 75 cm2 flask). Duplicate 25 cm2 flasks were used for each experimental point; half the cells scored were from each culture.
In the assay without metabolic activation, one day after culture initiation, the medium was replaced and cells were treated with test of control chemicals for 8 to 10 h. Medium was removed and fresh medium containing colcemid (final concentration 0.1 µg/ml) was added, and incubation continued for 2 to 2.5 h before fixation. In the assay with metabolic activation, cells were treated with test of control chemicals and S9 mix for 2 h. After the incubation with colcemid, cells were collected by mitotic shake-off and trated for up to 3 min at room temperature with hypotonic KCl (75 mM).

DURATION
- Exposure duration: 8 -10 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3 responses were analyzed for each trial

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were selected for scoring on the basis of good morphology and a chromosome number of 21 ± 2. For control of bias, all slides were coded before scoring. All slides from a test (control and doses) were read by the same reader. Slides were stained in Giemsa.

NUMBER OF CELLS EVALUATED: 100 cells per dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 of metaphase spreads were analysed per dose.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 occurence 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.

OTHER:
- The S9 fraction was prepared from Sprague-Dawley rats induced with Aroclor 1254. The same batch of liver homogenate was used in both labs and was stored at -80ºC. S9 fraction was thawed immediately before use, mixed with an NADPH-generating system, and added to medium to give the following final concentrations in culture: NADP (sodium salt), 2.4 mg/ml; isocitric acid (trisodium salt), 4.5 mg/ml, and S9 fraction, 15 µl/ml.

Evaluation criteria:

The evaluation reflects a blend of statistical analysis and biological intuition.The summary of judgement is shown in Table 1. For each test, an efferot has been made to recognize any response to the chemical under test while guarding against flase-positive evaluations that could arise by chance as a result of the multiple tests performed.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 2. Results of the chromosomic aberrations test for Choline chloride without metabolic activation.

		Dose µg/mL	Total Cells examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
				No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs
Vehicle control:	Water	0	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
Test chemical:	Choline chloride	1000	100	3	0.030	3.0	0	0.000	0.0	2	0.020	2.0	1	0.010	1.0
		2000	100	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0	0	0.000	0.0
		3000	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		4000	100	7	0.070	7.0	4	0.040	4.0	2	0.020	2.0	1	0.010	1.0
		5000	100	2	0.020	2.0	0	0.000	0.0	1	0.010	1.0	1	0.010	1.0
Positive control:	Triethylenemelamine	0.5	100	37	0.370	32.0	5	0.050	4.0	32	0.320	26.0	0	0.000	0.0
Trend:				1.591			1.600			0.451
Probability:				0.056			0.055			0.326

 

Table 3. Results of the chromosomic aberrations test for Choline chloride with metabolic activation (induced rat liver S9).

		Dose µg/mL	Total Cells examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
				No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs	No. of Abs	Abs per Cell	% Cells with Abs
Vehicle control:	Water	0	100	1	0.010	1.0	0	0.000	0.0	0	0.000	0.0	1	0.100	1.0
Test chemical:	Choline chloride	0.05	100	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0	0	0.000	0.0
		0.5	100	2	0.020	2.0	2	0.020	2.0	0	0.000	0.0	0	0.000	0.0
		5	100	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0	0	0.000	0.0
		50	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		500	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		5000	100	3	0.030	3.0	1	0.010	1.0	2	0.020	2.0	0	0.000	0.0
Positive control:	Cyclophosphamide	25	100	67	0.670	37.0	34	0.340	27.0	30	0.300	18.0	3	0.030	3.0
	Cyclophosphamide	50	100	96	0.960	49.0	34	0.340	29.0	58	0.580	30.0	4	0.040	4.0
Trend:				0.356			-0.224			2.124
Probability:				0.361			0.589			0.017

 

Conclusions:

Choline chloride does not induce chromosomal aberration in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as not mutagenic.

Executive summary:

In order to determine the ability of choline chloride to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 473. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity, 5000 µg/ml. Then, CHO cells were exposed to five concentrations of the test item for 8 -10 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation with colcemid (0.1  g/ml), cells were fixed and stained. Negative and positive controls were run in parallel. 100 cells were examined per dose level and the results for chromosome aberration assay with and without metabolic activation showed no evidence of the induction of structural aberrations in CHO cells by the test substance under test conditions. Therefore, the test item can be considered as not mutagenic.

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

100 well-spread methaphases, no metabolic activation

GLP compliance:

no

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Source: Japan Food Additives Association, Tokyo
Purity: 98.7 %

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cell line was originally stablished from the lung of a newborn female at the Cancer Research Institute, Tokyo (Konama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 h, as described elsewhere (Ishidate & Odashima, 1977)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Three different doses, maximal dose 2 mg/ml.
The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Method as previously described [Ishidate & Odashima (1977), see 'Attached background material']: Three different doses, including the 50% inhibition dose of each agent, which was estimated by a growth inhibition test, were prepared and separately added to 3-day-old cultures (about 10^5 cells/6-cm dish). Chromosome preparations were made, at 24 and 48 h. Cells were treated with colcemid (0.2 μg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KCl hypotonic solution for 15 min at 37ºC. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3
times. A few drops of the suspension were placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1.5% Giemsa's buffered solution.

DURATION
- Exposure duration: 24-48h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concn. 0.2 μg/ml)

STAIN (for cytogenetic assays): Giemsa solution (1.5%, at pH 6.8; E. Merck)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colcemid (final concn. 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens).

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth; cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)

OTHER EXAMINATIONS:
- Determination of polyploidy: The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.

Evaluation criteria:

The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0-9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relation ships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed.

Statistics:

The frequency of cells with exchange-type aberrations per unit dose (mg/ml) was calculated (TR value).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

Polyploid: 1%; Structural aberrations: 2% (48 h) at 2 mg/ml

Table 1. Mutagenicity of synthetic food additives in chromosomal aberration tests in vitro.

			Chromosome test
Additive	CAS no.	Purity (%)	Max dose* (mg/ml)	Solvent	Polyploid (%)	Structural aberations		Result
						(%)	(hr)
Sodium phosphate	7558-79-4	98.7	2	Phys. saline	1	2	(48)	Negative

Conclusions:

Disodium phosphate does not induce chromosomal aberration in Chinese Hamster Lung fibroblasts (CHL) without metabolic activation. Therefore, the test item can be considered as not mutagenic.

Executive summary:

A study to determine the ability of disodium phosphate to induce chromosomal aberrrations in Chinese Hamster Lung fibroblasts (CHL) was performed by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose, which was the dose needed for 50% cell-growth inhibiton (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (2.0 mg/plate) for 24 and 48h, without metabolic activation. Untreated cells and solvent treated cells served as negative controls (incidence of aberrations < 3.0%); 200 chemicals were tested, the positive results served as controls. A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens), and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each cultured plate. The results showed no evidence of the induction of structural aberrations in cultured lymphocytes by the test substance under test conditions. Therefore, the test item was not mutagenic.

Reference 12

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 25, 1981 - December 28, 1981

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 100 well-spread metaphases were observed

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: National Toxicology Program chemical repository

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With metabolic activation: 5000 - 4000 - 3000 - 2000 and 0 µg/ml
Without metabolic activation: 5000 - 4000 - 3000 and 0 µg/ml
The highest concentration was determined from the information on cell cycling and toxicity obtained from

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHO cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in air, in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), and L-glutamine (2mM). Penicillin (100 units/ml) and streptomycin (100 µg/ml) were used. Cultures were set up the day before treatment at a uniform cell density, chosen to allow treatment of exponentially growing cultures (eg, approximately 1E06 cells per 75 cm2 flask). Duplicate 25 cm2 flasks were used for each experimental point; half the cells scored were from each culture.
In the assay without metabolic activation, one day after culture initiation, the medium was replaced and cells were treated with test of control chemicals for 8 to 10 h. Medium was removed and fresh medium containing colcemid (final concentration 0.1 µg/ml) was added, and incubation continued for 2 to 2.5 h before fixation. In the assay with metabolic activation, cells were treated with test of control chemicals and S9 mix for 2 h. After the incubation with colcemid, cells were collected by mitotic shake-off and trated for up to 3 min at room temperature with hypotonic KCl (75 mM).

DURATION
- Exposure duration: 8 -10 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3 responses were analyzed for each trial

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were selected for scoring on the basis of good morphology and a chromosome number of 21 ± 2. For control of bias, all slides were coded before scoring. All slides from a test (control and doses) were read by the same reader. Slides were stained in Giemsa.

NUMBER OF CELLS EVALUATED: 100 cells per dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 of metaphase spreads were analysed per dose.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 occurence 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.

OTHER:
- The S9 fraction was prepared from Sprague-Dawley rats induced with Aroclor 1254. The same batch of liver homogenate was used in both labs and was stored at -80ºC. S9 fraction was thawed immediately before use, mixed with an NADPH-generating system, and added to medium to give the following final concentrations in culture: NADP (sodium salt), 2.4 mg/ml; isocitric acid (trisodium salt), 4.5 mg/ml, and S9 fraction, 15 µl/ml.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 2. Results of the chromosomic aberrations test for Choline chloride without metabolic activation.

		Dose	Total	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
		µg/mL	Cells Examined	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells
				Abs	Per	With	Abs.	Per	With	Abs.	Per	With	Abs.	Per	With
					Cell	Abs		Cell	Abs		Cell	Abs		Cell	Abs
Vehicle Control:	Negative (Not Specified)	0	100	1	0.010	1.0	0	0.000	0.0	1	0.010	1.0	0	0.000	0.0
Test Chemical:	Choline chloride	2000	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
		3000	100	5	0.050	5.0	4	0.040	4.0	1	0.010	1.0	0	0.000	0.0
		4000	100	4	0.040	4.0	2	0.020	2.0	2	0.020	2.0	0	0.000	0.0
		5000	100	3	0.030	2.0	2	0.020	1.0	1	0.010	1.0	0	0.000	0.0
Positive Control:	Mitomycin-C	0.25	100	26	0.260	22.0	7	0.070	7.0	15	0.150	14.0	4	0.040	4.0
Trend:				1.458			1.308			0.699
Probability:				0.072			0.095			0.242

 

Table 3. Results of the chromosomic aberrations test for Choline chloride with metabolic activation (induced rat liver S9).

		Dose	Total	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
		µg/mL	Cells Examined	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells
				Abs	Per	With	Abs.	Per	With	Abs.	Per	With	Abs.	Per	With
					Cell	Abs		Cell	Abs		Cell	Abs		Cell	Abs
Vehicle Control:	Negative (Not Specified)	0	100	3	0.030	2.0	1	0.010	1.0	2	0.020	1.0	0	0.000	0.0
	Water	0	0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
Test Chemical:	Choline chloride	3000	100	5	0.050	5.0	2	0.020	2.0	3	0.030	3.0	0	0.000	0.0
		4000	100	5	0.050	5.0	3	0.030	3.0	2	0.020	2.0	0	0.000	0.0
		5000	100	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0	0	0.000	0.0
Positive Control:	Cyclophosphamide	25	100	36	0.360	31.0	23	0.230	23.0	7	0.070	7.0	6	0.060	6.0
Trend:				-1.438			-0.440			-1.629
Probability:				0.925			0.670			0.948

 

Conclusions:

Choline chloride does not induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells with and without metabolic activation up to 5000 µg/mL.

Executive summary:

In order to determine the ability of the choline chloride to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 473. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to different concentrations of the test item (5000, 4000, 3000 and 2000 µg/mL without metabolic activation; 5000, 4000 and 3000 µg/mL with metabolic activation) for 8 -10 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation with colcemid (0.1  g/ml), cells were fixed and stained. Negative and positive controls were run in parallel. 100 cells were examined per dose level and the results for chromosome aberration assay with and without metabolic activation showed no evidence of the induction of structural aberrations in CHO cells by the test substance under test conditions. Therefore, the test item can be considered as not mutagenic.

Reference 13

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 18, 1981

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: National Toxicology Program chemical repository.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5000 - 1600 - 500 - 160 - 16 µg/ml
The highest concentration tested for cytogenetic activity was the concentration estimated to reduce cell numbers by about 50%; lower, nontoxic doses were also included.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHO cells were grown at 37ºC in a humidified atmosphere of 5% CO2 in air, in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), and L-glutamine (2mM). Penicillin (100 units/ml) and streptomycin (100 µg/ml) were used. Cultures were set up the day before treatment at a uniform cell density, chosen to allow treatment of exponentially growing cultures (eg, approximately 1E06 cells per 75 cm2 flask). Duplicate 25 cm2 flasks were used for each experimental point; half the cells scored were from each culture.
Assay without metabolic activation: One day after culture initiation, the medium was replaced and cells were treated with test of control chemicals for 2 h to allow interaction with cells before addition of BrdUrd. BrdUrd was added (final concentration 10 µM) and incubation continued for about 24 h. Medium was removed to allow an opportunity to wash off any test chemical precipitate that might interfere with cell fixation and to avoid harvest of cultures containing test chemicals. Fresh medium containing 10 µM BrdUrd and colcemid (final concentration 0.1 µg/ml) was added, and incubation continued for 2 to 3 h.
The total incubation time test chemical was thus about 26 h, and total time with BrdUrd was also about 26 h, beginning about 2 h after addition of test chemical.
Assay with metabolic activation: One day after culture initiation, medium was replaced with S9 mix in medium without fetal calf serum. After exposure for 2 h to test compound, cells were washed at least twice with buffered saline, and culture medium containing 10% FCS and 10 µM BrdUrd was added. Cells were incubated for a further 26 h, with colcemid (0.1 µg/ml) present for the final 2 to 3 h.
Two to three hours after addition of colcemid, cells were collected by trypsinization and treated for up to 3 min at room temperature with hypotonic KCl (75 mM). cell were then washed twice with fixative (3:1, methanol:glacial acetic acid, v/v), dropped onto slides, and air-dried.

DURATION
- Exposure duration: 26 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 28-29 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)

STAIN (for cytogenetic assays): Hoechst 33258 and Giemsa

NUMBER OF REPLICATIONS: 3 responses were analyzed for each trial

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were selected for scoring on the basis of good morphology and a chromosome number of 21 ± 2. For control of bias, all slides were coded before scoring. All slides from a test (control and doses) were read by the same reader. Slides were stained in Giemsa.

NUMBER OF CELLS EVALUATED: 50 cells per dose level

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 of metaphase spreads were analysed per dose.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: 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 occurence 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.

OTHER:
- The S9 fraction was prepared from Sprague-Dawley rats induced with Aroclor 1254. The same batch of liver homogenate was used in both labs and was stored at -80ºC. S9 fraction was thawed immediately before use, mixed with an NADPH-generating system, and added to medium to give the following final concentrations in culture: NADP (sodium salt), 2.4 mg/ml; isocitric acid (trisodium salt), 4.5 mg/ml, and S9 fraction, 15 µl/ml.

Statistics:

Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control . An sister chromatid exchange (SCE) frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01, the probability for such a chance occurred at two dese points is less than 0.001. An increase of 20%, or greater, at any single dose, was considered weak evidence of activity (weak positive), increases at two or more doses resulted in a determination that the trial was positive. A trend P-value of less than 0.005, in the absence of any responses reaching 20% above background, led to a call of equivocal for the trial. Positive and weak positive trials were repeated. The overall assay result was based on an evaluation of the responses in all trials within an activation condition.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Results of the sister chromatid exchange test for Choline chloride without metabolic activation.

		Dose	Number	Number	Total	SCE /	SCE /	Hours in	% Increase
		µg/mL	Cells	Chromosomes	Number	Chromosome	Cell	BRDU	Over Solvent
			Examined	Examined	SCEs				Control
Vehicle Control	Water	0	0	0	0	0.000	0.00	0.0	N/A
	Negative (Not Specified)	0	50	1046	368	0.352	7.36	26.0	0.000
Test Chemical	Choline chloride	16	50	1051	402	0.382	8.04	26.0	0.000
		160	50	1034	357	0.345	7.14	26.0	0.000
		500	50	1032	381	0.369	7.62	26.0	0.000
		1600	50	1040	366	0.352	7.32	26.0	0.000
		5000	50	1042	404	0.388	8.08	26.0	0.000
Positive Control	Mitomycin-C	0.005	50	1044	1572	1.506	31.44	26.0	0.000
Trend:					0.017
Probability:					0.493

Table 2. Results of the sister chromatid exchange test for Choline chloride with metabolic activation.

 

		Dose	Number	Number	Total	SCE /	SCE /	Hours in	% Increase
		µg/mL	Cells	Chromosomes	Number	Chromosome	Cell	BRDU	Over Solvent
			Examined	Examined	SCEs				Control
Vehicle Control	Water	0	0	0	0	0.000	0.00	0.0	N/A
	Negative (Not Specified)	0	50	1050	458	0.436	9.16	26.0	0.000
Test Chemical	Choline chloride	16	50	1036	461	0.445	9.22	26.0	0.000
		50	50	1042	429	0.412	8.58	26.0	0.000
		160	50	1038	447	0.431	8.94	26.0	0.000
		500	50	1046	468	0.447	9.36	26.0	0.000
		1600	50	1038	427	0.411	8.54	26.0	0.000
		5000	50	1035	466	0.450	9.32	26.0	0.000
Positive Control	Cyclophosphamide	1.5	50	1040	2076	1.996	41.52	26.0	0.000
Trend:					0.247
Probability:					0.403

Conclusions:

Choline chloride does not induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Therefore, the test item can be considered as not mutagenic.

Executive summary:

In order to determine the ability of choline chloride to induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells studies iwere performed by a method similar to OECD 479. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (5000, 1600, 500, 160 and 16 µg/mL) for 26 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation, the medium with test chemical was removed and replaced with fresh medium plus BedUrD and colcemid (0.1  g/ml), without test chemical, and incubated for further 2 hours. Then, cells were harvested, fixed and stained with Hoechdt 33258 and Giemsa. Negative and positive controls were run in parallel. 50 cells were examined per dose level and the results for sister chromatid exchange assay were negative.Under these test conditions, the test item was found to be non mutagenic.

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 30, 1975

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only three S. thyphimurium strains tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Fine white powder.

Target gene:

Histidine.

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

Saccharomyces cerevisiae

Remarks:

Strain D4

Metabolic activation:

with and without

Metabolic activation system:

Tissue homogenate fraction of Mouse-ICR random bred adult males; rat-Sprague-Dawley adult males, and primate-Maccaca mulatta adult males.

Test concentrations with justification for top dose:

0.75%
The top dose corresponds to the 1/2 50% dose calculated from the survival curve.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: the test compound was soluble under treatment conditions.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

2-nitrofluorene

N-dimethylnitrosamine

ethylmethanesulphonate

other: Quinacrine mustard

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate tests:
Without metabolic activation: approximately 10E09 cell of a log phase culture of the bacterial indicator strains were spread over the surface of a minimal plate, and a measured amount of the test chemical was placed in the center of the test plate.
With metabolic activation: the test chemical was added to the cells, and an aliquot of the mixture was spread on the surface of the test plate. The reaction mixture (0.1 ml) plus tissue extract was then spotted on the surface of the plate. Positive and solvent controls were included. All plates were incubated at 37ºC for four days.

Suspension tests:
Without metabolic activation: log-phase bacteria cultures were grown in complete broth, washed and resuspended in 0.9% saline to densities of 10E09 cells/ml, respectively. This constituted the working stock for tests of a group of test chemicals and their respective controls. Tests were conducted in plastic tissue culture plates. Cells plus appropiate volume(s) of the test chemical/solvent were added to the wells to give a final volume of 1.5 ml. Treatment was at 37ºC for 1 h for bacterial tests. All flasks were shaken during treatment. Following treatment, the plates were set on ice. Aliquots of cells were removed, diluted in sterile saline (4ºC) and plated on the appropiate complete media. Undiluted samples from flasks containing the bacteria were plated on minimal selective medium in reversion experiments. Samples from 10E-1 dilution of treated cells were plated on the selected media for enumeration of gene conversion with strain D4. Bacterial plates were scored after incubation for 48 h at 37ºC.
With metabolic activation: bacteria cells were grown and prepared as described in the nonactivation tests. Measured amounts of the test and control chemicals plus 0.25 ml of the stock-cell suspension were added to wells of the Limbro plate containing the appropriate tissue fraction and reaction mixture. All flasks were incubated at 37ºC in an oxygen atmosphere with shaking. The treatment times as well as the dilutions, plating procedures and scoring of the plates were the same as described for nonactivation tests.

DURATION
- Exposure duration: 1 h at 37 ºC
- Selection time (if incubation with a selection agent): 48 h at 37 ºC

SELECTION AGENT (mutation assays): lack of histidine in the media.

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: other: 50% survival dose
- Any supplementary information relevant to cytotoxicity:
The following concentrations of the test compound, 10, 1, 0.1, 0.01 and 0.001%, were tested for survival against the specific the specific indicator strains over a range of doses to determine the 50% survival dose. Bacteria and yeast strains were tested in phosphate buffer, pH 7.4, for 4 h on a shaker. The 50% survival curve and the 1/4 and 1/2 50% doses calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Summary of test results: Plate tests. Test compound concentration: 0.75%

			REVERTANTS/PLATE
	Species	Tissue	TA-1535		TA-1537		TA-1538
Non-activation
Solvent control	-	-	40	41	11	13	27	23
Positive control^1	-	-	>10E03	>10E03	183	115	98	128
Test compound	-	-	51	38	9	9	18	29
Activation
Negative control	-	-	8	12	7	7	6	18
Solvent control	-	-	13	4	15	16	23	21
Reaction Mixture Control	-	-	7	10	8	8	10	18
Positive control^2	Mouse	Liver	>10E03	>10E03	41	43	397	420
Positive control		Lung	11	13	5	12	72	30
Positive control		Testes	9	11	22	10	19	22
Positive control	Rat	Liver	>10E03	>10E03	41	45	327	340
Positive control		Lung	12	9	7	7	26	29
Positive control		Testes	9	11	16	10	14	11
Positive control	Monkey	Liver	390	329	44	41	363	310
Positive control		Lung	11	9	7	10	21	24
Positive control		Testes	9	12	16	6	17	12
Test compound	Mouse	Liver	22	28	15	17	23	19
Test compound		Lung	28	29	9	8	21	23
Test compound		Testes	27	24	9	8	19	16
Test compound	Rat	Liver	24	27	16	22	19	20
Test compound		Lung	27	29	9	6	22	20
Test compound		Testes	27	24	7	8	18	19
Test compound	Monkey	Liver	21	27	16	20	22	18
Test compound		Lung	26	29	12	7	20	19
Test compound		Testes	23	21	5	5	22	18

¹TA-1535 EMS 10 µl/plate; TA-1537 QM 20 µg/plate; TA-1538 NF 100 µg/plate

²TA-1535 DMNA 50 µM/plate; TA-1537 AAF 100 µg/plate; TA-1538 AAF 100 µg/plate

Table 2. Compound frequency summary report: Suspension tests (without metabolic activation)

	TA 1535 (His)	TA 1538 (His)	TA 1537 (His)	D4 (Ade)	D4 (Try)
Solvent control	5.93	8.10	2.32	1.21	1.69
Positive control	145.25	256.53	54.95	102.54	192.66
Dose level 1	5.24	7.00	2.32	1.71	3.89
Dose level 2	7.05	6.33	0.20	1.33	1.43

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

 

Table 3. Compound frequency summary report: Suspension tests (with metabolic activation: mouse tissue fraction)

		TA 1535 (His)	TA 1538 (His)	TA 1537 (His)	D4 (Ade)	D4 (Try)
Negative control		25.16	5.49	10.13	3.11	2.43
Tissue control		10.85	19.43	7.69	3.17	3.04
Solvent control		30.64	4.80	11.85	1.46	2.47
Positive control	Liver	99.45	96.04	20.85	4.07	7.05
	Lung	32.03	9.09	15.03	2.32	2.32
	Testes	23.13	12.00	7.90	3.94	1.69
Dose level 1	Liver	11.36	9.65	8.97	3.35	5.45
	Lung	20.17	4.90	18.18	2.85	2.48
	Testes	26.00	5.71	16.94	3.24	1.75
Dose level 2	Liver	13.12	8.78	12.77	5.28	5.13
	Lung	19.34	4.89	14.41	4.04	3.31
	Testes	29.16	3.23	15.84	1.99	1.77

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

 

Table 4. Compound frequency summary report: Suspension tests (with metabolic activation: rat tissue fraction)

		TA 1538 (His)	TA 1537 (His)	TA 1535 **(His)	D4 (Ade)	D4 (Try)
Negative control		5.08	25.00	4.13	2.42	1.16
Tissue control		11.94	16.99	19.55	1.31	1.19
Solvent control		4.90/2.85*	10.34	3.40	1.19	1.39
Positive control	Liver	25.88	23.46	655.87	6.42	8.88
	Lung	9.79	15.63	5.92	2.53	3.29
	Testes	7.41	13.52	11.07	2.51	2.16
Dose level 1	Liver	6.00	18.24	4.33	2.58	3.63
	Lung	8.37	11.47	5.49	1.80	2.30
	Testes	11.06	15.17	3.74	3.24	3.14
Dose level 2	Liver	16.44/10.48*	8.07	3.45	3.54	3.67
	Lung	4.50	7.68	4.53	3.41	3.29
	Testes	7.94	10.88	3.94	3.01	2.04

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

* The experiment was repeated because of increased frequencies. The repeat tests were negative.

** The TA 1535 culture used with mouse tissues had a high spontaneous background, but the data appeared acceptable.

 

Table 5. Compound frequency summary report: Suspension tests (with metabolic activation: monkey tissue fraction)

		TA 1538 (His)	TA 1537 (His)	TA 1535 **(His)	D4 (Ade)	D4 (Try)
Negative control		5.15	17.39	13.57	2.10	1.65
Tissue control		7.48	33.71	7.81	2.38	1.70
Solvent control		1.80/12.53*	15.22	8.46	1.31	2.15
Positive control	Liver	13.89	57.35	165.66	2.46	1.74
	Lung	3.05	18.75	7.04	2.78	2.16
	Testes	3.81	18.10	7.46	3.99	2.07
Dose level 1	Liver	4.15/2.88*	46.48	15.58	1.59	1.84
	Lung	1.79	8.00	10.49	2.79	1.74
	Testes	2.95	13.71	6.97	3.95	3.26
Dose level 2	Liver	6.36	17.83	8.89	2.85	2.37
	Lung	3.15	16.29	6.27	1.28	2.04
	Testes	4.99	14.97	8.91	2.29	1.02

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

* The experiment was repeated because of increased frequencies. The repeat tests were negative.

** The TA 1535 culture used with mouse tissues had a high spontaneous background, but the data appeared acceptable.

Conclusions:

Under the experimental conditions used, monocalcium phosphate was found to be non-mutagenic in Salmonella thyphimurium TA1535, TA1537 and TA1538.

Executive summary:

The ability of monocalcium phosphate to induce mutation was assessed by a similar protocol to the bacterial reverse mutation test (OECD 471). A preliminary toxicity study showed that the 1/2 50% dose calculated from the survival curve was 0.75%. Three histidine dependent strains of Salmonella typhimurium TA1535, TA1537 and TA1538 were exposed to monocalcium phosphate at 0.75%, with and without metabolic activation (tissue homogenate fraction of Mouse-ICR random bred adult males, rat sprague-Dawley adult males, and primate-maccaca mulatta adult males). Plate and suspension tests were performed. For both assays, negative and positive controls were carried out in parallel. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any bacterial strains tested, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 15

Endpoint:

genetic toxicity in vitro, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 30, 1975

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)

Deviations:

yes

Remarks:

number of concentrations tested; number of replicates; no statistical analysis

GLP compliance:

not specified

Type of assay:

other: in vitro gene mutation study in yeast

Specific details on test material used for the study:

Fine white powder.

Target gene:

Tryptophan or adenine.

Species / strain / cell type:

Saccharomyces cerevisiae

Remarks:

Strain D4

Metabolic activation:

with and without

Metabolic activation system:

Tissue homogenate fraction of Mouse-ICR random bred adult males; rat-Sprague-Dawley adult males, and primate-Maccaca mulatta adult males.

Test concentrations with justification for top dose:

5%
The top dose corresponds to the 1/2 50% dose calculated from the survival curve.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: the test compound was soluble under treatment conditions.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

2-nitrofluorene

N-dimethylnitrosamine

ethylmethanesulphonate

other: Quinacrine mustard

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Suspension tests:
Without metabolic activation: stationary-phase yeast cultures were grown in complete broth, washed and resuspended in 0.9% saline to densities of 5E07 cells/ml. This constituted the working stock for tests of a group of test chemicals and their respective controls. Tests were conducted in plastic tissue culture plates. Cells plus appropiate volume(s) of the test chemical/solvent were added to the wells to give a final volume of 1.5 ml. Treatment was at 30ºC for 4 h. All flasks were shaken during treatment. Following treatment, the plates were set on ice. Aliquots of cells were removed, diluted in sterile saline (4ºC) and plated on the appropiate complete media. Undiluted samples from flasks containing the bacteria were plated on minimal selective medium in reversion experiments. Samples from 10E-1 dilution of treated cells were plated on the selected media for enumeration of gene conversion with strain D4.The yeast plates were incubated at 30ºC for 3-5 days before scoring.
With metabolic activation: yeast cells were grown and prepared as described in the nonactivation tests. Measured amounts of the test and control chemicals plus 0.25 ml of the stock-cell suspension were added to wells of the Limbro plate containing the appropriate tissue fraction and reaction mixture. All flasks were incubated at 37ºC in an oxygen atmosphere with shaking. The treatment times as well as the dilutions, plating procedures and scoring of the plates were the same as described for nonactivation tests.

DURATION
- Exposure duration: 4 h at 30 ºC
- Selection time (if incubation with a selection agent): 3-4 days at 30 ºC

SELECTION AGENT (mutation assays): lack of tryptophan or adenine in the media.

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: other: 50% survival dose
- Any supplementary information relevant to cytotoxicity:
The following concentrations of the test compound, 10, 1, 0.1, 0.01 and 0.001%, were tested for survival against the specific the specific indicator strains over a range of doses to determine the 50% survival dose. Bacteria and yeast strains were tested in phosphate buffer, pH 7.4, for 4 h on a shaker. The 50% survival curve and the 1/4 and 1/2 50% doses calculated.

Key result

Species / strain:

Saccharomyces cerevisiae

Remarks:

Strain D4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Compound frequency summary report: Suspension tests (without metabolic activation)

	TA 1535 (His)	TA 1538 (His)	TA 1537 (His)	D4 (Ade)	D4 (Try)
Solvent control	5.93	8.10	2.32	1.21	1.69
Positive control	145.25	256.53	54.95	102.54	192.66
Dose level 1	5.24	7.00	2.32	1.71	3.89
Dose level 2	7.05	6.33	0.20	1.33	1.43

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

 

Table 2. Compound frequency summary report: Suspension tests (with metabolic activation: mouse tissue fraction)

		TA 1535 (His)	TA 1538 (His)	TA 1537 (His)	D4 (Ade)	D4 (Try)
Negative control		25.16	5.49	10.13	3.11	2.43
Tissue control		10.85	19.43	7.69	3.17	3.04
Solvent control		30.64	4.80	11.85	1.46	2.47
Positive control	Liver	99.45	96.04	20.85	4.07	7.05
	Lung	32.03	9.09	15.03	2.32	2.32
	Testes	23.13	12.00	7.90	3.94	1.69
Dose level 1	Liver	11.36	9.65	8.97	3.35	5.45
	Lung	20.17	4.90	18.18	2.85	2.48
	Testes	26.00	5.71	16.94	3.24	1.75
Dose level 2	Liver	13.12	8.78	12.77	5.28	5.13
	Lung	19.34	4.89	14.41	4.04	3.31
	Testes	29.16	3.23	15.84	1.99	1.77

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

 

Table 3. Compound frequency summary report: Suspension tests (with metabolic activation: rat tissue fraction)

		TA 1538 (His)	TA 1537 (His)	TA 1535 **(His)	D4 (Ade)	D4 (Try)
Negative control		5.08	25.00	4.13	2.42	1.16
Tissue control		11.94	16.99	19.55	1.31	1.19
Solvent control		4.90/2.85*	10.34	3.40	1.19	1.39
Positive control	Liver	25.88	23.46	655.87	6.42	8.88
	Lung	9.79	15.63	5.92	2.53	3.29
	Testes	7.41	13.52	11.07	2.51	2.16
Dose level 1	Liver	6.00	18.24	4.33	2.58	3.63
	Lung	8.37	11.47	5.49	1.80	2.30
	Testes	11.06	15.17	3.74	3.24	3.14
Dose level 2	Liver	16.44/10.48*	8.07	3.45	3.54	3.67
	Lung	4.50	7.68	4.53	3.41	3.29
	Testes	7.94	10.88	3.94	3.01	2.04

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

* The experiment was repeated because of increased frequencies. The repeat tests were negative.

** The TA 1535 culture used with mouse tissues had a high spontaneous background, but the data appeared acceptable.

 

Table 4. Compound frequency summary report: Suspension tests (with metabolic activation: monkey tissue fraction)

		TA 1538 (His)	TA 1537 (His)	TA 1535 **(His)	D4 (Ade)	D4 (Try)
Negative control		5.15	17.39	13.57	2.10	1.65
Tissue control		7.48	33.71	7.81	2.38	1.70
Solvent control		1.80/12.53*	15.22	8.46	1.31	2.15
Positive control	Liver	13.89	57.35	165.66	2.46	1.74
	Lung	3.05	18.75	7.04	2.78	2.16
	Testes	3.81	18.10	7.46	3.99	2.07
Dose level 1	Liver	4.15/2.88*	46.48	15.58	1.59	1.84
	Lung	1.79	8.00	10.49	2.79	1.74
	Testes	2.95	13.71	6.97	3.95	3.26
Dose level 2	Liver	6.36	17.83	8.89	2.85	2.37
	Lung	3.15	16.29	6.27	1.28	2.04
	Testes	4.99	14.97	8.91	2.29	1.02

Dose level 1: Bacteria strains = 0.375%; yeast strain = 2.5%.

Dose level 2: Bacteria strains = 0.75%; yeast strain = 5%.

* The experiment was repeated because of increased frequencies. The repeat tests were negative.

** The TA 1535 culture used with mouse tissues had a high spontaneous background, but the data appeared acceptable.

Conclusions:

Under the experimental conditions used, monocalcium phosphate was found to be non-mutagenic in Sacharomyces cerevisae D4.

Executive summary:

The ability of monocalcium phosphate to induce mutation was assessed by a similar protocol to the Saccharomyces cerevisae gene mutation test (OECD 480). A preliminary toxicity study showed that the 1/2 50% dose calculated from the survival curve was 5%. Saccharomyces cerevisae strain D4 was exposed to monocalcium phosphate at 5%, with and without metabolic activation (tissue homogenate fraction of Mouse-ICR random bred adult males, rat sprague-Dawley adult males, and primate-maccaca mulatta adult males). Suspension tests were performed and negative and positive controls were carried out in parallel. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in the yeast strain tested, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 16

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Species / strain / cell type:

S. typhimurium, other: TA 104

Details on mammalian cell type (if applicable):

Cells were obtained from Dr. B.N. Ames (University of California, Berkeley, CA).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 0.5, 1 and 2 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
The standard plate incorporation assay was carried out according to the procedure developed by Ames (1975) and Maron and Ames et al. (1983) [see attached background material]. Positive and negative control are also included in the assay. After incubation at 37ºC for 48 h, revertant colonies are counted.
S9 microsomal fraction was prepared from liver homogenates of CD-COBS rats after induction of cytochrome P-450. The protein concentration of liver homogenate S9 fraction was determined according to Kalcker (J. Biol. Chem., 147, 461-468, 1947).

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 3

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. His+ revertants/plate induced by various concentrations of phosphoric acid in the presence and absence of S9.

Phosphoric acid concentration (µg/plate)	S. thyphimuriumstrain
	TA 97		TA 98		TA 100		TA 104
	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9
0	47.3 ± 1.52	23.6 ± 2.30	40.0 ± 1.00	34.3 ± 2.08	160.0 ± 4.35	127.3 ± 2.51	448.0 ± 6.92	388.0 ± 4.00
0.5	43.6 ± 2.30	22.0 ± 3.46	32.6 ± 3.21	26.3 ± 1.52	140.0 ± 1.73	124.3 ± 3.51	522.0 ± 2.00	438.2 ± 3.51
1.00	45.3 ± 2.68	31.0 ± 1.00	36.0 ± 1.73	31.6 ± 2.08	145.3 ± 6.35	148.3 ± 1.51	536.0 ± 1.73	450.6 ± 5.13
2.00	43.3 ± 3.05	23.3 ± 2.08	40.3 ± 2.51	34.6 ± 0.57	130.0 ± 3.00	123.3 ± 3.51	489.0 ± 5.29	438.0 ± 3.60
2-AA (10 µg/plate)	426.0 ± 4.00	31.0 ± 1.00	440.6 ± 5.68	39.3 ± 0.57	576.0 ± 2.64	150.3 ± 1.52	977.3 ± 2.51	624.3± 4.04

2-AA: 2-aminoanthracene

Conclusions:

Phosphoric acid did not induce any mutagenic change in Salmonella typhimurium TA 97, TA 98, TA 100 and TA 10, with and without metabolic activation up to 2 µg/plate.

Executive summary:

The ability of phosphoric acid to induce mutation was assessed by a similar protocol to the bacterial reverse mutation test (OECD 471). Four histidine dependent strains of Salmonella typhimurium TA97, TA98, TA100 and TA104 were exposed to phosphoric acid 75 -85% solution in the presence and absence of S9 metabolic activation (induced rat liver homogenates), by the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In-vitro gene mutation in bacteria:

- Key study. The ability of choline chloride to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to a National Toxicology Program standardised method, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to exposed to 0, 333, 1000, 3333, 10000 and 20830 µg/ml of test item in the presence and absence of S9 metabolic activation (Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Key study: The ability of choline chloride to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the a National Toxicology Program standarised method, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Key study: The ability of choline chloride to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to ta National Toxicology Program standardised method, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Key study: The ability of monocalcium phosphate to induce mutation was assessed by a similar protocol to the bacterial reverse mutation test (OECD 471). A preliminary toxicity study showed that the 1/2 50% dose calculated from the survival curve was 0.75%. Three histidine dependent strains of Salmonella typhimurium TA1535, TA1537 and TA1538 were exposed to monocalcium phosphate at 0.75%, with and without metabolic activation (tissue homogenate fraction of Mouse-ICR random bred adult males, rat sprague-Dawley adult males, and primate-maccaca mulatta adult males). Plate and suspension tests were performed. For both assays, negative and positive controls were carried out in parallel. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any bacterial strains tested, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Key study: The ability of disodium phosphate to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames (1975), similar to OECD 471. Six histidine dependent strains of Salmonella typhimurium (TA92, TA1535, TA97, TA98, TA100 and TA94) were exposed to exposed to six concentrations of test item up to a maximal dose of 100 mg/plate of test item in the presence and absence of S9 metabolic activation (Kanchlor KC-400- induced rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Key study: The ability of phosphoric acid to induce mutation was assessed by a similar protocol to the bacterial reverse mutation test (OECD 471). Five histidine dependent strains of Salmonella typhimurium TA1535, TA97, TA98, TA100 and TA102 were exposed to phosphoric acid 75 -85% solution in the presence and absence of S9 metabolic activation (Aroclor 1254- induced rat liver homogenates), by the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants, either with or without metabolic activation. Therefore, the test item was not mutagenic.

- Key study: The ability of phosphoric acid to induce mutation was assessed by a similar protocol to the bacterial reverse mutation test (OECD 471). Four histidine dependent strains of Salmonella typhimurium TA97, TA98, TA100 and TA104 were exposed to phosphoric acid 75 -85% solution in the presence and absence of S9 metabolic activation (induced rat liver homogenates), by the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants, either with or without metabolic activation. Therefore, the test item was not mutagenic.

In-vitro gene mutation in yeast:

- Key study: The ability of monocalcium phosphate to induce mutation was assessed by a similar protocol to the Saccharomyces cerevisae gene mutation test (OECD 480). A preliminary toxicity study showed that the 1/2 50% dose calculated from the survival curve was 5%. Saccharomyces cerevisae strain D4 was exposed to monocalcium phosphate at 5%, with and without metabolic activation (tissue homogenate fraction of Mouse-ICR random bred adult males, rat sprague-Dawley adult males, and primate-maccaca mulatta adult males). Suspension tests were performed and negative and positive controls were carried out in parallel. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in the yeast strain tested, either with or without metabolic activation. Therefore, the test item was not mutagenic.

In-vitro cytogenicity study in mammalian cells:

- Key study: In order to determine the ability of choline chloride to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 473. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity, 5000 µg/ml. Then, CHO cells were exposed to five concentrations of the test item for 8 -10 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation with colcemid (0.1  g/ml), cells were fixed and stained. Negative and positive controls were run in parallel. 100 cells were examined per dose level and the results for chromosome aberration assay without metabolic activation. The results showed no evidence of the induction of structural aberrations in CHO cells by the test substance under test conditions. Therefore, the test item can be considered as not mutagenic.

- Key study: In order to determine the ability of the choline chloride to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 473. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (500, 50, 0.5, 0.05 and 0.005 µg/mL) for 8 -10 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation with colcemid (0.1  g/ml), cells were fixed and stained. Negative and positive controls were run in parallel. 100 cells were examined per dose level and the results for chromosome aberration assay without metabolic activation, there was a single questionable positive, that it was contributed by simple breaks. Under these test conditions, the test item was found to slightly induce chromosomal aberrations in CHO cells without metabolic activation but not with metabolic activation.

- Supporting study: In order to determine the ability of the choline chloride to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 473. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to different concentrations of the test item (5000, 4000, 3000 and 2000 µg/mL without metabolic activation; 5000, 4000 and 3000 µg/mL with metabolic activation) for 8 -10 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation with colcemid (0.1  g/ml), cells were fixed and stained. Negative and positive controls were run in parallel. 100 cells were examined per dose level and the results for chromosome aberration assay with and without metabolic activation showed no evidence of the induction of structural aberrations in CHO cells by the test substance under test conditions. Therefore, the test item can be considered as not mutagenic.

- Key study: A study to determine the ability of disodium phosphate to induce chromosomal aberrations in Chinese Hamster Lung fibroblasts (CHL) was performed by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose, which was the dose needed for 50% cell-growth inhibition (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (2.0 mg/plate) for 24 and 48h, without metabolic activation. Untreated cells and solvent treated cells served as negative controls (incidence of aberrations < 3.0%); 200 chemicals were tested, the positive results served as controls. A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens), and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each cultured plate. The results showed no evidence of the induction of structural aberrations in cultured lymphocytes by the test substance under test conditions. Therefore, the test item was not mutagenic.

- Key study: In order to determine the ability of choline chloride to induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 479. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (5000, 500, 50, 5, 0.5 and 0.05 µg/mL) for 26 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation, the medium with test chemical was removed and replaced with fresh medium plus BedUrD and colcemid (0.1  g/ml), without test chemical, and incubated for further 2 hours. Then, cells were harvested, fixed and stained with Hoechdt 33258 and Giemsa. Negative and positive controls were run in parallel. 50 cells were examined per dose level and the results for sister chromatid exchange assay were negative. Under these test conditions, the test item was found to be non mutagenic.

- Key study: In order to determine the ability of choline chloride to induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 479. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (5000, 500, 50, 5, 0.5 and 0.05 µg/mL) for 26 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation, the medium with test chemical was removed and replaced with fresh medium plus BedUrD and colcemid (0.1  g/ml), without test chemical, and incubated for further 2 hours. Then, cells were harvested, fixed and stained with Hoechdt 33258 and Giemsa. Negative and positive controls were run in parallel. 50 cells were examined per dose level and the results for sister chromatid exchange assay were weakly positive.Under these test conditions, the test item was found to be slightly mutagenic.

- Supporting study: In order to determine the ability of choline chloride to induce sister chromatid exchange in Chinese Hamster Ovary (CHO) cells studies were performed by a method similar to OECD 479. A preliminary toxicity test was performed to choose the highest concentration tested for cytogenetic activity. Then, CHO cells were exposed to five concentrations of the test item (5000, 1600, 500, 160 and 16 µg/mL) for 26 h in the assay without metabolic activation and for 2 h in the assay with metabolic activation with S9 mix. After incubation, the medium with test chemical was removed and replaced with fresh medium plus BedUrD and colcemid (0.1  g/ml), without test chemical, and incubated for further 2 hours. Then, cells were harvested, fixed and stained with Hoechdt 33258 and Giemsa. Negative and positive controls were run in parallel. 50 cells were examined per dose level and the results for sister chromatid exchange assay were negative.Under these test conditions, the test item was found to be non mutagenic.

In-vitro mammalian cell gene mutation test using the thymidine kinase gene:

Key study: The potential of 2 -trimethylazaniumylethyl phosphate chloride, tetrahydrate to induce mutations at the mouse lymphoma thymidine kinase locus was studied using the cell line L5178Y according to OECD 490, following GLP. A cytotoxicity test included in the gene mutation test was performed on 8 doses. According to the results, four doses were selected, in all treatments with and without metabolic activation the doses tested were 500, 250, 125 and 62.5 µg/ml. Negative and a positive controls were performed in parallel. Positive controls led to an increase in mutation frequency at least 300 x 10E-6 with at least 40% in small colony, which validate the test. The frequencies of mutations observed for the test item were statistically different from those observed for the negative control. Under these experimental conditions, the test item is considered genotoxic with the mouse lymphoma assay. In addition, this genotoxic effect is more mutagenic than clastogenic.

Justification for classification or non-classification

Based on the available information, the substance is not classified for mutagenicity according to CLP Regulation (EC) no.1272/2008.