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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Choline chloride

EC Number:

200-655-4

EC Name:

Choline chloride

Cas Number:

67-48-1

Molecular formula:

C5H14NO.Cl

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

Method

Target gene:

Histidine

Species / strain

Species / strain 1

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 2

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 3

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 4

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

Controls

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).

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.