Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-12 until 1992-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test substance is not specified on composition (content of 2-phosphonobutane-1,2,4-tricarboxylic acid).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-phosphonobutane-1,2,4-tricarboxylic acid

EC Number:

253-733-5

EC Name:

2-phosphonobutane-1,2,4-tricarboxylic acid

Cas Number:

37971-36-1

Molecular formula:

C7H11O9P

IUPAC Name:

2-phosphonobutane-1,2,4-tricarboxylic acid

Details on test material:

- Storage condition of test material: The test substance was stored tightly closed in a dark, dry and cool place.

Method

Target gene:

No target gene. The biological target is the mammalian cell chromosome.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture media: 500mL Ham's F-10 medium, 50 mL FCS, 100 µg/mL streptomycin, 100IU/mL penicillin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9- mix

Test concentrations with justification for top dose:

For the cytotoxicity assay with and without S9-mix: 50, 100, 200, 400, 600, 800, 1000, 1250, 2500, 5000 µg 2-phosphonobutane-1,2,4-tricarboxylic acid / mL
For the chromosome aberration assay with metabolic activation: 625, 1250, 2500 µg 2-phosphonobutane-1,2,4-tricarboxylic acid /mL
For the chromosome aberration assay without metabolic activation: 125, 250, 500 µg 2-phosphonobutane-1,2,4-tricarboxylic acid /mL

Vehicle / solvent:

Ham's F-10 medium.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Ham's F medium, the solvent of the test compound, served as the negative control.

Negative solvent / vehicle controls:

yes

Remarks:

See above

True negative controls:

yes

Remarks:

See above

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

This positive control substance requires no metabolic activation.

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

This positive control is used in the presence of a metabolic system (S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI)

For the main assay (chromosome aberration test):
DURATION
- Preincubation period: No
- Exposure duration: Without S9-mix-21 hours, with S9-mix-3 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): Without S-9 mix- 21 hours fixation time. For samples with S-9 mix two fixation times-18 hours and 21 hours.

SPINDLE INHIBITOR (cytogenetic assays): Yes (colcemid)
STAIN (for cytogenetic assays): Yes

NUMBER OF REPLICATIONS: The study was carried out in two independent experiments.

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per treatment group and experiment were examined for structural chromosome aberrations.


OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: Yes
- Other: Gaps, breaks, chromatid type exchanges, chromosome type exchanges, aneuploidy, atypical chromosomes, complete metaphase pulverisation.

Evaluation criteria:

- As it is generally accepted, a substance has chromosome damaging activity when parallel cultures at one dose level repeatedly produce aberrations in more than 10% of analyzed cells (gaps excluded). Dose dependency may provide further evidence for clasogenicity.
- If only gaps are increased, and this in single test group without any dose relation, the result can be considered to be negative; in this case gaps express cytotoxicity rather than genotoxic effects because there is no unequivocal mechanistic explanation for the origin of gaps.
- Though it is difficult to estimate the background level of exchanges and endoreplication, in most of these cases there is also a similar increase in gaps and breaks.

Statistics:

In general, up to now no equivocal statistical methods have been developed for evaluating chromosomal aberrations. In addition, the kind of the accepted results demanded no statistical analysis.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: The results of the cytotoxicity assay indicated and limited the concentrations of the test substance for the main study (chromosomal activation, with and without metabolic activations), as were written above (in the concentration part).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cultures treated with the test substance with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable.

Any other information on results incl. tables

No remarks.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Under the study conditions, the test substance did not induce structural chromosomal aberrations (with/without activation).

Executive summary:

Cultures treated with 2-phosphono-butane-1,2,4-tricarboxylic acid with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable. Hence, under the test conditions, 2-phosphonobutane-1,2,4-tricarboxylic acid does not induce structural chromosomal aberrations in cultured mammalian somatic cells (V79) tested with and without an exogenous metabolic system. Following a cytotoxicity assay, test substance concentrations were selected for the chromosomal aberration assay, with and without metabolic activation (S9 -mix). Results of cultures with and without S9 -mix showed no biologically significant increase in the number of breaks, exchanges or other aberrations in cultures from two independent experiments.