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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test was performed with 2-phosphono-butane-1,2,4-tricarboxylic acid, at nominal concentrations of 16; 80; 400; 2500 and 12,500 µg /plate. Under the experimental conditions, the test item did not induce gene mutations by frameshift or base-pair substitution strains used and is therefore to be considered as non-mutagenic in this bacterial reverse mutation assay. The assays was performed in four strains (TA1535, TA 100, TA 1537, TA98).

In addition, tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate was investigated in an Ames test to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In conclusion, the test was negative.
In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of 2-phosphonobutane- 1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.
Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid is justified.

In two further in-vitro assays, Mammalian Chromosome Aberration and V79 -HPRT Forward Mutation assay, both with and without metabolic activation, 2-phosphono-butane-1,2,4-tricarboxylic acid was found to be non-mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471: Bacterial Reverse Mutation Test, corrected June 26, 2020

Principles of method if other than guideline:

This study was performed to investigate the potential of Tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The Salmonella typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
No precipitation of the test item occurred up to the highest investigated dose

Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

Deionized water or DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 μg/plate

Experimental Performance
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar

Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37 °C ± 1.5 °C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL Bacteria suspension
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification, the plates were incubated upside down for at least 48 hours at 37 °C ± 1.5 °C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Rationale for test conditions:

Pre-Experiment for Toxicity

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 μg/plate

Evaluation criteria:

Acceptability of the Assay

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Current historical control data are available.

Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.

An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Biometry

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

other: Bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

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:

No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only four instead of at least five strains of bacteria were used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26 May 1983

Deviations:

yes

Remarks:

Only four instead of at least five strains of bacteria were used.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Directive 84/449/EEC

Deviations:

yes

Remarks:

Only four instead of at least five strains of bacteria were used.

Principles of method if other than guideline:

Only four instead of at least five strains of bacteria were used. It has to be mentioned that the test was performed in 1979, the current guideline was adopted in 1997.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. All stains show increased sensitivity due to "deep rough" (one polysaccharide chain is missing, hence, also large molecules can penetrate into the bacteria cells ) and UV-repair-deficiency.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

NA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Nominal concentrations: 20; 100; 500; 2500; 12.500 µg /plate.

Vehicle / solvent:

- DMSO (BAHIBIT AM; Trypaflavin)
- demineralised water (Endoxan)

Untreated negative controls:

yes

Remarks:

0 µg BAHIBIT AM /plate / demineralised water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Substance name: Endoxan; cytostatic agent; the active agent cyclophosphamid is a well-known pro-mutagen; 217. 5 µg equivalent to 150 µg cyclophosphamid; only for strains 1535 and TA 100

Negative solvent / vehicle controls:

yes

Remarks:

demineralised water

Positive controls:

yes

Positive control substance:

other: Trypaflavin

Remarks:

Active substance of Panflavin and Rivanols; pro-mutagen for frameshift mutations; 200 µg; only for strains 1537 and TA 98

Details on test system and experimental conditions:

Incubation for 48h at 37°C.
4 plates / substance / strain
Negative controls:
4 plates / DMSO / strain
4 plates / demineralised water / stain
Additionally, the total germ account was determined for 2 plates/group.

Evaluation criteria:

A positive result requires a reproducible dose-related duplication of mutants compared with the negative controls for at least one strain.
The colony numbers for negative controls, positive controls and the test plates were determined, the mean values were calculated.

Statistics:

Mean values

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: only weak bacteriotoxic effects for concentrations >500 µg/ plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

NA

No remarks.

Conclusions:

Interpretation of results: negative

BAYHIBIT AM containing 45-50% (w/w) 2-phosphonobutane-1,2,4-tricarboxylic acid in water was found to be non-mutagenic under test conditions.

Executive summary:

The Ames test was performed with BAYHIBIT AM containing 45-50% (w/w) 2-phosphonobutane-1,2,4-tricarboxylic acid in water, at nominal concentrations of 16; 80; 400; 2500 and 12,500 µg /plate. Strain-specific bacteriotoxicity was observed at doses >500 µg/ plate and higher. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution strains used. Hence, BAYHIBIT AM containing 45-50% (w/w) 2-phosphonobutane-1,2,4-tricarboxylic acid is considered non-mutagenic in this bacterial reverse mutation assay.

Reference 3

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).

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

Target gene:

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

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 1,2,4-Butanetricarboxylic acid, 2-phosphono / mL.
For the chromosome aberration assay with metabolic activation: 625, 1250, 2500 µg 1,2,4-Butanetricarboxylic acid, 2-phosphono /mL.
For the chromosome aberration assay without metabolic activation: 125, 250, 500 µg 1,2,4-Butanetricarboxylic acid, 2-phosphono /mL.

Vehicle / solvent:

Ham's F-10 medium.

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.

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.

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.

No remarks.

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 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. Hence, under the test conditions, 1,2,4-Butanetricarboxylic acid, 2-phosphono 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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-11-12 until 1997-03-20

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

The study was also performed according to the following guidelines: 1- EEC Directive 88/302/EEC Mutagenicity. In Vitro Mammalian Cell- Gene Mutation Test. 2- Health Effects Test Guidelines, June 1996. (U.S) Environmental Protection Agency Washington DC

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Type of assay:

other: Chinese hamster lung cells (V79) forward mutation assay.

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene locus in Chinese hamster lung cells (V79).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

NA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Nine concentrations of test substance (Bayhibit AM) ranging from 19.5 µg/mL to 5000µg/mL. Concentration range was chosen according to the results of the cytotoxicity test with the test substance.

Vehicle / solvent:

demineralised water

Untreated negative controls:

yes

Remarks:

Untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

Not exceeding 1 % (v/V) vehicle in control medium. Examined with and without metabolic activation (S9)

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Done without S9 mix. The mutagen does not require exogenous activation.

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

Done with S9 mix. The mutagen requires metabolic activation by microsomal enzymes.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6-7 days

SELECTION AGENT (mutation assays): 10 µg/mL 6-TG
SPINDLE INHIBITOR (cytogenetic assays): No
STAIN (for cytogenetic assays): Yes, stained with Giemsa


NUMBER OF REPLICATIONS: 8 dishes for each substance concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency; relative total growth; other: Osmolality and pH values

Evaluation criteria:

- At least two independent repetitions are done for the activation and the nonactivation assays.
- the average cloning efficiency of the negative and the vehicle controls should be at least 50%.
-Cytotoxicity is determined after treatment with the test substance where the highest concentration should produce a low level of relative survival (0-30%) and in the lowest concentration the survival should approximate the negative control.
- The background mutant frequency (average value for vehicle controls) in a trial should not exceed 25x10-6 cells.
- Mutant frequencies are determined for at least four concentrations of the test substance.
- Mutant frequencies are normally derived from sets of 8 dishes per parallel-culture of each dose level.
- An assay will be considered positive if a significant dose-dependent is observed for at least 3 doses.
- A significant mutagenic response to the substance should be at least two to three times that of the highest negative or vehicle control value observed in the same trial. If this is reproducible and observed for a single dose near the highest testable concentration, the test substance is also considered mutagenic. Same is with one or more doses that induce a reproducible, significant mutant frequency in all assays but do not exhibit dose-dependency.

Statistics:

The statistical analysis relies on the mutation frequencies rather than on individual plate counts which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981., Arlett et al., 1989).
In addition, all groups are included in the weighted analysis of variance followed by pairwise comparisons to the vehicle control on a nominal significance level of =0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual dose levels thereby omitting the positive, negative and vehicle controls. If there is a significant increase of the mutation frequency with dose (=0.05) in the main analysis the highest dose group will be dropped and the analysis will be repeated until P> 0.05. Dose levels eliminated in that way are flagged correspondingly.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No cytotoxicity was observed up to 3000 µg/mL. At and above 4000 µg/mL all cells died.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect
- Effects of osmolality: Was examined. A decrease in osmolality was observed in the highest concentration examined.
- Evaporation from medium: NA
- Water solubility: A clear solution was obtained up to a concentration of 500mg/ml.
- Precipitation: No
- Other confounding effects: No


RANGE-FINDING/SCREENING STUDIES: Yes, a preliminary cytotoxicity test was conducted, its results were used to select concentrations of Bayhibit AM for the mutation experiments.


COMPARISON WITH HISTORICAL CONTROL DATA: In line with the test results

ADDITIONAL INFORMATION ON CYTOTOXICITY: NA

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

No remarks.

Conclusions:

Interpretation of results: negative

Non-mutagenic

Executive summary:

No cytotoxic effects were observed up to a concentration of 3000 µg/mL. No significant dose-related or increase in mutant frequency above that of the negative controls was observed, on the contrary to positive controls which induced clear mutagenic effect. Hence, the test substance Bayhibit AM is considered to be non-mutagenic in the V79-HPRT forward mutation assay, both with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An Ames test was performed with 2-phosphono-butane-1,2,4-tricarboxylic acid, at nominal concentrations of 16; 80; 400; 2500 and 12,500 µg /plate. Under the experimental conditions, the test item did not induce gene mutations by frameshift or base-pair substitution strains used and is therefore to be considered as non-mutagenic in this bacterial reverse mutation assay. The assays was performed in four strains (TA1535, TA 100, TA 1537, TA98).
In addition, tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate was investigated in an Ames test to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In conclusion, the test was negative.
In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of 2-phosphonobutane- 1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.
Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid is justified.

In an in-vitro Mammalian Chromosome Aberration Assay with 2-phosphono-butane-1,2,4-tricarboxylic according to OECD Guideline 473 with Chinese hamster lung fibroblasts (V79)) cultures that were 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. Hence, under the test conditions, 1,2,4 -Butanetricarboxylic acid, 2 -phosphono 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 (May, 1996).
In a Chinese hamster lung cells (V79) forward mutation assay with 2-phosphono-butane-1,2,4-tricarboxylic, no cytotoxic effects were observed up to a concentration of 3000 µg/mL (Brendler; Schwaab, 1997), no significant dose-related or increase in mutant frequency above that of the negative controls was observed; on the contrary to positive controls which induced clear mutagenic effect.
Thus, based on the available data derived from four in-vitro tests, 2-phosphono-butane-1,2,4-tricarboxylic is considered as non-mutagenic.

Justification for classification or non-classification

2-phosphono-butane-1,2,4-tricarboxylic acid was investigated in an Ames test in four Salmonella typhimurium strains (TA1535, TA 100, TA 1537, TA98). In addition, tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate was investigated in an Ames test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
Both assays were negative.

In addition, 2-phosphono-butane-1,2,4-tricarboxylic was regarded as negative in an in-vitro Mammalian Chromosome Aberration Assay and In a Chinese hamster lung cells (V79) forward mutation assay.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.