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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 01 february 18 February 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, well conducted on the similar substance Reaction mass of dipotassium phosphonate and Phosphonic acid, potassium salt (1:1). Rationale for Read Across is attached at point 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: potassium phosphonate KH2PO3/K2HPO3
- Substance type: inorganic
- Physical state: limpid fluid colourless solution
- Storage condition of test material: room temperature
- Purity: 50%

Method

Target gene:
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
Species / strain
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):
- Source: American Type Culture Collection, Rockville, Maryland
- Storage: in liquid nitrogen and subcultures are prepared from the frozen stocks for experimental use
- Cleaning before use: prior to use cells were cleansed of pre-existing mutants
- Observation: the generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Concentrations were expressed in terms of active constituents (excluding water content, 50%). - All test item solutions were used within 4 hours and 27 minutes from the initial formulation. No assay of the test item stability, nor its concentration and homogeicity in solvent were undertaken.
1580, 500, 158, 50.0 µg/mL
Maximum dose level: 5000 µg/mL

Vehicle / solvent:
sterile injectable water and DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
sterile injectable water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Absence of S9 metabolism
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Absence of S9 metabolism
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive control substance:
2-nitrofluorene
Remarks:
Absence of S9 metabolism
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive control substance:
other: 2-Aminoanhracene
Remarks:
Presence of S9 metabolism
Negative solvent / vehicle controls:
yes
Remarks:
sterile injectable water
Positive control substance:
methylmethanesulfonate
Remarks:
Absence of S9 metabolism
Details on test system and experimental conditions:
S9 TISSUE HOMOGENATE
- Source: Trinova Biochem GmbH (producer: MOLTOX, Molecular Toxicology, Inc., USA)
- Species: rat
- Strain: Sprague Dawley
- Sex: male
- Tissue: liver
- Inducing Agents: Phenobarbital-5,6-Benzoflavone
- Storage: - 80 °C
S9 MIX (10 ml)
S9 tissue fraction, NADP, G-6-P, MgCl2,KCl, Phosphate buffer, Distilled water
POSITIVE CONTROLS TREATMENT, STRAINS AND S9
Strain TA1535: sodium azide (1 µg/plate) in absence of S9; 2-aminoanthracene (1 µg/plate) in presence of S9;
Strain TA100: sodium azide (1 µg/plate) in absence of S9; 2-aminoanthracene (1 µg/plate and 2 µg/plate) in presence of S9;
Strain TA1537: 9-amino-acridine (50 µg/plate) in absence of S9; 2-aminoanthracene (1 µg/plate) in presence of S9;
Strain TA98: 2-nitrofluorene (2 µg/plate) in absence of S9; 2-aminoanthracene 1 µg/plate and 2 µg/plate) in presence of S9;
GROWTH MEDIA
Nutrient broth, Nutrient Agar, Minimal Agar, Top Agar.

Evaluation criteria:
Results show that mean plate counts for untreated and positive control plates fell within the RTC acceptance criteria based on historical control data included in Appendix 1 (confidence interval: mean value ± 2 standard deviations).
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. On the basis of the stated criteria it must be concluded that the test item KH2PO3/K2HPO3 is not mutagenic to S. typhimurium or E. coli, under the reported experimental conditions.
CRITERIA FOR OUTCOME OF ASSAY
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Regression line by the equation Y= a+bx
y= transformed revertant number
a= intercept
b= slope value
x= dose level

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It can be concluded that the test item KH2PO3/K2HPO3 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item KH2PO3/K2HPO3 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item was used as a solution in sterile water for injection. Dose levels are expressed as active constituents.

The test item KH2PO3/K2HPO3 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000 µg/plate and at four lower dose levels spaced by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

As no relevant increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item KH2PO3/K2HPO3 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.