2,5-dihydroxy-3,6-dimethyl-3,6-bis(dihydroxyphosphinyl)-1,4,2,5-dioxadiphosphorane-2,5-dioxide, hexasodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-8 to 2016-03-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

hexasodium 3,6-dimethyl-2,5-dioxo-3,6-diphosphonato-1,4,2λ⁵,5λ⁵-dioxadiphosphinane-2,5-bis(olate)

EC Number:

928-304-7

Molecular formula:

C4H6O12P4Na6

IUPAC Name:

hexasodium 3,6-dimethyl-2,5-dioxo-3,6-diphosphonato-1,4,2λ⁵,5λ⁵-dioxadiphosphinane-2,5-bis(olate)

Constituent 2

Reference substance name:

ADPA Dimer Na Salt

IUPAC Name:

ADPA Dimer Na Salt

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): ADPA Dimer Na Salt (wet cake)
- Physical state: White crystalline solid
- Storage condition of test material: At room temperature, in the original plastic, opaque, container

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

52 to 5000 μg/plate

Vehicle / solvent:

Milli-Q water (Millipore Corp., Bedford, MA., USA).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: Sodium azide (TA1535), ICR-191 (TA1537), 2-Nitrofluorene (TA98), Methyl methanesulphonate (TA 100) and 4-nitroquinoline N-oxide (WP2uvrA); With metabolic activation: 2-aminoanthracene (all strains).

Details on test system and experimental conditions:

METHOD OF APPLICATION
Direct incorporation method: 0.1 ml of a fresh bacterial culture, 0.1 mL or 0.2 mL of a dilution of the test item in Milli-Q water and 0.5 ml S9-mix (in case of activation assay) or 0.5 ml phosphate buffer (in case of non-activation assays) were successively added to 3 ml molten top agar. After agitation the mix was poured onto a selective agar plate.
DURATION
- Preincubation period: not applicable
- Exposure duration: 48 ± 4 hours at 37°C in the dark
NUMBER OF REPLICATES
- 3 plates/dose/strain.
- Two independent experiments were performed. In the first experiment ADPA Dimer Na Salt was tested both in the absence and presence of 5% (v/v) S9-mix inall tester strains. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
DETERMINATION OF TOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of microcolonies and the reduction of revertant colonies were examined.

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the vehicle control values of the strains are within the laboratory historical range, if the results of the positive controls meet the criteria for a positive response within the laboratory historical range and if the selected dose range includes a clearly toxic concentration or is extended to 5 mg/plate. No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

A test substance is considered positive (mutagenic) in the test if the total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control. A positive or negative response should be reproducible in at least one independently repeated experiment.

Statistics:

No statistical analysis was performed.

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:

other: yes for one strain, see details on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
ADPA Dimer Na Salt (wet cake) was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
Precipitate: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.
Toxicity: A biologically relevant decrease in the number of revertants was observed in tester strain TA98 in the absence of S9-mix at the highest concentration tested. In strains TA1537 (presence of S9-mix) and TA98 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
Mutagenicity: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

MUTATION EXPERIMENT 2:
A second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.
Toxicity: A biologically relevant decrease in the number of revertants was observed in tester strain WP2uvrA in the absence of S9-mix at the highest concentration tested. In strain TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is morelikely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
Mutagenicity: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

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

Based on the results of this study it is concluded that ADPA Dimer Na Salt is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of ADPA Dimer Na Salt was investigated in theSalmonella typhimuriumreverse mutation assay and the Escherichia colireverse mutation assay according to the OECD Testing Guideline 471 and under GLP.

The test substance was tested in theSalmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535 TA1537, TA100 and TA98) and in theEscherichia colireverse mutation assay with one tryptophan-requiring strain of Escherichia coli(WP2uvrA) in two independent experiments.

ADPA Dimer Na Salt was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that ADPA Dimer Na Salt (wet cake) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.