[cyclohexane-1,2-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid, sodium salt

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:

3 November 2016 to 25 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

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:

up 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 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 ALBRITE CIX(N) 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
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
The test item precipitated on the plates at dose levels of 17 μg/plate and upwards, except in tester strain WP2uvrA in the presence of S9-mix, where the test item already precipitated at 5.4 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in both tester strains. Results of this dose range finding test were reported as part of the first mutation assay.

MUTATION EXPERIMENT (1, 1A, 1B)
Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 0.055 to 17 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix at the highest tested concentration.
Since in the first mutation experiment, no dose level with toxicity or precipitate on the plates was observed and the results of the dose range finding test were different from the results seen in the first mutation experiment, an additional mutation experiment was performed. In this additional experiment (1A), the test item was tested at a concentration range of 1.7 to 164 μg/plate in the absence and presence of 5% (v/v) S9-mix in all five tester strains. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all tester strains. With the exception of TA100 in the absence and presence of S9-mix and WP2uvrA in the presence of S9-mix.
Since in the additional first mutation experiment (1A), no dose level with obvious toxicity or precipitate on the plates was observed in the tester strains TA1535, TA100 and WP2uvrA, an additional mutation experiment was performed. In this additional experiment (1B), the test item was tested at a concentration range of 17 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in in the tester strains TA1535, TA100 and WP2uvrA. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all three tester strains

MUTATION EXPERIMENT (2, 2A, 2B)
In a follow-up experiment with additional parameters, the test item was tested up to a maximum concentration of 512 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all tester strains. With the exception of TA1537 and TA98 in the presence of S9-mix.
Since in the second mutation experiment, no dose level with toxicity or precipitate on the plates was observed in the tester strains TA1537 and TA98 in the presence of S9-mix, an additional mutation experiment was performed. In this additional experiment (2A), the test item was tested at a concentration range of 0.55 to 164 μg/plate in the presence of S9-mix in the tester strains TA1537 and TA98. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537.
Since in the additional second mutation experiment (2A), no dose level with toxicity or precipitate on the plates was observed in the tester strain TA98, an additional mutation experiment was performed. In this additional experiment (2B), the test item was tested at a concentration range of 17 to 1600 μg/plate in the presence of S9-mix in tester strain TA98. The test item did not precipitate on the plates. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that ALBRITE CIX(N) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of ALBRITE CIX(N) 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 the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535 TA1537, TA100 and TA98) and in the Escherichia colireverse mutation assay with one tryptophan-requiring strain of Escherichia coli(WP2uvrA) in two independent experiments.

ALBRITE CIX(N) 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 ALBRITE CIX(N) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.