Nitrilotrimethylenetris(phosphonic acid)

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:

07 November 2017 to 23 November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only one test strain of E. coli was used to detect mutations via cross-linking.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

Only one test strain of E. coli was used to detect mutations via cross-linking.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

[nitrilotris(methylene)]trisphosphonic acid, sodium salt

EC Number:

243-900-0

EC Name:

[nitrilotris(methylene)]trisphosphonic acid, sodium salt

Cas Number:

20592-85-2

Molecular formula:

General formula C3H12NO9P3.xNa where x=3-5 ATMP-3Na C3H9NNa3O9P3 ATMP-4Na C3H8NNa4O9P3 ATMP-5Na C3H7NNa5O9P3

IUPAC Name:

Sodium salt of [nitrilotris(methylene)]trisphosphonic acid (3-5Na:1)

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: On the day of the experiment, ATMP-H was dissolved in deionised water to form the sodium salt. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
- Preliminary purification step: The dose selection was adjusted to the purity of 33%.
- Final dilution of a dissolved solid, stock liquid or gel: Not applicable
- Final preparation of a solid: Not applicable

Method

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

To evaluate the toxicity of the test item, a pre-experiment was performed. 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). In the pre-experiment, the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed, 5000 µg/plate were selected as the maximal concentration.
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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was selected because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

no treatment

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

ACTIVATION: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approximately 10 % (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C in the dark

SELECTION AGENT (mutation assays): Selective agar

NUMBER OF REPLICATIONS: Triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertants
- Any supplementary information relevant to cytotoxicity: Not specified

Rationale for test conditions:

To evaluate the toxicity of the test item a pre-experiment was performed. 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). In the pre-experiment, the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed, 5000 µg/plate were selected as the maximal concentration.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding 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:

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

Results and discussion

Test results

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not specified
- Effects of osmolality: Not specified
- Evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation: Not observed

RANGE-FINDING/SCREENING STUDIES: 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 relevant toxic effects were observed, 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Within the range of positive historical control data
- Negative (solvent/vehicle) historical control data: Within the range of negative historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Number of revertants

Any other information on results incl. tables

Table 1: Summary of experiment 1

Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
Without Activation		WP2 uvrA
Deionised water		48 ± 8
Untreated		48 ± 4
ATMP-H	3 µg	43 ± 5
	10 µg	48 ± 8
	33 µg	44 ± 8
	100 µg	48 ± 10
	333 µg	43 ± 9
	1000 µg	47 ± 11
	2500 µg	40 ± 7
	5000 µg	36 ± 5
MMS	2.0 µL	953 ± 87
With Activation
Deionised water		55 ± 3
Untreated		56 ± 11
ATMP-H	3 µg	51 ± 8
	10 µg	57 ± 8
	33 µg	51 ± 13
	100 µg	49 ± 15
	333 µg	51 ± 7
	1000 µg	55 ± 7
	2500 µg	39 ± 9
	5000 µg	47 ± 13
2-AA	10.0 µg	534 ± 112

Table 2: Summary of experiment 2

Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ± SD)
Without Activation			WP2 uvrA
Deionised water			32 ± 5
Untreated			34 ± 4
ATMP-H	33 µg		36 ± 5
	100 µg		35 ± 5
	333 µg		32 ± 6
	1000 µg		31 ± 2
	2500 µg		33 ± 4
	5000 µg		19 ± 2
MMS	2.0 µL		945 ± 40
With Activation
Deionised water			42 ± 2
Untreated			49 ± 12
ATMP-H	33 µg		34 ± 3
	100 µg		43 ± 2
	333 µg		37 ± 4
	1000 µg		36 ± 4
	2500 µg		35 ± 5
	5000 µg		28 ± 3
2-AA	10.0 µg		495 ± 16

MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

In a valid bacterial reverse mutation assay (reliability 2), conducted according to OECD Test Guideline 471 and in compliance with GLP, ATMP-xNa has been tested using E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that ATMP-xNa is negative for mutagenicity to bacteria under the conditions of the test.