[[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic 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:

14-Jan-2003 to 17-Jan-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Standards for mutagenicity tests using microorganisms (Notification no. 77, 1988, and Notification no. 67, 1997); Amendment of the reporting form of the results of the mutagenicity tests using microorganisms (Notification no. 653, 1997)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only duplicate plates used)

GLP compliance:

no

Remarks:

Japanese guideline notification no. 76

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid

EC Number:

239-931-4

EC Name:

[[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid

Cas Number:

15827-60-8

Molecular formula:

C9H28N3O15P5

IUPAC Name:

[(bis{2-[bis(phosphonomethyl)amino]ethyl}amino)methyl]phosphonic acid

Method

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:

phenobarbital- and 5,6-benzoflavone-induced male rat liver S9

Test concentrations with justification for top dose:

10, 20, 39, 78, 156, 313, 625, 1250 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance supplied in water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

-S9; TA100, WP2 uvrA, 0.01 µg/plate; TA98, 0.1 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9; TA1535, 0.5 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191 (2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

Remarks:

-S9; TA1537, 1.0 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9; TA100, TA98, TA1537, 5 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+S9; TA1535, 2 µg/plate; WP2 uvrA, 10 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: duplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition

Evaluation criteria:

"If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was judged positive."

Statistics:

Not done

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

>=313 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=625 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: test substance supplied as a solution in water
- Precipitation: no precipitate seen
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations tested: 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate without S9
- Growth inhibition in all S. typhimurium TA strains at 313 µg/plate and above and in WP2 uvrA at 1250 µg/plate and above
- No precipitate
- Highest concentrations selected for the main test: 313 µg/plate for all S. typhimurium TA strains and 1250 µg/plate for WP2 uvrA
- Other concentrations obtained by 1:2 dilutions to provide 6 concentrations for each strain

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Any other information on results incl. tables

Table 1 Dose finding experiment: Revertants per plate (mean of two plates)

Concentration (µg/plate)	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	134	128	23	14	19	27	18	39	16	23
1.2	137	131	21	13	20	30	19	35	16	26
4.9	127	142	18	16	24	26	16	33	17	22
20	114	127	28	16	20	34	18	32	19	17
78	132	130	18	13	29	30	18	31	14	22
313	46*	114	8*	8	21	18	11*	20	5*	12
1250	0*	125*	0*	4*	0*	17*	0*	10*	0*	4*
5000	0*	0*	0*	0*	0*	0*	0*	0*	0*	0*
Positive control	486	842	461	344	135	595	511	226	1767	82

* The growth inhibition of tested bacterium by the test substance was observed.

Table 2 Main experiment: Revertants per plate (mean of two plates)

Concentration (µg/plate)	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	117	137	17	13	27	26	17	43	8	22
10	137	NT	21	NT	NT	NT	15	NT	9	NT
20	124	NT	15	NT	NT	NT	21	NT	11	NT
39	124	125	9	11	22	27	22	37	9	23
78	125	136	15	10	27	31	11	37	12	15
156	116	133	16	17	20	26	17	39	6*	13
313	50*	115	6*	9	16	19	11*	32	6*	12
625	NT	106*	NT	4*	10*	21	NT	23*	NT	5*
1250	NT	108*	NT	2*	0*	13*	NT	3*	NT	5*
Positive control	496	810	451	321	160	563	588	200	1739	69

* The growth inhibition of tested bacterium by the test substance was observed.

Applicant's summary and conclusion

Conclusions:

In a reliable study, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653), no genotoxicity was seen in a bacterial mutagenicity assay at up to 1250 µg/plate. The study was performed in compliance with Japanese guideline notification no. 76.