Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7:1)

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:

02-Jul-2001 to 26-Jul-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report Date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only 2-AA used as positive control +S9)

Test guideline 2

Qualifier:

according to

Guideline:

other: Japanese Guidelines on Industrial Chemicals

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Remarks:

brown liquid

Details on test material:

The tested salt was reported to be the 7-sodium salt.

Method

Target gene:

histidine (Salmonella typhimurium)
tryptophan (Escherichia coli)

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 rat liver S9

Test concentrations with justification for top dose:

Test 1-1: 78-5000 µg mixed sodium salts/plate -S9, 156-5000 µg/plate +S9
Test 1-2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium
Test 2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium, 78-5000 µg mixed sodium salts/plate -S9 for E coli, 156-5000 µg mixed sodium salts/plate +S9
20-5000ug/plate -S9; 156-5000ug/plate +S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; TA98, 0.1 µg/plate; TA100, 0.01 µg/plate

Positive control substance:

furylfuramide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 ; TA1535, 0.5 µg/plate

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; TA1537, 80 µg/plate

Positive control substance:

9-aminoacridine

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9, WP2 uvrA, 2 µg/plate

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9; TA98, 0.5 µg/plate; TA100, 1 µg/plate; TA1535, TA1537, 2 µg/plate; WP2 uvrA, 10 µg/plate

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition

Evaluation criteria:

Posltive: mean number of revertant colonies more than double the negatlve control value and signlflcant concentration-dependent Increase

Statistics:

No statistical analysis performed

Results and discussion

Test results

Test results 1

Key result

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:

>= 625 µg mixed sodium salts/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250 µg mixed sodium salts/plate -S9

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: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations of 5-5000 µg/plate
- No growth inhibition in any strain
- Bacteriostatics observed at 1250 µg/plate -S9 in all strains
- No precipitation

COMPARISON WITH HISTORICAL CONTROL DATA:
- Negative/solvent control: within the range of historical data
- Positive controls: within the range of historical data

Any other information on results incl. tables

All treated cultures had less than 2-fold increase in mutant frequency and no evidence of a dose-response.  All positive controls gave a greater than 2-fold increase in mutant numbers.

Table 1 Experiment 1 Mean number of revertants (average of 3 plates)

 

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	102	10	23	14	7
78	-	102	8	21	16	9
156	-	88	7	18	16	6
313	-	71	5	18	8	1
625	-	0	0	7	0	0
1250	-	0	0	0	0	0
2500	-	0	0	0	0	0
5000	-	0	0	0	0	0
Positive control	-	528	422	631	559	194
Control	+	111	11	25	24	6
156	+	133	11	30	24	10
313	+	148	11	21	22	6
625	+	140	9	23	17	6
1250	+	122	6	21	11	3
2500	+	108	4	15	9	3
5000	+	98	5	9	8	2
Positive control	+	501	260	648	446	154
Mean number or revertants 16-19.07.2001

Table 2 Experiment 2 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	104	11	-	16	7
20	-	101	6	-	16	8
39	-	94	8	-	12	8
78	-	106	8	-	14	8
156	-	97	9	-	14	5
313	-	81	6	-	14	4
1250	-	0	0	-	0	0
5000	-	0	0	-	0	0
Positive control	-	522	411	-	604	211

 

Table 3 Experiment 3 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	99	8	20	21	7
20	-	105	10	-	18	7
39	-	107	11	-	19	6
78	-	110	10	22	19	6
156	-	103	11	14	17	7
313	-	86	8	16	15	4
625	-	-	-	13	-	-
1250	-	0	0	0	0	0
2500	-	-	-	0	-	-
5000	-	0	0	0	0	0
Positive control	-	510	419	655	601	183
Control	+	127	10	21	23	8
156	+	130	9	29	26	9
313	+	132	12	17	19	7
625	+	127	12	22	18	6
1250	+	114	8	15	14	7
2500	+	119	6	13	6	3
5000	+	103	6	12	3	2
Positive control	+	679	304	758	517	152

 

Applicant's summary and conclusion

Conclusions:

In a reliable study, conducted according to OECD guideline 471, no genotoxicity was seen in a bacterial mutagenicity assay. The study was performed in compliance with GLP.