A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate

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:

From 9 september 1992 To 15 october 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate

EC Number:

410-800-5

EC Name:

A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate

Cas Number:

143239-08-1

Molecular formula:

There is no molecular formula available, as the reference substance is a multi- constituent reaction mass. The molecular formulae of the constituents are documented in section 1.2.

IUPAC Name:

dotriacontasodium (1R,2S,3R)-1-phosphonatobutane-1,2,3,4-tetracarboxylate (1R,2S,3S)-1-phosphonatobutane-1,2,3,4-tetracarboxylate (1S,2R,3S)-1-phosphonatobutane-1,2,3,4-tetracarboxylate (1S,2S,3S)-1-phosphonatobutane-1,2,3,4-tetracarboxylate (2R)-2-phosphonatobutanedioate (2S)-2-phosphonatobutanedioate

Details on test material:

- Physical state: white powder
- Storage condition of test material: room temperature under silica gel

Method

Target gene:

Histidine gene (S. Typhimurium)
Tryptophane gene (E. Coli (WP2uvrA-))

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98, and TA100. Escherichia coli: WP2uvrA-

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from induced Aroclor rat liver

Test concentrations with justification for top dose:

0, 8, 40, 200, 1000, and 5000 µg/plate (experiment 1)
0, 312.5, 625, 1250, 2500 and 5000 µg/plate (experiment 2)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not specified

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: N-Ethyl-N'-nitro-N-nitrosoguanidine 3 µg/plate for TA100 and 5 µg/plate for TA1535, 9-aminoacridine 80 µg/plate for TA1537,4-Nito-o-phenylenediamine 5 µg/plate for TA 1538 and 4-Nitroquinoline-1-oxide 0.2 µg/plate for TA98. + S9: 2-Aminoanthracene a

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method:
The cytotoxicity of the test material was determined using TA 100 or WP2 uvrA-. Five doses(0, 8, 40, 200, 1000, and 5000 µg/plate ) of ITC 288 were tested in duplicate. After 48 hours incubation at 37°C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100

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:

not applicable

Positive controls validity:

valid

Additional information on results:

none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1:Number of revertants per plate (mean of 3 plates), (TA 100, TA 1535 and WP2uvrA- +/- S9 in experiment 1)

	[Strain TA 100]			[Strain TA 1535]			WP2uvrA-
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	170	186.3	no	17	17.7	no	17.7	18.3	no
8.0	156.7	162.0	no	16.3	20.0	no	16.3	18.7	no
40	167.7	153.7	no	18.7	15.0	no	10.3	14.0	no
200	151.7	149.0	no	17.7	17.3	no	17.7	14.0	no
1000	165.7	144.0	no	21.3	15.7	no	11.3	13.3	no
5000	170.7	168.7	no	14.7	13.0	no	15	14.7	no
Positive control	695	421.7	no	348.7	306.7	no	447.7	187.0	no

*solvent control with water.

Table 7.6.1/2:Number of revertants per plate (mean of 3 plates), (TA98 and TA 1537 +/- S9 in experiment 1)

	[Strain TA 98]			[Strain TA 1537]
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	20	28	no	14.3	11.7	no
8.0	18.3	23.7	no	9.0	14.7	no
40	21.7	23.3	no	13.7	10.3	no
200	20.3	26.7	no	12.3	11.3	no
1000	16.7	31.0	no	9.7	11.7	no
5000	16.3	21.3	no	13.0	12.0	no
Positive control	183.3	263.3	no	454	144.0	no

*solvent control with water.

Table 7.6.1/3:Number of revertants per plate (mean of 3 plates), (TA 100, TA 1535 and WP2uvrA- +/- S9 in experiment 2)

	[Strain TA 100]			[Strain TA 1535]			WP2uvrA-
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	160.0	174.7	no	11.0	12.0	no	16	11.7	no
312.5	154.7	141.7	no	14.0	9.5	no	13.7	11.3	no
625	127.3	159.0	no	14.3	9.0	no	11.0	15.3	no
1250	163.5	148.0	no	13.0	11.7	no	13.0	15.7	no
2500	124.3	169.3	no	14.0	13.3	no	15.7	11.7	no
5000	158.3	136.7	no	11.0	12.3	no	13.0	15.0	no
Positive control	508.3	436.3	no	161.3	142	no	599.0	111.3	no

*solvent control with water.

Table 7.6.1/4:Number of revertants per plate (mean of 3 plates), (TA98 and TA 1537 +/- S9 in experiment 2)

	[Strain TA 98]			[Strain TA 1537]
Conc. [µg/plate]	— MA	+ MA	Cytotoxicity	— MA	+ MA	Cytotoxicity
0*	17.3	22.3	no	6.0	9.3	no
312.5	22.3	20.0	no	8.7	6.7	no
625	25.0	19.7	no	10.7	13.0	no
1250	22.0	17.7	no	7.7	4.0	no
2500	22.0	22.7	no	8.0	8.3	no
5000	20	17.7	no	8.7	7.7	no
Positive control	203.7	158.0	no	301	161.0	no

*solvent control with water.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of this test, ITC 288 was found to be non-mutagenic in Bacteria.

Executive summary:

In a reverse gene mutation assay in bacteria (Thompson, 1992), Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with ITC 288 by the Ames plate incorporation method at five dose levels, in triplicate, both with and without S9. The dose range determined in a preliminary toxicity assay was 8 to 5000 µg/plate using TA100 or WP2 uvrA-. In the mutation study, The dose range used were 8 to 5000 µg/plate in the first experiment and 312.5 to 5000 µg/plate in the second experiment. The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. ITC 288 caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of salmonella and escherichia used with and without S9. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of ITC 288, either with or without metabolic activation (experiment 1 + experiment 2). Under the conditions of this test, ITC 288 was found to be non-mutagenic in Bacteria.

This study is classified as acceptable and satisfies the requirement for test Guideline OECD 471 for in vitro mutagenesis (bacterial reverse gene mutation) data.