phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium 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:

The study was conducted between 05 September 2018 and 27 September 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

These deviations are considered to have no impact on either the result or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

These deviations are considered to have no impact on either the result or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

EC Number:

828-215-2

Cas Number:

172796-84-8

Molecular formula:

C8H12NO7P2Na

IUPAC Name:

phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-, monosodium salt

impurity 1

Reference substance name:

4-aminophenylacetic acid

EC Number:

214-828-7

EC Name:

4-aminophenylacetic acid

Cas Number:

1197-55-3

Molecular formula:

C8H9NO2

IUPAC Name:

4-aminophenylacetic acid

impurity 2

Reference substance name:

EBP dimer

Molecular formula:

C16H22N2O12P4

IUPAC Name:

EBP dimer

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”)

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CHPC071917EBP
- Expiration date of the lot/batch: 01 July 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

OTHER SPECIFICS:
- Physical state/Appearance: Beige coloured powder
- Purity: >98%

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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 homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water at a maximum concentration of 12.5 mg/mL, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Absence of S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

Untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Study Controls
The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (tested in singular prior to Experiment 1).

Microsomal Enzyme Fraction
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized
in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 25 May 2018 was used in this study.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 7193746 04/2022) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 48648 10/2018 and 48698 10/2018).

Test System and Supporting Information
Bacteria
The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
• Trinova Biochem GmbH on 27 June 2017
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2104309 04/2022) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. To aid the suspension of the test item in sterile distilled water, each formulation was prepared at concentrations four times less than required on Vogel-Bonner agar plates. To compensate for this discrepancy, each formulation was dosed using 0.4 mL (400 µL) aliquots. Formulated concentrations were adjusted to allow for the stated water/impurity content (2%) of the test item.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.4 mL aliquot of the appropriate concentration of test item or solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.4 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Typical ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
These values will also be confirmed against current in-house historical control profiles to further validate acceptability. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (S9-mix).
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
123		20		22		28		8
120	(121)	21	(21)	15	(22)†	27	(28)	10	(10)
121		21		29		30		11

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
99		18		31		22		14
111	(112)	24	(19)	43	(35)	19	(22)	6	(9)
126		16		32		26		7

†Experimental procedure repeated at a later date (without metabolic activation (S9-mix)) due to controls not achieving the expected criteria in the original test. There are no untreated controls (spontaneous mutation rates) available for part of this experiment (date of testing 10 September 2018

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 10 September 2018 24 September 2018†					To: 13 September 2018 27 September 2018†
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA†			TA98		TA1537
	Solvent Control (Water)	131 82 108	(107) 24.5#	22 24 32	(26) 5.3	32 21 29		(27) 5.7	22 28 26	(25) 3.1	11 16 17	(15) 3.2
	1.5 µg	116 120 115	(117) 2.6	24 22 16	(21) 4.2	21 28 24		(24) 3.5	27 25 27	(26) 1.2	21 17 22	(20) 2.6
	5 µg	97 105 82	(95) 11.7	28 15 9	(17) 9.7	14 22 29		(22) 7.5	21 33 22	(25) 6.7	5 12 7	(8) 3.6
	15 µg	73 117 112	(101) 24.1	14 12 18	(15) 3.1	24 36 18		(26) 9.2	35 27 33	(32) 4.2	14 12 21	(16) 4.7
	50 µg	119 108 127	(118) 9.5	13 17 21	(17) 4.0	27 22 25		(25) 2.5	35 18 24	(26) 8.6	10 15 13	(13) 2.5
	150 µg	91 119 114	(108) 14.9	11 18 19	(16) 4.4	28 23 28		(26) 2.9	29 23 21	(24) 4.2	13 14 10	(12) 2.1
	500 µg	115 111 113	(113) 2.0	12 10 12	(11) 1.2	24 31 29		(28) 3.6	25 40 22	(29) 9.6	12 12 15	(13) 1.7
	1500 µg	90 135 79	(101) 29.7	18 10 12	(13) 4.2	23 24 36		(28) 7.2	24 32 25	(27) 4.4	14 11 18	(14) 3.5
	5000 µg	97 120 98	(105) 13.0	12 9 12	(11) 1.7	18 27 20		(22) 4.7	20 23 26	(23) 3.0	11 8 8	(9) 1.7
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		474 420 454	(449) 27.3	307 544 512	(454) 128.6	1323 1180 1151		(1218) 92.1	138 171 173	(161) 19.7	146 297 167	(203) 81.8

†                Experimental procedure repeated at a later date due to controls not achieving the expected criteria in the original test

ENNG       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

#               Standard deviation

Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 17 September 2018					To: 20 September 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	125 126 128	(126) 1.5#	15 18 24	(19) 4.6	29 43 36		(36) 7.0	19 25 23	(22) 3.1	11 13 10	(11) 1.5
	15 µg	99 127 93	(106) 18.1	14 16 22	(17) 4.2	33 35 17		(28) 9.9	19 21 21	(20) 1.2	19 16 8	(14) 5.7
	50 µg	126 115 119	(120) 5.6	9 26 33	(23) 12.3	23 27 29		(26) 3.1	30 20 12	(21) 9.0	19 10 11	(13) 4.9
	150 µg	135 110 98	(114) 18.9	18 17 14	(16) 2.1	31 33 37		(34) 3.1	14 17 23	(18) 4.6	18 13 17	(16) 2.6
	500 µg	106 135 119	(120) 14.5	13 13 13	(13) 0.0	27 30 31		(29) 2.1	31 23 21	(25) 5.3	16 18 15	(16) 1.5
	1500 µg	118 116 93	(109) 13.9	19 14 22	(18) 4.0	32 26 31		(30) 3.2	26 26 22	(25) 2.3	10 9 8	(9) 1.0
	5000 µg	115 92 94	(100) 12.7	11 11 21	(14) 5.8	37 17 35		(30) 11.0	27 19 34	(27) 7.5	17 12 8	(12) 4.5
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		601 592 584	(592) 8.5	280 291 347	(306) 35.9	992 1112 1025		(1043) 62.0	248 274 269	(264) 13.8	336 244 247	(276) 52.3

ENNG       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

#               Standard deviation

Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 17 September 2018					To: 20 September 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	106 136 104	(115) 17.9#	14 9 24	(16) 7.6	26 43 37		(35) 8.6	28 27 29	(28) 1.0	18 17 13	(16) 2.6
	15 µg	114 125 100	(113) 12.5	9 18 15	(14) 4.6	38 34 29		(34) 4.5	34 29 44	(36) 7.6	13 10 17	(13) 3.5
	50 µg	106 123 132	(120) 13.2	16 12 16	(15) 2.3	38 44 39		(40) 3.2	29 26 28	(28) 1.5	18 13 12	(14) 3.2
	150 µg	126 149 90	(122) 29.7	11 17 18	(15) 3.8	27 30 40		(32) 6.8	29 32 43	(35) 7.4	15 12 12	(13) 1.7
	500 µg	91 123 116	(110) 16.8	9 11 17	(12) 4.2	33 29 36		(33) 3.5	23 35 19	(26) 8.3	19 20 12	(17) 4.4
	1500 µg	108 116 139	(121) 16.1	10 12 11	(11) 1.0	31 44 44		(40) 7.5	30 34 25	(30) 4.5	9 19 12	(13) 5.1
	5000 µg	128 104 136	(123) 16.7	9 13 12	(11) 2.1	42 31 31		(35) 6.4	28 27 23	(26) 2.6	10 11 10	(10) 0.6
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1154 1189 1489	(1277) 184.1	199 219 237	(218) 19.0	188 205 210		(201) 11.5	94 107 111	(104) 8.9	197 246 248	(230) 28.9

BP          Benzo(a)pyrene

2AA       2-Aminoanthracene

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”) did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”) was considered to be non-mutagenic.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxicity of the test item following the change in test methodology.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). 

Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). 

Conclusion

Phosphonic acid, [2-(4-aminophenyl)-1-hydroxyethylidene]bis-,monosodium salt (“EBP”)was considered to be non-mutagenic under the conditions of this test.