Phosphonic acid, monoethyl ester, sodium salt (1:1)

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:

23 Feb - 7 Apr 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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1997) and current version of 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 1993)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

(adopted 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-960-2

EC Name:

-

Cas Number:

39148-16-8

Molecular formula:

C2H6O3P.Na

IUPAC Name:

sodium ethyl phosphonate

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Range finding experiment (with and without metabolic activation, performed only in TA100):
1.6, 8, 40, 200, 1000 and 5000 µg/plate
Experiment I: (with and without metabolic activation):
1.6, 8, 40, 200, 1000 and 5000 µg/plate
Experiment II: (with and without metabolic activation):
156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

Range Finding Experiment and Main Experiments:

METHOD OF APPLICATION: in agar (plate incorporation, range finding experiment, experiment I and experiment II without S9 mix) and pre-incubation (experiment II with S9 mix)

DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: triplicates (test item and positive controls), quintuplicate (solvent control) in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method:other: inspection of the background lawn

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

A test item is considered as mutagenic if:
- the assay is considered as valide
- the results revealed statistical significance (p ≤ 0.01) and a significant dose-relation ship was present
- positive responses were reproducible

Acceptability
The assay is considered valid if the following criteria were met:
1. the mean negative control counts fell within the range of historical controls
2. the positive control substances induce clear increases in revertants confirming discrimination between different strains and an active S9-mix
3. no more than 5% of the plates were lost through contamination or some other unforeseen event

Statistics:

Individual plate counts were determined separately and the mean and standard deviations of plate counts for each treatment were calculated.
Statistical analysis were performed by m-statistic (to prove Poisson-distribution) and Dunnett's test (comparison of counts from each dose with the control) and linear regression analysis (prove of dose-response).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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 examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

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 examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

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 examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

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 examined

Positive controls validity:

valid

Test results 5

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was completely soluble in water.
- Precipitation: No precipitation was observed at any concentration.

RANGE-FINDING/SCREENING STUDIES:
A range finding study was performed initially in tester strain TA100 to determine cytotoxicity. As the study design of experiment I was similar to the range finding test, the obtained mutagenicity data were included in experiment I (please refer to Table 1).

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants obtained for solvent and positive controls lay within the range of historical control data. Thus, the study was considered as valide.

Any other information on results incl. tables

Table 1. Test results of experiment I 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate ± SD
		Base-pair substitution type			Frameshift type
		TA 100^1	TA 1535	WP2 uvrA	TA 98	TA 1537
–	water (100 µL)	108 ± 14	23 ± 9	19 ± 5	34 ± 13	11 ± 3
–	1.6	115 ± 16	27 ± 7	17 ± 5	65 ± 3	13 ± 1
–	8	96 ± 12	23 ± 6	15 ± 6	63 ± 10	15 ± 3
–	40	105 ± 10	25 ± 6	16 ± 4	59 ± 18	20 ± 7
–	200	125 ±32	20 ± 4	18 ± 10	45 ± 7	13 ± 3
–	1000	108 ± 13	17 ± 7	20 ± 2	61 ± 4	12 ± 5
–	5000	113 ± 7	20 ± 6	18 ± 8	47 ± 17	19 ± 3
–	Positive controls	NaN3	NaN3	NQO	2NF	AAC
	Mean No. of colonies/plate ± SD	700± 58	598± 17	559± 66	953± 22	194 ± 50
+ (1.6%)	water (100 µL)	112 ± 10	21 ± 4	25 ± 6	36 ± 7	14 ± 3
+ (1.6%)	1.6	104 ± 14	22 ± 8	32 ± 6	36 ± 9	13 ± 2
+ (1.6%)	8	106 ± 8	20 ± 4	25 ± 1	50 ± 7	12 ± 2
+ (1.6%)	40	114 ± 7	25 ± 8	22 ± 3	46 ± 5	7 ± 5
+ (1.6%)	200	115 ± 23	22 ± 2	27 ± 8	39 ± 6	12 ± 2
+ (1.6%)	1000	113 ± 1	23 ± 5	28 ± 3	41 ± 4	11 ± 4
+ (1.6%)	5000	127 ± 8	26 ± 5	23 ± 3	33 ± 8	13 ± 4
+ (1.6%)	Positive controls	AAN	AAN	AAN	B[a]P	AAN
	Mean No. of colonies/plate ± SD	2218 ± 71	234 ± 10	54 ± 20	288 ± 40	281 ± 14

1: Mutagenicity data for TA100 were provided by the range finding test.

NaN3 = sodium azide

AAC = 9-aminoacridine

4NQO = 4-nitroquinoline 1-oxide

2NF = 2-nitro-fluorene

B[a]P = Benzo(a)pyrene

AAN = 2-aminoanthracene

SD = standard deviation

 

Table 2. Test results of experiment II 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate ± SD
		Base-pair substitution type			Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
–	water (100 µL)	89 ± 9	24 ± 4	11 ± 3	26 ± 8	12 ± 5
–	156.25	100 ± 3	21 ± 2	10 ± 1	25 ± 5	10 ± 3
–	312.5	100 ± 15	16 ± 0	9 ± 1	23 ± 7	11 ± 2
–	625	100 ± 8	17 ± 7	11 ± 6	34 ± 15	12 ± 2
–	1250	83 ± 7	24 ± 11	6 ± 3	27 ± 4	13 ± 2
–	2500	100 ± 10	13 ± 5	10 ± 5	23 ± 5	9 ± 7
–	5000	80 ± 8	17 ± 4	10 ± 3	36 ± 7	13 ± 4
–	Positive controls	NaN3	NaN3	NQO	2NF	AAC
	Mean No. of colonies/plate ± SD	683± 36	634± 20	922± 88	1081± 115	141 ± 8
+ (1.6%)	water (100 µL)	97 ± 17	16 ± 3	15 ± 5	34 ± 8	11 ± 3
+ (1.6%)	156.25	111 ± 8	15 ± 1	17 ± 5	36 ± 6	13 ± 1
+ (1.6%)	312.5	110 ± 4	14 ± 2	15 ± 6	28 ± 12	14 ± 3
+ (1.6%)	625	111 ± 5	19 ± 1	9 ± 1	31 ± 9	16 ± 4
+ (1.6%)	1250	100 ± 16	15 ± 4	10 ± 2	39 ± 11	19 ± 12
+ (1.6%)	2500	93 ± 1	17 ± 5	11 ± 1	30 ± 3	15 ± 5
+ (1.6%)	5000	88 ± 26	19 ± 7	11 ± 5	35 ± 6	14 ± 2
+ (1.6%)	Positive controls	AAN	AAN	AAN	B[a]P	AAN
	Mean No. of colonies/plate ± SD	161 ± 26	68 ± 12	75 ± 22	140 ± 37	101 ± 27

NaN3 = sodium azide

AAC = 9-aminoacridine

4NQO = 4-nitroquinoline 1-oxide

2NF = 2-nitro-fluorene

B[a]P = Benzo(a)pyrene

AAN = 2-aminoanthracene

SD = standard deviation

 

In experiment II, only a weak positive control response was observed in strain TA100 in the presence of S9 mix for AAN. However, concurrent control treatments with this strain in the absence of S9 (as well as the negative controls in the presence of S9), and other strains in the presence of S9, served to otherwise confirm the correct strain and assay functioning, together with the metabolic activity of the S9 mix. It was therefore considered that this weak positive control response was not indicative of any strain or assay failure, and the data from these treatments were therefore accepted as valid.

 

 

Table 3: Historical control values

	Strain	S9	N	Revertant colonies per plate ± SD	Range
					Lower	Upper
Solvent controls	TA98	-	20	47.4 ± 10	33.6	61.4
		+	20	44.3 ± 8.4	31.6	55.0
	TA100	-	20	117.1 ± 19.4	92.8	156.2
		+	20	125.1 ± 21.0	97.2	153.4
	TA1535	-	20	19.5 ± 5.5	14.0	26.2
		+	20	19.3 ± 5.1	14.0	25.6
	TA1537	-	19	14.4 ± 5.6	7.4	25.0
		+	18	14.2 ± 5.1	7.2	19.0
	WP2 uvrA	-	20	16.4 ± 4.5	11.8	21.0
		+	20	20.4 ± 7.4	10.0	27.2
Positive controls	TA98	- (2NF)	18	303.6 ± 197.0	147.1	947.5
		+ (B[a]P)	20	141.2 ± 75.5	80.0	377.1
	TA100	- (NaN3)	20	603.4 ± 87.9	368.9	694.2
		+ (AAN)	19	1886.7 ± 368.1	1330.7	2548.2
	TA1535	- (NaN3)	20	604.4 ± 77.4	448.5	744.9
		+ (AAN)	20	157.6 ± 46.6	90.2	253.0
	TA1537	- (AAC)	19	196.2 ± 95.6	52.5	314.7
		+ (AAN)	18	241.6 ± 75.2	87.5	354.0
	WP2 uvrA	- (NQO)	19	793.2 ± 131.3	601.3	1056.8
		+ (AAN)	20	127.5 ± 69.1	43.9	267.3

1: Mutagenicity data for TA100 were provided by the range finding test.

NaN3 = sodium azide

AAC = 9-aminoacridine

4NQO = 4-nitroquinoline 1-oxide

2NF = 2-nitro-fluorene

B[a]P = Benzo(a)pyrene

AAN = 2-aminoanthracene

SD = standard deviation

Applicant's summary and conclusion

Conclusions:

Under the conditions chosen, the test substance was not mutagenic in this bacterial reverse mutation assay.