Vinylphosphonic 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:

7 February 2017- 9 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vinylphosphonic acid

EC Number:

217-123-2

EC Name:

Vinylphosphonic acid

Cas Number:

1746-03-8

Molecular formula:

C2H5O3P

IUPAC Name:

vinylphosphonic acid

Test material form:

other: Liquid: Clear colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: VPS9016001
- Expiration date of the lot/batch: 27.06.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C)

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test.
±S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate in Experiment I (Initial Mutation Test) and Experiment II (Confirmatory Mutation Test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; ultrapure water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation, TA1537, 50 µg

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, TA100 and TA1535, 2 µg

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, E.coli WP2 uvrA, 2 µL

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with metabolic activation, all of Salmonella strains (2 µg), E.coli strain (50 µg)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

without metabolic activation, TA98, 4 µg

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours in the dark at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and induction of mutations with 3 plates each.

Evaluation criteria:

The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined by manual counting, the mean values, standard deviations and the mutation rates were calculated.

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

None

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No. In the main experiments the pH of the test system varied between 6.21 and 7.45.
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration levels (±S9 Mix).

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and induction of mutations with 3 plates each. In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
Slightly lower revertant colony numbers (within the biological variability range of the applied test system) were obtained in S. typhimurium TA98 at 5000, 1600, 160 μg/plate in the absence and presence (±S9 Mix) and at 500 μg/plate, in the presence of exogenous metabolic activation (+S9 Mix).
The revertant colony numbers were above the vehicle control data range (within the historical control data and biological variability range) in S. typhimurium TA100 at 5000 and 16 μg/plate (-S9 Mix) and in TA98 at 16 μg/plate (+S9 Mix).

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

	strain TA 98		strain TA 100		Strain TA 1535		strain TA 1537		E. coli WP2 uvrA
conc. [µg]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Untreated control	19.0	23.3	81.7	101.0	11.7	13.7	10.3	8.0	25.7	31.3
DMSO Control	15.0	23.3	–	99.0	–	10.7	7.7	8.7	–	28.7
Water control	23.3	24.0	76.0	110.0	11.7	11.7	8.3	8.7	23.7	30.0
5000	24.7	21.0	90.7	80.0	12.0	10.0	9.7	6.3	25.0	30.7
1600	23.7	20.0	92.0	114.7	11.0	11.0	9.7	8.3	31.3	28.3
500	25.0	23.0	90.7	105.3	10.3	13.0	8.0	10.3	26.0	39.3
160	16.7	23.0	88.0	108.0	10.7	10.0	9.7	7.3	35.0	33.7
16	23.0	24.3	84.7	99.3	12.0	13.7	9.0	7.7	28.3	40.7
NPD (4µg)	17.7	24.7	90.0	110.0	9.7	12.7	9.0	10.0	27.0	27.3
SAZ (2µg)	344.0	–	–	–	–	–	–	–	–	–
9AA (50 µg)	–	–	1072.0	–	1037.3	–	–	–	–	–
MMS (2µL)	–	–	–	–	–	–	450.0	–	–	–
2AA (2µg)	–	–	–	–	–	–	–	–	810.7	–
2AA (50 µg)	–	1597.3	–	3014.7	–	171.3	–	153.3	–	–

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

	strain TA 98		strain TA 100		Strain TA 1535		strain TA 1537		E. coli WP2 uvrA
conc. [µg]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Untreated control	15.7	28.7	81.7	106.7	11.7	11.7	8.0	9.0	30.0	33.7
DMSO Control	17.0	25.3	–	111.3	–	13.3	8.3	8.3	–	38.7
Water control	19.3	30.0	82.3	104.0	12.0	14.3	8.0	8.7	34.3	51.0
5000	15.0	17.3	81.7	99.3	11.3	17.7	5.7	9.7	30.3	41.3
1600	17.7	22.3	90.3	93.0	11.3	10.0	9.3	9.3	35.3	49.3
500	18.0	25.3	120.3	90.0	10.7	12.0	6.7	9.7	40.0	49.0
160	19.3	21.3	90.3	94.7	12.7	11.7	10.3	12.0	34.3	37.7
50	19.7	22.3	94.3	90.3	10.3	12.3	9.3	10.7	33.0	38.3
16	16.3	24.0	90.3	102.7	10.7	19.3	9.0	10.7	24.7	36.7
NPD (4µg)	187.3	–	–	–	–	–	–	–	–	–
SAZ (2µg)	–	–	607.3	–	984.0	–	–	–	–	–
9AA (50 µg)	–	–	–	–	–	–	170.7	–	–	–
MMS (2µL)	–	–	–	–	–	–	–	–	898.7	–
2AA (2µg)	–	2496.0	–	2546.7	–	229.3	–	333.0	–	–
2AA (50 µg)	–	–	–	–	–	–	–	–	–	280.0

 

Applicant's summary and conclusion

Conclusions:

The test item did not increase the frequency of revertant colonies in bacteria in the presence and absence of the metabolic activation system and thus is considered to be non-mutagenic in bacteria.

Executive summary:

The mutagenic potential of the test item was investigated according OECD TG 471 in Salmonella typhimurium strains TA 98, TA100, TA 1535, TA 1537, and TA 1538 and in E. coli WP2 uvrA. A plate incorporation test and a pre-incubation test were conducted with and without metabolic activation using S9 fraction of Phenobarbital and β-naphthoflavone (BNF) induced rat liver. The following concentrations were examined in triplicate: ±S9 Mix: 5000, 1600, 500; 160; 50 and 16 μg/plate. Positive and negative (vehicle) controls were run concurrently.

No substantial increases were observed in revertant colony numbers following treatment with the test item, neither in the presence nor in the absence of metabolic activation. Sporadic increases in revertant colony numbers compared to the vehicle control remained within the corresponding historical control data ranges. Control plates without mutagen showed no increase in revertant colonies. The positive controls showed the expected, biological relevant increases in revertant colonies in all tests. No cytotoxicity was observed in either tested strains with or without metabolic activation.

In conclusion, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without metabolic activation at any dose level investigated.