Vanadyl pyrophosphate

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:

16 January to 4 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary GLP guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vanadyl pyrophosphate

EC Number:

406-260-5

EC Name:

Vanadyl pyrophosphate

Cas Number:

58834-75-6

Molecular formula:

(VO)2 P2 O7

IUPAC Name:

divanadium(4+) (phosphonatooxy)phosphonate dioxidandiide

Details on test material:

FV100 catalyst, a grey powder, batch no. 248, stored at room temperature (10-30°C) in the dark. Stability and purity data can be provided by the study sponsor if required.

Method

Target gene:

Reversion to histidine independence.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other:

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver post-mitochondrial fraction from Aroclor 1254 induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Range-finding study (strain TA100 only) and Mutation Experiment 1: 1.6, 8, 40, 200, 1000, and 5000 µg/plate.
Mutation Experiment 2: 51.2, 128, 320, 800, 2000, and 5000 µg/plate.

Vehicle / solvent:

The test article was suspended in 0.5% (w/v) aqueous methylcellulose (0.5% MC). Aqueous methycellulose was chosen as the vehicle because the test article had been shown to be poorly soluble in a wide range of commonly used vehicle substances.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA102 without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 with S9

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA1535, TA1537, TA102 with S9

Details on test system and experimental conditions:

An initial range-finding study was carried out with strain TA100 only, with and without S9. The two mutation experiments were conducted with all 5 strains with and without S9. Bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubtion was carried out with shaking in an anhydric incubator. All experimentation commenced within 2 hours of the end of the incubation period. The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics.
Triplicate plates with and without S9 were used. Vehicle and positive controls were included in quintuplicate and triplicate respectively (with and without S9). In all cases, 0.1 ml bacterial culture was added to 2.5 ml molten agar at 46±1°C followed by 0.1 ml test article suspension or control, and 0.5 ml 10% S9 or buffer. The mixture was rapidly mixed and poured on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C in the dark for 3 days. Following incubation the plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

As the results of the Mutation Experiment 1 were negative, treatments in the presence of S9 in Experiment 2 included a preincubation step. Quantities of test article/control, bacteria and S9 mix (as above) were mixed together and incubated for 1 hour at 37±1°C, before the addition of 2.5 ml molton agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

Evaluation criteria:

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually if necessary. The background lawn was inspected for signs of toxicity. For valid data, the test article was considered mutagenic if Dunnett's test gave a significant response (p≤0.01) which was dose related, and the positive trend/effects were reproducible.

Statistics:

The mean and SD of the plate counts were determined for each treatment. Dunnett's test was used to compare the counts at each concentration with the control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No precipitation of the test article was observed. Although the test article was treated as a suspension, test particles were only visible using a microscope on plates treated at the highest concentrations. In the range-finding study, evidence of toxicity in the form of a marked reduction in the number of revertant colonies was observed at 5000 µg/plate with and without S9.
A reduction in revertant numbers was also observed at 1000 µg/plate in strain TA98 with S9 in Experiment 1. In Experiment 2, signs of toxicity were apparent in strain TA98 at concentrations of 320 µg/plate and above without S9. In the presence of S9, toxicity was apparent in strains TA98, TA100 and TA1537 at 5000 µg/plate and in TA102 at 800 µg/plate and above.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test article did not cause statistically significant increases in the numbres of revertant colonies following exposure of the tester strains in the absence or presence of S9 metabolic activation. Results are summarised below.

Experiment 1
	TA98		TA100		TA1535		TA 1537		TA102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	19.2	34.2	111.8	115.2	21.6	15.8	8.4	17.8	275.6	259.0
1.6	23.7	34.3	35.7	111.7	16.0	15.0	8.7	19.7	312.7	208.3
8	21.0	32.0	128.7	106.0	16.3	15.0	14.3	26.0	287.0	214.0
40	22.3	32.0	149.0	94.0	19.0	14.0	11.7	21.3	306.0	233.3
200	22.0	36.3	112.3	112.3	14.3	14.7	9.7	20.0	281.0	232.7
1000	9.0	28.3	133.0	95.0	16.3	11.7	8.0	17.0	284.3	233.7
5000	0.7	2.0	3100	67.3	3.3	4.3	0.3	2.7	195.3	190.0
+control	769.3	444.0	705.0	1310	604.0	242.3	108.7	134.0	640.7	1619
Experiment 2
0	22.4	40.2	117.4	129.2	13.2	15.0	10.6	22.8	310.4	280.8
51.2	21.0	31.0	139.3	113.7	20.3	15.7	6.7	25.0	295.3	242.7
128	23.7	38.7	128.3	120.7	15.0	17.0	9.7	23.3	309.3	246.3
320	9.3	41.0	110.3	119.3	15.7	16.7	6.3	19.0	294.7	250.0
800	9.3	29.7	110.7	106.3	18.0	15.7	9.7	17.3	288.3	206.7
2000	0.3	26.7	101.7	110.7	19.0	13.7	3.3	17.0	242.7	181.3
5000	0.0	1.3	35.7	92.0	4.3	22.3	2.0	11.0	169.7	161.0
+control	716.7	237.7	771.0	1216	642.7	288.3	82.7	219.3	706.0	1459

Applicant's summary and conclusion

Conclusions:

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

The test article was found to be non-mutagenic under the conditions of this study.

Executive summary:

The mutagenic potential of FV100 catalyst was assayed for mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102). The test article proved poorly soluble in a range of commonly used vehicles therefore it was administered as a suspension in 0.5% (w/v) aqueous methylcellulose (0.5% MC). Evidence of toxicity was apparent at concentrations of 1000 and/or 5000 µg/plate with and without S9 activation. No signs of test substance precipitation were observed. No statistically significant increases in revertant numbers were observed in any strain with and without S9 activation, at concentrations ranging from 1.6 to 5000 µg/plate. Therefore it was concluded that FV100 catalyst did not induce mutation in 5 strains of S. typhimurium under the conditions of this study.