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Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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:
vanadyl pyrophosphate
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other:
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.
Controlsopen allclose all
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
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
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
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
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
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 resultsopen allclose all
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
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
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.