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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May to 06 Jun 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA 712-C-98-247
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EC Commission Directive 2000/32/EC Annex 4D-B.13/14. No. L 136/57
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United kingdom, London, England
Type of assay:
bacterial reverse mutation assay

Test material

Method

Target gene:
his operon (for S. typhimurium) and trp operon (for E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male SD rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
First experiment: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was not sufficiently soluble in distilled water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation assay); pre-incubation.

DURATION
- Preincubation period: when used during the second test: at 37 °C for 30 mins with shaking
- Exposure duration: 37 °C for 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Method inspection of the background bacterial lawn
Rationale for test conditions:
The test material caused no reduction in the growth of the background bacterial lawn at any dose level. The test material therefore, was tested up to the maximum recommended dose level of 5000 µg/plate
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative' response, even after the additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical analyses used are those described by Mahon et al (1989) and are usually followed by Dunnett's test, then if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Negative (solvent/vehicle) historical control data:
- Positive historical control data:

DMSO (Negative Control) historical control: mean revertant colony counts

[ TA100 ] [ TA1535 ] [ WP2 uvrA (pKM101) ] [ TA98 ] [ TA1537 ]
S9 mix - + - + - + - + - +
Mean 138 152 20 20 128 157 38 48 15 28
SD 25 27 5 4 27 29 6 9 5 8
Values 656 663 643 647 592 592 662 666 633 636
Min 70 56 9 5 60 78 17 17 6 9
Max 226 249 43 38 280 262 75 85 50 54

Positive Controls

[ TA100 ] [ TA1535 ] [ WP2 uvrA (pKM101) ] [ TA98 ] [ TA1537 ]
S9 mix - - + + - - + + - - + - - + - +
PC NaN3 NaN3 B(a)P AAN NaN3 NaN3 AAN AAN AF-2 NQO AAN 2NF 2NF B(a)P AAC B(a)P
µg/plate 0.5 2 5 5 0.5 2 2 5 0.05 2 10 1 2 5 50 5
Mean 571 950 755 1913 371 1093 160 290 917 776 699 460 430 495 848 246
SD 177 278 174 736 159 302 112 100 447 354 400 522 107 222 440 117
Values 750 270 746 273 748 264 744 263 51 10 57 757 275 1028 736 988
Min 208 425 212 424 49 325 40 52 268 272 130 112 178 93 73 64
Max 1534 2440 1373 4328 1130 2306 1145 952 2081 1262 1451 4280 792 1287 3307 2115

PC = Positive Control
NaN3 = Sodium azide
AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide
2NF = 2-Nitrofluorene
AAC = 9-Aminoacridine
B(a)P = Benzo(a)pyrene
AAN = 2-Aminoanthracene
NQO = 4-Nitroquinoline-1-oxide

Any other information on results incl. tables

 Test Results from Experiments 1 and 2

EXPERIMENT 1 (Standard Plate Test, SPT)

S9-Mix

Without

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA(pKM101)

TA98

TA1537

VC

169.3±30.0

28.0±1.0

146.7±25.0

33.7±3.8

11.0±1.0

5

182.0±5.3

19.0±3.6

159.3±25.1

33.0±5.3

12.3±4.9

15

216.7±70.6

11.7±3.8

159.7±10.1

31.0±13.2

12.3±3.2

50

170.7±20.8

21.0±7.9

196.3±118.6

32.3±5.1

13.7±2.1

150

128.0±14.0

19.7±3.2

163.7±48.6

31.7±17.6

18.3±1.5

500

150.0±14.2

20.0±1.7

142.0±24.9

35.7±3.1

12.3±0.6

1500

165.0±46.4

26.0±7.5

156.7±41.4

25.3±3.2

10.7±2.1

5000

159.0±18.7

19.3±7.8

195.7±55.1

34.0±10.6

10.0±3.6

PC (mg/plate)

NaN3(2)

NaN3(2)

NQO (2)

2NF (2)

AAC (50)

965.3±125.5

1428.0±25.5

2171.0±180.4

309.3±75.7

770.0±444.4

S9-Mix

With

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA(pKM101)

TA98

TA1537

VC

211.3±26.8

18.3±2.9

171.0±13.1

39.60±5.5

28.0±3.6

5

180.7±10.1

21.0±3.0

200.7±21.0

38.0±6.6

32.0±10.8

15

193.7±16.9

24.3±9.1

204.0±21.4

33.0±4.6

34.3±13.1

50

163.0±26.9

16.3±1.2

164.7±24.6

41.7±2.5

32.0±2.6

150

159.3±17.6

17.0±1.7

175.0±9.86

35.3±6.5

31.7±6.4

500

187.7±13.8

19.3±9.1

186.7±24.0

41.7±2.9

27.7±4.7

1500

201.7±6.8

18.3±6.4

166.0±22.1

47.7±3.5

19.7±3.8

5000

184.0±17.1

19.0±2.6

196.3±22.5

39.7±7.5

18.0±1.7

PC (mg/plate)

AAN (5)

AAN (5)

AAN (10)

B(a)P (5)

B(a)P (5)

2845.3±597.9

315.3±11.0

437±16.7

179.3±31.5

304±36.1

VC = Vehicle control; PC = Positive control

NaN3= Sodium azide

2NF = 2-Nitrofluorene

NQO = 4-Nitroquinoline-1-oxide

AAC = 9-Aminoacridine

AAN = 2-Aminoanthracene

B(a)P = Benzo(a)pyrene


 

 

EXPERIMENT 2 (Standard Plate Test with Incubation)

S9-Mix

Without

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA(pKM101)

TA98

TA1537

VC

163.3±12.9

26.7±2.3

197.7±5.5

46.3±4.2

13.3±1.5

50

155.3±22.0

23.3±3.1

185.3±19.7

47.7±5.5

16.3±1.2

150

140.0±5.3

27.7±1.5

214.7±16.3

43.7±4.0

13.0±3.0

500

136.3±5.9

25.3±2.9

190.3±12.7

41.7±3.2

15.3±4.5

1500

173.0±1.7

24.7±2.3

132.0±31.0

40.7±3.2

11.7±1.5

5000

148.7±16.3

23.0±2.6

163.3±10.2

46.7±6.4

15.7±3.2

PC (mg/plate)

NaN3(2)

NaN3(2)

NQO (2)

2NF (2)

AAC (50)

1062.3±76.7

1128.3±54.3

2138.0±493.1

326.0±32.0

436.0±12.1

S9-Mix

With

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA(pKM101)

TA98

TA1537

VC

194.0±13.7

28.3±0.6

220.3±18.5

56.3±6.8

34.3±4.2

50

214.0±18.5

19.7±2.3

221.3±23.0

57.3±9.1

34.3±3.5

150

180.7±11.0

18.0±5.2

213.7±16.5

60.3±2.9

28.3±5.1

500

179.0±28.2

22.3±2.1

212.0±29.7

60.7±4.2

33.0±4.0

1500

205.0±12.3

23.0±1.7

233.3±44.5

52.0±11.5

27.7±1.5

5000

223.3±10.0

18.7±2.9

243.7±45.6

57.0±3.6

27.0±6.1

PC (mg/plate)

AAN (5)

AAN (5)

AAN (10)

B(a)P (5)

B(a)P (5)

2785.0±271.6

312.3±15.3

707.3±143.9

319.3±42.5

267.7±69.6

VC = Vehicle control; PC = Positive control

NaN3= Sodium azide

2NF = 2-Nitrofluorene

NQO = 4-Nitroquinoline-1-oxide

AAC = 9-Aminoacridine

AAN = 2-Aminoanthracene

B(a)P = Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
Negative (for mutation) with metabolic activation.
Negative (for mutation) without metabolic activation.

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