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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to an approved guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The maximum concentration tested in this study was 5495mg/plate, which exceeded the maximum concentration recommended by the guidelines which this assay follows of 5000 mg/plate.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
2-Propanol, titanium (4+) salt (TIPT), (also known as Vertec TIPT, titanium tetraisopropanolate, EC number 208-909-6), batch number V0303339, was a yellow liquid. It was received into Covance Laboratories Ltd on 1 April 2010 and stored at 15 to 25ºC under desiccant in the dark and from 20 April 2010 it was also stored under nitrogen. Purity was initially stated as ≥90% and the expiry date was given as 7 April 2011. Subsequent to this, following completion of the study, information was received from the Sponsor detailing the following purity profile for the batch: 2-propanol, titanium (4+) salt >99%, Heptanes (naphtha) 0.56%, Iso-propanol <0.5%.

Method

Target gene:
histidine locus in selected strains
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 fraction
Test concentrations with justification for top dose:
Range-Finder Experiment, Mutation Experiment 1 and additional Mutation Experiment 1 treatments of strain TA102 -S-9:
1.758, 8.792, 43.96, 219.8, 1099 and 5495 microgram/plate TIPT
Mutation experiment 2: 171.8, 343.4, 686.9, 1374, 2748 and 5495 microgram/plate TIPT
Vehicle / solvent:
DMSO
Justification for vehicle: preliminary solubility data indicated that TIPT was soluble in anhydrous analytical grade DMSO at concentrations of at least 100 mg/mL.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Strain: TA98

Migrated to IUCLID6: 2NF
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strain: TA100 and TA1535

Migrated to IUCLID6: NaN3
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain: TA1537

Migrated to IUCLID6: AAC
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Strain: TA102

Migrated to IUCLID6: MMC
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Strain: TA98

Migrated to IUCLID6: B[a]P
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (AAN)
Remarks:
Strain: TA100, TA1535 and TA1537
Details on test system and experimental conditions:
Bacteria
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. All the tester strains, with the exception of strain TA102, were originally obtained from the UK NCTC. Strain TA102 was derived from a culture obtained from Glaxo Group Research Limited. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 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 (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline). Checks were carried out according to Maron and Ames and De Serres and Shelby.

Maron D M and Ames B N (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173-215
De Serres F J and Shelby M D (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Research 64, 159-165
Evaluation criteria:
Evaluation criteria
For valid data, the test article was considered to be mutagenic if:
1. Dunnett's test gave a significant response (p<= 0.01) which was concentration related
2. the positive trend/effects described above were reproducible.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 microgram/plate).

Results and discussion

Test results
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
Remarks:
evidence of toxicity seen in a reduction of revertants at 5495 microgram/plate in strains TA98 and TA102 in absence of S9.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation was observed on the test plates at the highest one or two concentrations (1099 and 5495 µg/plate) in the Range-Finder and Experiment 1. Precipitation was observed on the test plates at the highest two concentrations in the absence of S-9 (2748 and 5495 µg/plate) and the highest three concentrations in the presence of S-9 (1374 to 5495 µg/plate) in Experiment 2.

Mutation
Following treatments of all the test strains in the absence and presence of S-9, only Experiment 1 treatments of strain TA102 in the absence of S-9 at 1.758, 8.792 and 219.8 ug/plate resulted in increases in revertant numbers that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. The increases were small in magnitude and occurred at intermediate concentrations with no indication of a concentration relationship and were not reproducible in two further experiments. Accordingly, these increases were considered to have been due to normal biological variability and not evidence of mutagenic activity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Raw plate counts and calculated mutagenicity data Experiment 1

Experiment 1, TA98 +S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

31, 29, 34, 34, 31

31.8

5

 

2.2

 

 

TIPT

1.758

31, 25, 29

28.3

3

0.9

3.1

-1.08

NS

TIPT

8.792

30, 26, 23

26.3

3

0.8

3.5

-1.74

NS

TIPT

43.96

29, 34, 19

27.3

3

0.9

7.6

-1.51

NS

TIPT

219.8

33, 29, 26

29.3

3

0.9

3.5

-0.77

NS

TIPT

1099

28, 23, 34

28.3

3

0.9

5.5

-1.12

NS

TIPT

5495

28 P M, 25 P M, 22 P M

25.0

3

0.8

3.0

-2.18

NS

B[a]P

10

328, 376, 355

353.0

3

11.1

24.1

 

 

 Experiment 1,TA98 –S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

25, 24, 29, 23, 21

24.4

5

 

3.0

 

 

TIPT

1.758

20, 28, 23

23.7

3

1.0

4.0

-0.27

NS

TIPT

8.792

19, 19, 28

22.0

3

0.9

5.2

-0.90

NS

TIPT

43.96

19, 16, 20

18.3

3

0.8

2.1

-2.24

NS

TIPT

219.8

23, 19, 21

21.0

3

0.9

2.0

-1.21

NS

TIPT

1099

23, 15, 15

17.7

3

0.7

4.6

-2.57

NS

TIPT

5495

13 P M, 16 P M, 9 P M

12.7

3

0.5

3.5

-4.78

NS

2NF

5

1299, 1116, 1347

1254.0

3

51.4

121.9

 

 

 Experiment 1, TA100 -S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

108, 110, 122, 123, 125

117.6

5

 

8.0

 

 

TIPT

1.758

130, 124, 130

128.0

3

1.1

3.5

1.18

NS

TIPT

8.792

125, 124, 107

118.7

3

1.0

10.1

0.12

NS

TIPT

43.96

142, 124, 155

140.3

3

1.2

15.6

2.47

NS

TIPT

219.8

128, 134, 127

129.7

3

1.1

3.8

1.36

NS

TIPT

1099

155, 144, 118

139.0

3

1.2

19.0

2.31

NS

TIPT

5495

141 P M, 107 P M, 107 P M

118.3

3

1.0

19.6

0.04

NS

NaN3

2

915, 783, 752

816.7

3

6.9

86.6

 

 

Experiment 1, TA100 +S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

122, 139, 135, 163, 149

141.6

5

 

15.4

 

 

TIPT

1.758

152, 159, 134

148.3

3

1.0

12.9

0.78

NS

TIPT

8.792

142, 115, 113

123.3

3

0.9

16.2

-2.18

NS

TIPT

43.96

124, 130, 127

127.0

3

0.9

3.0

-1.69

NS

TIPT

219.8

137, 154, 140

143.7

3

1.0

9.1

0.27

NS

TIPT

1099

132, 129, 148

136.3

3

1.0

10.2

-0.59

NS

TIPT

5495

128 P M, 126 P M, 123 P M

125.7

3

0.9

2.5

-1.85

NS

AAN

5

1481, 1784, 1581

1615.3

3

11.4

154.4

 

 

Experiment 1, TA1535 -S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

10, 15, 9, 18, 18

14.0

5

 

4.3

 

 

TIPT

1.758

11, 14, 23

16.0

3

1.1

6.2

0.52

NS

TIPT

8.792

10, 21, 11

14.0

3

1.0

6.1

-0.04

NS

TIPT

43.96

14, 15, 21

16.7

3

1.2

3.8

0.76

NS

TIPT

219.8

19, 20, 15

18.0

3

1.3

2.6

1.12

NS

TIPT

1099

11, 24, 11

15.3

3

1.1

7.5

0.29

NS

TIPT

5495

19 P M, 11 P M, 11 P M

13.7

3

1.0

4.6

-0.08

NS

NaN3

2

677, 618, 640

645.0

3

46.1

29.8

 

 

Experiment 1, TA1535 +S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

14, 9, 16, 11, 16

13.2

5

 

3.1

 

 

TIPT

1.758

8, 15, 23

15.3

3

1.2

7.5

0.47

NS

TIPT

8.792

25, 14, 9

16.0

3

1.2

8.2

0.64

NS

TIPT

43.96

14, 20, 15

16.3

3

1.2

3.2

0.89

NS

TIPT

219.8

16, 26, 16

19.3

3

1.5

5.8

1.61

NS

TIPT

1099

18, 16, 11

15.0

3

1.1

3.6

0.52

NS

TIPT

5495

10 P M, 15 P M, 13 P M

12.7

3

1.0

2.5

-0.14

NS

AAN

5

128, 160, 119

135.7

3

10.3

21.5

 

 

Experiment 1, TA1537 -S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

18, 13, 13, 13, 13

14.0

5

 

2.2

 

 

TIPT

1.758

14, 9, 8

10.3

3

0.7

3.2

-1.54

NS

TIPT

8.792

14, 9, 23

15.3

3

1.1

7.1

0.32

NS

TIPT

43.96

14, 16, 15

15.0

3

1.1

1.0

0.39

NS

TIPT

219.8

19, 16, 18

17.7

3

1.3

1.5

1.33

NS

TIPT

1099

10, 10, 19

13.0

3

0.9

5.2

-0.49

NS

TIPT

5495

9 P M, 11 P M, 7 P M

9.0

3

0.6

2.0

-2.12

NS

AAC

50

71, 80, 76

75.7

3

5.4

4.5

 

 

 Experiment 1, TA1537 +S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

16, 16, 21, 20, 14

17.4

5

 

3.0

 

 

TIPT

1.758

17, 15, 20

17.3

3

1.0

2.5

-0.01

NS

TIPT

8.792

24, 17, 14

18.3

3

1.1

5.1

0.31

NS

TIPT

43.96

24, 27, 24

25.0

3

1.4

1.7

2.72

*

TIPT

219.8

17, 27, 21 M B

21.7

3

1.2

5.0

1.54

NS

TIPT

1099

22, 24, 16

20.7

3

1.2

4.2

1.20

NS

TIPT

5495

13 P M, 8 P M, 13 P M

11.3

3

0.7

2.9

-2.63

NS

AAN

5

62, 44, 51

52.3

3

3.0

9.1

 

 

 Experiment 1, TA102 -S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

242, 216, 219, 210, 214

220.2

5

 

12.6

 

 

TIPT

1.758

299, 279, 269

282.3

3

1.3

15.3

3.55

**

TIPT

8.792

317, 325, 292

311.3

3

1.4

17.2

5.07

**

TIPT

43.96

222, 212, 266

233.3

3

1.1

28.7

0.76

NS

TIPT

219.8

241, 345, 312

299.3

3

1.4

53.1

4.37

**

TIPT

1099

234, 243, 258

245.0

3

1.1

12.1

1.47

NS

TIPT

5495

99 P M, 118 P M, 96 P M

104.3

3

0.5

11.9

-8.37

NS

MMC

0.2

838, 680, 764

760.7

3

3.5

79.1

 

 

Experiment 1, TA102 +S-9

Compound

Concentration (μg/plate)

Revertant numbers/plate

Mean

N

Fold Increase

Standard Deviation

Dunnett’s t‑value

Significance

DMSO

 

192, 192, 195, 293, 243

223.0

5

 

44.7

 

 

TIPT

1.758

231, 219, 219

223.0

3

1.0

6.9

0.06

NS

TIPT

8.792

217, 194, 247

219.3

3

1.0

26.6

-0.09

NS

TIPT

43.96

207, 198, 207

204.0

3

0.9

5.2

-0.62

NS

TIPT

219.8

251, 222, 214

229.0

3

1.0

19.5

0.26

NS

TIPT

1099

272, 229, 125

208.7

3

0.9

75.6

-0.63

NS

TIPT

5495

196 P M, 184 P M, 232 P M

204.0

3

0.9

25.0

-0.64

NS

AAN

20

1817, 1141, 996

1318.0

3

5.9

438.2

 

 

P - precipitation of test article observed, M plate counted manually, B bubbles or split in agar, NS not significant, * p<0.05, ** p<0.01

Applicant's summary and conclusion