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Genetic toxicity: in vitro

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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Citric acid, titanium salt
EC Number:
923-927-0
Molecular formula:
C30H32O35.Ti2
IUPAC Name:
Citric acid, titanium salt
Constituent 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Dihydrogen oxide
Specific details on test material used for the study:
(1) Product name : CTL Ti638 UP
(2) CAS No. : Not available
(3) Lot No. : 110200/51
(4) Appearance : Orange, particulate free liquid
(5) Purity : 54.7 wt.% (Ti content 4.97 wt.%)
(6) Solubility : Water soluble
(7) Stability : Not available
(8) Storage condition : Room temperature
(9) Supplier : Catalytic Technologies Limited

Method

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
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary range-finding test was carried out on 0.06, 0.19, 0.56, 1.67, 5 µl/plate (three-fold serial dilutions). As a result of preliminary range-finding test, cytotoxicity was not observed at any dose levels in the presence and absence of metabolic activation system. Based on the results of preliminary range-finding test, the main test was performed on dose levels of :
- In the absence of metabolic activation system (S9 mix(-)) 0, 0.06, 0.19, 0.56, 1.67, 5 µl/plate
- In the presence of metabolic activation system (S9 mix(+)) 0, 0.06, 0.19, 0.56, 1.67, 5 µl/plate
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (CAS No.3688-53-7) and 2-Aminoanthracene (CAS No.613-13-8)
Details on test system and experimental conditions:
Composition
Bacterial suspension 0.1 µl
Test substance 0.05 µl
0.1M Phosphate buffered saline (direct method) 0.5 µl
S9 mix (metabolic activation method) 0.5 µl
Top agar 2.0 µl

Preincubation
Temperature 37 °C
Time 20 min

Incubation
Temperature 37 °C
Time 48 hours

No of replicants: 3 plates per dose
Evaluation criteria:
Method of observation, measurement and analysis
(1) Colony counting
The number of revertants were counted only inside area of plate (diameter : 90 mm) by manual measurement using electronic register (Model 570, SUNTEX, Taiwan, MAI-035-01).
(2) Test method of growth inhibition
It was tested that all plate was observed with the naked eye.
(3) Measurement of bacterial concentration
It was performed by step-dilution method according to the Standard Operation Procedure, KCL.
(4) Sterility test
0.1 ml of S9 mix and test substance were mixed with top agar respectively, and then, it was added onto minimum glucose agar plate. The plate was incubated at 37 °C for 48 hours.
4) Evaluation and interpretation of results

The result was judged as ‘Positive’, if there is a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Statistics:
The statistical method for analysis of result was not applied in this study.

Results and discussion

Test resultsopen allclose all
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 applicable
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 applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 applicable
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 applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. Test results (Main study)

Metabolic activation

Dose (µl /plate)

Number of colony/plate

Base pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9 Mix(-)

0

100

111

102

10

5

5

21

23

25

8

7

8

4

6

6

Mean±SD

104±5.9

7±2.9

23±2.0

8±0.6

5±1.2

0.06

95

101

100

6

5

8

26

26

26

10

5

12

6

7

5

Mean±SD

99±3.2

6±1.5

26±0.0

9±3.6

6±1.0

0.19

115

105

115

8

7

7

27

19

22

8

11

7

4

6

3

Mean±SD

111±5.5

7±0.6

23±4.0

9±2.1

4±1.5

0.56

111

106

117

5

8

6

26

30

23

13

17

9

5

5

6

Mean±SD

113±8.2

6±1.5

26±3.5

13±4.0

5±0.6

1.67

102

116

117

5

7

7

27

28

33

10

8

8

5

5

3

Mean±SD

112±8.4

6±1.2

29±3.2

9±1.2

4±1.2

5

73

68

67

4

6

8

22

22

26

5

5

11

6

5

6

Mean±SD

69±3.2

6±2.0

23±2.3

7±3.5

6±0.6

S9 Mix(+)

0

109

97

116

9

10

6

22

21

28

19

19

19

 

8

7

14

Mean±SD

107±9.6

8±2.1

24±3.8

19±0.0

10±3.8

0.06

118

107

106

8

9

4

21

25

27

17

17

15

8

9

9

Mean±SD

110±6.7

7±2.6

24±3.1

16±1.2

9±0.6

0.19

105

110

101

6

7

6

26

25

32

16

12

15

8

6

9

Mean±SD

105±4.5

6±0.6

28±3.8

14±2.1

8±1.5

0.56

116

95

96

8

8

11

23

23

18

16

18

16

10

9

9

Mean±SD

102±11.8

9±1.7

21±2.9

17±1.2

9±0.6

1.67

104

104

92

8

6

5

33

22

27

13

22

17

7

8

12

Mean±SD

100±6.9

6±1.5

27±5.5

17±4.5

9±2.6

5

96

125

94

6

12

9

22

32

24

19

21

19

10

7

9

Mean±SD

105±17.3

9±3.0

26±5.3

20±1.2

9±1.5

S9 Mix (-)

Positive controls

AF-2

NaN3

AF-2

AF-2

9-AA

Dose (/plate)

0.01

0.5

0.01

0.1

80

Number of Colony

516

534

493

218

230

206

210

255

256

395

404

448

2682

3005

2933

Mean±SD

514±20.6

218±12.0

240±26.3

416±28.4

2873±169.6

S9 Mix (+)

Positive controls

2-AA

2-AA

2-AA

2-AA

2-AA

Dose (/plate)

1.0

2.0

10

0.5

2.0

Number of colony

666

634

631

183

182

191

296

271

276

192

214

219

142

175

148

Mean±SD

644±19.4

185±4.9

281±13.2

208±14.4

155±17.6

Applicant's summary and conclusion

Conclusions:
Four histidine-requring strains of Samonella typhimurium (TA100, TA1535, TA98 and TA1537) and one tryptophan-requring strain of Escherichia coli (WP2uvrA) were used to evaluate the mutagenic potential of the test substance CTL Ti638 UP in the presence and absence of metabolic activation system (S9 mix). Under the conditions of this test, the results were as follows:
TA100, TA1535, WP2uvrA, TA98 and TA1537 :
Regardless of metabolic activation system, the number of revertant colony did not show increase compared with the number of revertant colony of negative control group
Executive summary:

To evaluate the mutagenic potential of CTL Ti638 UP in bacteria, a bacterial reverse mutation study was performed with preincubation method using four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the presence and absence of metabolic activation system (S9 mix). The test substance was dissolved in distilled water and then it was applied to the test strains at the following dose levels:

In the absence of metabolic activation system (S9 mix(-)) 0, 0.06, 0.19, 0.56, 1.67, 5 µl/plate

In the presence of metabolic activation system (S9 mix(+)) 0, 0.06, 0.19, 0.56, 1.67, 5 µl/plate

No increase in the number of revertant colonies was seen in TA98, TA100, TA1535, TA1537 and WP2uvrA strain compared with negative control groups in the presence and absence of the metabolic activation system.

Considering these findings, it is concluded that the test substance CTL Ti638 UP does not induce reverse mutation in the 5 bacterial strains which were used in this study.