Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(NPP No. 237, No. 306, No. 302, No. 303 on Mar. 31, 1984)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot number: 10705, manufactured by Tama Chemical Industry Co., Ltd.
- Purity: 99.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature shielded from light


Method

Target gene:
Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
Escherichia coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (liver rat)
Test concentrations with justification for top dose:
- Each strain was tested at six concentrations within the following: 0, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate.
- Justification: A preliminary test was carried out in the range of 50 to 5000 µg/plate with only one replicate per strain. Antibacterial activity was observed at 5000 µg/plate in all strains except for Escherichia coli WP2 with and without metabolic activation, at 1500 µg/plate without metabolic activation in TA100, TA 1535 and TA1537 and with metabolic activation in TA100 and TA1537. On this basis, the highest dose in the first study was 2500 µg/plate for Salmonella typhimurium TA1535, T98 and T100 and 5000 µg/plate in Escherichia coli WP2 with and without metabolic activation. For 1537 the highest dose was set at 1250 µg/plate without metabolic activation and 2500 µg/plate with it. A second test was performed at the same doses used in the first test except the maximum dose of the metabolic activation test in TA1537 which was changed to1250 μg / plate as in the non-activation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO) from Wako Pure Chemical Industries, Ltd, lot number: DSL5887
- Justification for choice of solvent/vehicle: a stability test in DMSO solution was conducted. Dicyclohexylcarbodiimide was prepared to be 50 mg / ml in DMSO and further diluted with the same solvent in a ratio of 2 to 3. The product was immediately used for the test. Two concentrations at the highest concentration (50 mg / ml) and at the lowest concentration (0.25 mg / ml) used in this test were carried out. The average content of the samples after 3 hours of preparation was 99.7 and 100%, respectively, with respect to the average (0 hour) of the initial value. These values satisfied the laboratory criteria (over 90% of the initial measurement average value).
The stability of the substance at the concentrations prepared for this test was also checked. For the 50 mg / ml solution were obtained values between 97.4 to 101%, 106 to 107% for the 0.781 mg / ml solution and 91.1-103% for the 0.391 mg / ml solution. These values also met the criteria of the standard laboratory operating procedure (the mean content is more than 85% of the aggregate quantity).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation method, with and without metabolic activation. 2 ml of top agar, 0.1 ml of test solution, 0.5 ml of phosphate buffer solution (0.5 ml of S9 mixture solution in metabolic activation test) and 0.1 ml of assay bacterial solution were mixed in a small test tube and then placed on a synthetic medium flat plate. After solidification, plates were incubated at 37ºC for 48 hours. As a control group, DMSO or positive control substance solutions were used instead of the test substance preparation solution. After the incubation period, the number of revertant mutant colonies generated was calculated and their average value and standard deviation were obtained, respectively.

DURATION
- Preincubation period: no preincubation period.
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays):
No specific selection agent was used. The lack of amino-acid in the medium allowed only mutants to grow because of their ability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS:
Three replicates per strain, dose and substance (vehicle, positive control substances and test substance).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.
Evaluation criteria:
The substance was considered to be mutagenic if:
- the number of revertant colonies counted in the plate containing the test substance was increased more than twice as compared with the counts of the negative control
- a dose dependence was recognized in the increase in counts.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 and 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1250 and 2500 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 625 and 1250 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: due to the antibacterial activity observed in the preliminary study, the highest dose selected for each strain in both tests was known to have antibacterial activity. This was confirmed in both tests: the highest dose for each strain showed inhibition against growth of the bacteria, in some cases in the two high doses, but although this antibacterial activity was observed, no increase in the number of mutant colonies dose-dependent was detected.

HISTORICAL CONTROL DATA: not specified.

Any other information on results incl. tables

Table 1. Number of revertants in the preliminary test.

Group

dose

(μg/plate)

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

Solvent control

-

-

138 ± 7.2

12 ± 2.1

11 ± 8.1

26 ± 2.6

8 ± 1.7

Test substance

50

-

76

9

8

21

8

150

-

82

5

14

13

8

500

-

62

11

10

10

7

1500

-

59*

5*

9

7

0*

5000

-

2*

0*

9

6*

0*

Positive controls

Substance and dose

 

AF2

0.01

SA

0.5

AF2

0.01

AF2

0.1

9AA

80

 

 

-

669 ± 30.1

319 ± 16.8

139 ± 15.9

561 ± 15.5

2329 ± 590.8.

Solvent control

-

+

139 ± 7.0

15 ± 3.8

15 ± 2.1

39 ± 3.1

11 ± 2.1

Test substance

50

+

115

15

17

21

7

150

+

138

13

22

22

12

500

+

88

8

24

28

6

1500

+

55*

8

13

8

5*

5000

+

22*

2*

8

3*

0*

Positive controls

Substance and dose

 

2AA

1

2AA

2

2AA

10

2AA

0.5

2AA

2

 

 

+

798 ± 52.2

245 ± 6.1

498 ± 24.7

189 ± 17.2

182 ± 18.2

* inhibition was observed against growth of the bacteria

Table 2. Number of revertants (mean ± SD) in the first test.

Group

dose

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

Solvent control

-

-

124 ± 7.5

15 ± 2.1

13 ± 3.8

22 ± 3.0

10 ± 0.6

Test substance

39.1

-

 

 

 

 

8 ± 1.5

78.1

-

118 ± 6.5

11 ± 3.2

 

19 ± 6.1

7 ± 2.6

156.3

-

108 ± 11.5

8 ± 3.6

13 ± 0.6

20 ± 4.5

10 ± 1.0

312.5

-

103 ± 8.0

10 ± 1.7

15 ± 4.6

20 ± 4.0

7 ± 1.5

625

-

105 ± 6.1

11 ± 2.9

13 ±3.1

18 ± 2.5

6 ± 1.0

1250

-

96 ± 11.5

9 ± 1.2 *

11 ± 3.1

16 ± 5.0

2 ± 2.9*

2500

-

85 ± 8.1 *

6 ± 1.2 *

11 ± 4.4

11 ± 2.1*

 

5000

-

 

 

6 ± 3.8*

 

 

Positive controls

Substance and dose

 

AF2

0.01

SA

0.5

AF2

0.01

AF2

0.1

9AA

80

 

 

-

626 ± 20.2

228 ± 15.2

152 ± 8.9

560 ± 54.0

2965 ± 56.0

Solvent control

-

+

157 ± 26.3

13 ± 2.6

17 ± 6.2

41 ± 3.8

14 ± 4.2

Test substance

78.1

+

128 ± 10.7

17 ± 7.0

 

35 ± 1.0

10 ± 4.5

156.3

+

125 ± 12.7

13 ± 0.6

12 ± 2.0

27 ± 2.1

8 ± 1.5

312.5

+

124 ± 5.0

8 ± 4.5

13 ± 0.00

30 ± 1.5

8 ± 1.5

625

+

117 ± 16.8

11 ± 5.7

14 ± 2.9

41 ± 2.1

6 ± 1.0

1250

+

96 ± 16.9*

7 ± 1.2

11 ± 3.2

28 ± 3.1

3 ± 0.0*

2500

+

101 ± 2.6*

8 ± 1.2*

15 ± 3.2*

20 ± 8.7*

2 ± 2.1*

5000

+

 

 

9 ± 0.6*

 

 

Positive controls

Substance and dose

 

2AA

1

2AA

2

2AA

10

2AA

0.5

2AA

2

 

 

+

750 ± 26.1

212 ± 4.6

273 ± 22.9

226 ± 9.7

164 ± 10.7

* inhibition was observed against growth of the bacteria

Table 3. Number of revertants (mean ± SD) in the second test.

Group

dose

S9 mix

TA100

TA1535

WP2 uvrA

TA98

TA1537

Solvent control

-

-

150 ± 15.3

16 ± 2.1

15 ± 4.0

21 ± 1.0

8 ± 1.0

Test substance

39.1

-

 

 

 

 

10 ± 4.9

78.1

-

123 ± 27.2

11 ± 5.5

 

21 ± 5.6

6 ± 2.9

156.3

-

125 ± 7.0

11 ± 2.1

16 ± 8.7

21 ± 6.1

5 ± 3.5

312.5

-

122 ± 19.2

9 ± 1.2

14 ± 4.4

24 ± 1.0

4 ± 0.6

625

-

115 ± 13.5

10 ± 2.3

13 ± 1.5

21 ± 4.0

5 ± 1.5*

1250

-

127 ± 3.0

7 ± 0.6*

12 ± 2.9

19 ± 1.2

4 ± 2.5*

2500

-

101 ± 7.3*

5 ± 2.3*

7 ± 4.47*

17 ± 4.5*

 

5000

-

 

 

3 ± 2.6*

 

 

Positive controls

Substance and dose

 

AF2

0.01

SA

0.5

AF2

0.01

AF2

0.1

9AA

80

 

 

-

709 ± 19.5

175 ± 11.2

162 ± 10.8

759 ± 15.3

4064 ± 478.1

Solvent control

-

+

160 ± 16.5

16 ± 4.5

17 ± 0.6

50 ± 5.3

12 ± 2.6

Test substance

39.1

+

 

 

 

 

7 ± 1.5

78.1

+

167 ± 20.6

17 ± 4.6

 

33 ± 5.1

10 ± 1.2

156.3

+

147 ± 5.5

12 ± 3.1

15 ± 1.7

34 ± 6.8

7 ± 1.5

312.5

+

138 ± 10.5

14 ± 3.1

15 ± 3.5

34 ± 7.5

5 ± 2.0

625

+

147 ± 11.8

13 ± 2.0

16 ± 4.0

31 ± 3.6

5 ± 1.5

1250

+

110 ± 7.5

10 ± 1.5

17 ± 2.9

24 ± 2.6

3 ± 2.6*

2500

+

126 ± 1.0*

8 ± 1.5*

 15 ± 1.5

24 ± 4.2*

 

5000

+

 

 

6 ± 5.3*

 

 

Positive controls

Substance and dose

 

2AA

1

2AA

2

2AA

10

2AA

0.5

2AA

2

 

 

+

922 ± 19.1

228 ± 14.0

711 ± 51.6

304 ± 26.3

203 ± 6.1

* inhibition was observed against growth of the bacteria

Applicant's summary and conclusion

Conclusions:
The test item was not mutagenic under the test conditions, with or without metabolic activation.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test) according to OECD Guidelines No. 471 and 472 and the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and tryptophan requiring Escherichia coli WP2 uvrA were tested in triplicate at six concentrations of test item in the range 40 - 5000 μg/plate, with and without metabolic activation (S9 mix), based on the results of a preliminary range-finding test. The plate incorporation method was used; vehicle (DMSO) and positive controls were run in parallel. Although inhibitory activity was observed in all strains tested in one or two of the higher dose groups, no increase in the number of the relevant colonies related with the dose were observed under the experimental conditions applied. Based on the above results, the test item was considered non-mutagenic.