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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 28 February, 2001 to 26 March, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well conducted in accordance with OECD 471, EU B.13/14, EPA OPPTS 870.5100 guidelines as well as in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solution
Details on test material:
- Physical state: Clear liquid
- Analytical purity: >93%
- Impurities (identity and concentrations): 0.8% Free Amine + Amine hydrochloride and ≤ 0.1% AAS
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in the dark
- pH (1% water): 6.5

Method

Target gene:
The specific target genes of Salmonella Typhimurium tester strains are as follows: TA1535, TA1537, TA102, TA98 and TA100
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (With and without metabolic activation)
Main Experiment: Experiment 1 & 2: 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate (With and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(concurrent untreated)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Positive control substance:
other: Nitroquinoline-1-oxide (4NQO)
Remarks:
Without metabolic activation
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation
Positive control substance:
other: 1,8-Dihydroxyanthraquinone (DANTHRON)
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (direct plate incorporation)

NUMBER OF REPLICATIONS: Triplicates

Evaluation criteria:
The test substance was considered positive in the test system if the following criteria were met:

The test substance should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- The test substance caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains both with and without S9-mix beginning at 15µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between experiment number, strain type and exposures with or without S9-mix. The test substance was, therefore, tested up to the toxic limit. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation.

- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table -1. Cytotoxicity (Number of revertant colonies)

 

 

Test substance concentration (µg/plate)

With/ Without

S9-mix

Strain

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

Without

TA100

91

91

81

83

64

17S

0T

0T

0T

0T

0T

With

TA100

97

105

103

103

83

50S

0T

0T

0T

0T

0T

S=sparse bacterial background lawn

T= toxic, no bacterial lawn

Table-2. Genotoxicity (Mean number of revertant colonies)

Strain

TA100

TA1535

TA102

TA98

TA1537

Test substance concentration (mg/plate)

 

 

 

 

 

With S9

 

 

 

 

 

+ve control type (concentration (mg/plate))

2AA (1)

2AA (2)

DAN (10)

BP (5)

2AA (2)

Test number

1

2

1

2

1

2

1

2

1

2

+ve control

1772

2317

287

135

886

723

229

251

582

336

-ve control

143

137

17

17

349

308

36

25

12

22

0.15

129

137

13

13

357

315

26

24

13

18

0.5

134

11

10

14

346

313

33

25

15

14

1.5

129

127

15

14

373

307

32

26

16

14

5

142

143

10

13

369

341

32

23

17

16

15

132

0

12

2

363

151

37

11

17

6

50

28

0

10

0

276

0

22

0

10

0

Without S9

 

 

 

 

 

+ve control type (concentration (mg/plate))

ENNG (3)

ENNG (5)

MMC (0.5)

4NQO (0.2)

9AA (80)

Test number

1

2

1

2

1

2

1

2

1

2

+ve control

621

464

603

430

854

961

142

126

656

716

-ve control

153

134

12

18

316

336

38

22

16

17

0.15

150

123

13

15

340

308

29

18

18

19

0.5

132

114

11

21

331

339

30

18

17

18

1.5

154

111

19

16

339

232

25

14

15

11

5

145

111

9

10

326

325

28

19

12

18

15

84

0

11

0

320

0

23

0

0

0

50

0

0

0

0

0

0

0

0

0

0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was found to be non-mutagenic under the conditions of this test.
Executive summary:

The mutagenic potential of C12-16 ADBAC was investigated inS. typhimurium strains A1535, TA1537, TA102, TA98 and TA100. Six dose levels of the test substance for each bacterial strain were tested in triplicate with and without a metabolic activation system. The dose range was determined in a preliminary toxicity assay and was 0.15 to 50 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels were included in both experiments to allow for test substance induced toxicity and to ensure there were a minimum of four non-toxic doses plated out. The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9-mix. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. The test substance was found to be non-mutagenic under the conditions of this test (Thompson PW, 2001).