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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:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
and OECD Guideline 472 (Gene Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-aminoanthracene as positive control with metabolic activation)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test substance (as cited in study report): 1, 8-Diazabicyclo (5, 4, 0) undecen-7
- Analytical purity: 97.7%
- Physical state: liquid
- Batch number: 13-0948
- Test substance number: 96/661
- Storage: room temperature
- Stability: The stability of the test substance throughout the study period has been verified by reanalysis.

Method

Target gene:
His- and Trp-Operon
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:
Aroclor-induced rat liver S9-mix
Test concentrations with justification for top dose:
- Standard plate test: 20, 100, 500, 2,500 and 5,000 µg/plate
- Preincubation test: 4, 20, 100, 500 and 2,000 µg/plate
Vehicle / solvent:
Water
Controls
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 2-aminoanthracene (all tester strains); without S9: N-methyl-N'-nitro-N-nitrosoguanidine (TA 1535, TA 100), 4-nitro-o-phenylendiamine (TA 98), 9-aminoacridine (TA 1537), N-ethyl-N'-nitro-N-nitrosoguanidine (E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION (standard plate test): Experiment 1

DURATION
- Exposure duration: 48 - 72 h, 37 °C

METHOD OF APPLICATION (preincubation test): Experiment 2
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h, 37 °C

NUMBER OF REPLICATIONS: 3 per dose per expermiment

DETERMINATION OF CYTOTOXICITY
- Method: 1) reduction of the number of revertants compared to negative control; 2) clearing of background lawn; 3) reduction of titer
Evaluation criteria:
The test subtance is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9-mix or after adding a metabolizing system.

Generally, the experiment is to be considered valid if the following criteria are met :
• The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
• The titer of viable bacteria was >= 10^9/ml .
Statistics:
Not generally required for this assay

Results and discussion

Test resultsopen allclose all
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:
Cytotoxicity was observed at 5000 µg/plate in all tester strains (in TA 100 and TA 2537 at 2500 µg/plate) in the standard plate test and at 2000 µg/plate in the preincubation test (all tester strains).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at 5000 µg/plate in the standard plate test and at 2000 µg/plate in the preincubation test (only without S9-mix).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Because of contamination the preincubation test was repeated for the E. coli strain in a third experiment.
- Precipitation: no precipitation occurred
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Standard plate test:

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

    E. coli WP2 uvrA

 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

-S9

 +S9

-S9 

+S9 

 0

20±4

19±1

117±15

137±6

12±2

12±1

29±3

37±4

31±3

36±5

20 

17±2

17±1

123±13

132±5

9±1

12±1

25±1

33±2

33±3

32±4

 100

17±1

18±4

114±7

107±7

9±1

11±2

26±2

33±3

24±3

32±3

 500

14±2

12±2

141±12

120±11

9±2

8±2

22±1

26±1

28±7

19±6

 2500

15±2

11±1

110±9x

116±4x

7±1x

5±2x

19±3x

21±1x

18±2

20±4

 5000

5±3x

7±1x

0x

67±6x

0x

4±2x

1±1x

7±2x

10±2x

12±2x

 2 -AA

-

141±8

-

1423±

216

 -

154±22

1006±

25

 -

271±

11

 MNNG

1022±

86

-

1085±

11

-

-

 -

-

 -

-

-

 AAC

-

-

-

-

654±51

-

-

-

-

-

 NOPD

 -

-

-

-

-

-

940±

9

-

 ENNG

 -

-

-

-

-

-

-

-

974±

46

-

Mean ± SD

 

Preincubation-test:

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

 E. coli WP2

uvrA   

 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

-S9

 +S9

 -S9

+S9 

 0

19±1

19±1

111±14

111±4

10±1

11±1

25±2

39±3

32±4

38±7

4

18±1

18±1

106±8

117±16

10±1

9±1

23±2

37±2

25±3

34±4

 20

17±2

15±2

100±6

120±26

11±1

9±1

24±3

31±3

30±3

31±4

 100

17±2

15±3

98±3

109±5

8±1

9±1

24±5

30±2

27±5

33±5

 500

18±2

12±3

105±12

94±3

9±1

8±3

17±6

35±3

29±5

34±4

 2000

1±2x

12±1x

0x

96±9x

0x

4±2x

0x

19±3x

8±1x

24±5

2-AA

-

203±5

-

829±

64

-

127±22

-

797±

36

-

188±3

 MNNG

1060±

43

-

913±90

-

-

-

-

-

-

-

 AAC

-

-

-

-

436±10

-

-

-

-

-

NOPD

-

-

-

-

-

-

901±43

-

-

-

 ENNG

-

 -

 -

 -

-

 -

-

753±56

 -

Mean ± SD

X: reduced background growth

2-AA: 2-aminoanthracene

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NOPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

 

 

According to the results of the study, the test substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen here. The positive controls gave the expected results.

 

Applicant's summary and conclusion

Conclusions:
No evidence of any induction of reverse mutation was seen under the conditions of this assay.
Executive summary:

DBU was investigated for the ability to induce reverse mutation in Salmonella typhimurium strains TA98, TA100, TA 1535 and TA 1535 and in Eschericia coli strain WP2uvrA. Strains were exposed to concentrations up to the limit concentration of 5000 ug/plate in the absence and presence of an exogenous metabolic activation system (Aroclor 1254 -induce rat liver S9 fraction) in an initial plate-incorporation assay and at concentrations of up to 2000 ug/plate in a confirmatory pre-incubation assay. Cytotoxicity was apparent at the highest concentrations used. No evidence of any induction of reverse mutation was seen under the conditions of this assay; the sensitivity of the assay was confirmed by appropriate responses to positive control compounds.