1,8-diazabicyclo[5.4.0]undec-7-ene

Genetic toxicity: in vitro

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

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

Test material information

Constituent 1

Reference substance name:

1,8-diazabicyclo[5.4.0]undec-7-ene

EC Number:

229-713-7

EC Name:

1,8-diazabicyclo[5.4.0]undec-7-ene

Cas Number:

6674-22-2

Molecular formula:

C9H16N2

IUPAC Name:

2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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 results

Test results 1

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

Test results 2

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.