N,N-dibutylformamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(July 21,1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(December 1992)

Deviations:

no

GLP compliance:

yes

Remarks:

Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dibutylformamide

EC Number:

212-090-0

EC Name:

N,N-dibutylformamide

Cas Number:

761-65-9

Molecular formula:

C9H19NO

IUPAC Name:

N,N-dibutylformamide

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Probe 527
- Purity test date: 18 January 2000

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

OTHER SPECIFICS:
- main component + 1.76% formic acid

Method

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 1254-induced rat liver S-9 mix

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate (standard plate test)
4 µg - 2500 µg/plate (preincubation test)

Vehicle / solvent:

Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation, for all strains

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix: strain TA 1535, TA 100

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S-9 mix: strain TA 98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix: strain TA 1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S-9 mix: strain E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Exposure duration: Preincubation Test: 20 min
- Expression time: Standard plate test and Preincubation Test: 48-72 hours in the dark at 37°C

SELECTION AGENT (mutation assays): Standard plate test: Salmonella typhimurium, 0.5 mM histidine + 0.5 mM biotin; Escherichia coli 0.5 mM tryptophan

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
Method:
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer

Evaluation criteria:

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 S9 mix induced a significant increase in the number of revertant colonies within the range of the historical control
- The titer of viable bacteria was >10E9/ mL

The test chemical 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 .
A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the standard plate test depending on the strain and test conditions at doses >= 2500 µg/plate. A bacteriotoxic effect was observed in the preincubation assay at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. No test substance precipitation was found .

Any other information on results incl. tables

Table 1: Number of revertants per strain (mean from three plates)- Standard Plate Test

	Strain TA 1535		Strain TA 100		Strain TA 1537		Strain TA 98		E.coli WP2 uvrA
Conc. [µg/plate]	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix
Solvent control	18	18			10	10	38	29
Positive control*	131	525	1210	649	173	525	1130	1040	252	607
20	18	20	115	114	9	10	38	29	37	37
100	16	19	108	106	8	7	35	26	39	34
500	11	18	109	102	9	9	32	26	38	29
2500	8	12	75	75	6	6	24	19	29	24
5000	6	7	59	55	3	3	15	12	24	19

*without S9 mix: MNNG (5 µg/plate): TA1535, TA 100; AAC (100 µg/plate): TA 1537; NOPD (10 µg/plate): TA98, 4-NQO (5 µg/plate): E.coli WP2 uvrA

*with S9 mix: 2-AA (2.5 µg/plate): TA1535, TA 100; TA 1537, TA 98, E.coli WP2 uvrA (60 µg/plate)

 

Table 2: Number of revertants per strain (mean from three plates)- Preincubation Test

	Strain TA 1535		Strain TA 100		Strain TA 1537		Strain TA 98		E.coli WP2 uvrA
Conc. [µg/plate]	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix
Solvent control	17	18	120	106	9	10	31	27	41	31
Positive control*	126	834	942	609	162	443	941	1063	218	550
4	16	19	118	110	9	9	30	26	37	29
20	15	16	111	110	9	9	26	25	36	27
100	15	15	107	106	7	7	32	20	36	33
500	10	14	111	91	4	4	22	15	33	27
2500	7	9	22	74	1	1	17	6	31	25

*without S9 mix: MNNG (5 µg/plate): TA1535, TA 100; AAC (100 µg/plate): TA 1537; NOPD (10 µg/plate): TA98, 4-NQO (5 µg/plate): E.coli WP2 uvrA

*with S9 mix: 2-AA (2.5 µg/plate): TA1535, TA 100; TA 1537, TA 98, E.coli WP2 uvrA (60 µg/plate)