N,N-bis(2-methylpropyl)formamide

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:

key study

Study period:

1997-02-04 to 1997-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-methylpropyl)formamide

EC Number:

219-983-4

EC Name:

N,N-bis(2-methylpropyl)formamide

Cas Number:

2591-76-6

Molecular formula:

C9H19NO

IUPAC Name:

N,N-bis(2-methylpropyl)formamide

Test material form:

other: liquid

Details on test material:

- Name of the test substance used in the study report: N,N-Diisobutylformamid
- Purity: 99.8 area-% (GC)

Method

Target gene:

S. typhimurium and E. coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

Standard plate test: 20, 100, 500, 2500 and 5000 µg/plate
Preincubation test: Standard plate test: 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: DMSO had been demonstrated to be suitable in bacterial reverse mutation tests and historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2-AA

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix (all tester strains)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

MNNG

Positive control substance:

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

Remarks:

without S9 mix (TA 1535, TA100)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

NOPD

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S9 mix (TA 98)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

AAC

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA 1537)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

ENNG

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix (WP2 uvrA)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 hours in the dark

NUMBER OF REPLICATIONS: 3

TOXICITY:
Toxicity detected by a
- 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:

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 S-9 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

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No Precipitation of the test substance was found.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants for all tester strains were within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the standard plate test a slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions at doses equal to or greater than 2500 µg/plate.
In the preincubation test bacteriotoxicity (reduced backgrown growth, decrease in the number of revertants, reduction in the titer) was observed from about 100 µg - 500 µg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Preincubation Test

Dose/plate	TA 1535		TA 100		TA 1537		TA 98		E. coli WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Negative control
DMSO	19	19	103	106	11	12	28	37	37	43
Test item
20 µg	18	16	97	102	10	9	29	33	34	31
100 µg	16	11	97	82	10	7	25	27	32	26
500 µg	11	9	94	82	9	8	20	23	27	21
2500 µg	12	10	71	51	7	-	14	20	21	15
5000 µg		-	-	-	-	-	10	9	15	7
Positive control
5.0 µg MNNG	1289		1091
2.5 µg 2-AA		406		795		173		1050
100 µg AAC					711
10 µg NOPD							995
10 µg ENNG									1216
60 µg 2-AA										235

Table 2: Standard Plate Test

Dose/plate	TA 1535		TA 100		TA 1537		TA 98		E. coli WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Negative control
DMSO	21	20	105	110	12	12	27	38	35	42
Test item
20 µg	20	17	107	123	9	10	33		40	47
100 µg	17	16	105	123	12	13	26		34	46
500 µg	16	16	108	105	7	10	27		39	45
2500 µg	14	11	104	109	7	7	29		31	45
5000 µg	11	10	104	85	6	7	22		29	41
Positive control
5.0 µg MNNG	1010		1368
2.5 µg 2-AA		158		1195		135		845
100 µg AAC					612
10 µg NOPD							649
10 µg ENNG									537
60 µg 2-AA										250

DMSO: dimethyl sulfoxide

2 -AA: 2 -aminoanthracene

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine

NOPD: 4 -nitro-o-phenylendiamine

AAC: 9-aminoacridine

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine