3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-06-20 to 2006-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile

EC Number:

300-496-1

EC Name:

3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile

Cas Number:

93940-97-7

Molecular formula:

C16H28N4

IUPAC Name:

3-[(3-{[(2-cyanoethyl)amino]methyl}-3,5,5-trimethylcyclohexyl)amino]propanenitrile

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: V3186/11
- Date of manufacture: December 7, 2004

Letter of Confirmation: CAS No 90530-15-7 and 93940-97-7 refer to the same substance and substance composition.

Method

Target gene:

his; trp

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:

S9 mix of Aroclor induced rat liver

Test concentrations with justification for top dose:

both experiments:
0; 20; 100; 500; 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: 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:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 2

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Controls 3

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Controls 4

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Controls 5

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

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

Rationale for test conditions:

according to guidelines

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

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: not soluble in water
- Precipitation: no
- Other confounding effects: none

HISTORICAL CONTROL DATA
please refer to "any other informations on results"

Any other information on results incl. tables

 Experiment 1 Standard plate test

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli
	TA 100	TA 98	TA 1535	TA 1537	WP2 uvrA
	Results without S9
DMSO	111 ± 8	31 ± 5	16 ± 3	8 ± 2	31 ± 4
20	108 ± 4	24 ± 6	17 ± 1	8 ± 1	27 ± 5
100	108 ± 6	28 ± 1	19 ± 3	8 ± 2	34 ± 3
500	102 ± 4	24 ± 4	15 ± 2	9 ± 2	29 ± 1
2500	102 ± 11	26 ± 1	16 ± 1	7 ± 1	31 ± 2
5000	107 ± 7	17 ± 4	16 ± 2	6 ± 1	32 ± 5
MNNG (5)	648 ± 36	-	634 ± 43	-	-
NOPD (10)	-	504 ± 26	-	-	-
AAC (100)	-	-	-	429 ± 20	-
4-NQO (5)	-	-	-	-	556 ± 24
	Results with S9 (1:9)
DMSO	114 ± 9	35 ± 3	16 ± 3	13 ± 2	35 ± 5
20	117 ± 12	30 ± 3	16 ± 2	11 ± 3	32 ± 6
100	116 ± 11	35 ± 11	15 ± 2	10 ± 2	35 ± 3
500	105 ± 4	33 ± 3	18 ± 3	8 ± 1	38 ± 5
2500	101 ± 4	30 ± 5	13 ± 3	9 ± 3	40 ± 6
5000	114 ± 18	31 ± 3	14 ± 3	7 ± 1	33 ± 2
2- AA (2.5; 60)	828 ± 27	593 ± 39	130 ± 21	133 ± 8	231 ± 18

 

Experiment 2 Preincubation test

Dose (µg/plate)	Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli
	TA 100	TA 98	TA 1535	TA 1537	WP2 uvrA
	Results without S9
DMSO	110 ± 4	30 ± 1	17 ± 2	8 ± 1	31 ± 5
20	108 ± 6	27 ± 3	15 ± 1	8 ± 3	29 ± 6
100	101 ± 10	28 ± 3	17 ± 2	8 ± 2	29 ± 3
500	103 ± 2	28 ± 4	16 ± 1	9 ± 5	27 ± 6
2500	101 ± 7	23 ± 4	15 ± 1	8 ± 2	25 ± 4
5000	99 ± 3	14 ± 2	15 ± 1	5 ± 3	21 ± 0
MMNG (5)	674 ± 50	-	553 ± 24	-	-
NOPD (10)	-	422 ± 29	-	-	-
AAC (100)	-	-	-	397 ± 25	-
4-NQO (5)	-	-	-	-	620 ± 19
	Results with S9 (1:9)
DMSO	107 ± 6	31 ± 2	17 ± 3	9 ± 2	35 ± 6
20	104 ± 12	32 ± 3	15 ± 2	11 ± 3	30 ± 6
100	104 ± 1	31 ± 3	17 ± 2	11 ± 4	30 ± 6
500	101 ± 5	25 ± 3	15 ± 3	7 ± 2	27 ± 2
2500	99 ± 6	22 ± 3	14 ± 4	7 ± 1	27 ± 6
5000	96 ± 11	13 ± 2	12 ± 1	5 ± 3	24 ± 4
2- AA (2.5)	631 ± 21	598 ± 78	141 ± 19	120 ± 20	225 ± 22

 

Table 3 Historical control values for revertants/plate: Standard Plate Test

		Bacterial strains
Historical control data of DMSO control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	29	108	18	10	32
		SD	4	8	2	2	5
		Minimum	19	87	11	5	25
		Maximum	48	140	25	16	53
	+S9 (2 -AA)		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	36	110	17	11	35
		SD	5	9	2	2	5
		Minimum	21	91	10	5	25
		Maximum	50	154	24	18	58
Historical control data of positive control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	750	953	853	440	671
		SD	125	258	205	94	159
		Minimum	332	506	502	300	502
		Maximum	1200	1795	1582	1042	1478
	+S9 (2-AA		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	786	957	139	136	323
		SD	157	225	29	26	28
		Minimum	504	573	85	200	153
		Maximum	1745	1878	254	136	324

 

Table 4 Historical control values for revertants/plat: Preincubation test

		Bacterial strains
Historical control data of DMSO control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	28	109	17	10	32
		SD	4	9	2	2	5
		Minimum	17	84	12	5	25
		Maximum	40	148	23	17	51
	+S9 (2 -AA)		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	32	110	17	10	33
		SD	5	9	2	2	5
		Minimum	20	147	10	5	25
		Maximum	49	91	24	16	50
Historical control data of positive control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	739	842	747	420	599
		SD	123	170	145	72	77
		Minimum	388	511	502	278	500
		Maximum	1129	1583	1830	842	1150
	+S9 (2-AA)		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	665	740	122	115	230
		SD	103	143	21	20	27
		Minimum	501	500	80	80	150
		Maximum	1120	1380	258	200	307