N'-[3-(dimethylamino)propyl]-N,N-dimethylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable key in vitro studies are available. All three studies demonstrated a negative result for mutagenicity::

- Bacterial reverse mutation assay: performed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A (BASF AG, 1998)

- Read-across in vitro mammalian chromosome aberration test: performed according to OECD Guideline 473 and EU Method B.10 in human lymphocytes (Mc Enaney, 1991)

- Read-across mammalian cell gene mutation test: Mouse lymphoma mutagenicity assay: performed according to a method equivalent to OECD Guideline 476 in Mouse lymphoma L5178Y cells (Covance Laboratories, Ltd, 2010)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial 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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Bis (3-dimethylaminopropyl) amin
- Physical state: Colourless liquid
- Analytical purity: 98.4%
- Lot/batch No.: 94-0675
- Storage condition of test material: Room temperature

Target gene:

his operon and trp operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: standard plate test 1: 0; 20; 100; 500; 2500 and 5000 µg/plate (with and without S9-mix); all strains
Experiment 2: standard plate test 2: 2000, 3000, 4000, 5000, 6000 µg/plate (with and without S9-mix); (TA100, E.coli WP2 uvrA)
Experiment 3: preincubation test : 0; 20; 100; 500; 2500 and 5000 µg/plate (with and without S9-mix); all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

water control

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (+ S9): 2-aminoanthracene, 2-AA (all strains); (- S9): N-methyl-N'-nitro-N-nitroso-guanidine, MNNG (TA 100, TA 1535); 4-nitro-o-phenylendiamine, NOPD (TA 98); 9-aminoacridine, AAC (TA 1537); N-ethyl-N'-nitro-N-nitrosoguanidin, ENNG (E.coli WP2 uvrA)

Remarks:

2-AA: 2 µg (TA100, TA 98, TA 1537, TA 1535), 60 µg (E. coli WP2 uvrA); MNNG: 5 µg; NOPD: 10 µg; AAC: 100 µg; ENNG: 10 µg; solvent for all positive control substances: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate test (first experiment), preincubation test (second experiment)

DURATION
- Preincubation period: 20 min (only preincubation test)
- Exposure duration: 48 - 72 h (in the dark, 37 °C)

NUMBER OF REPLICATIONS: 3 plates/concentration/experiment; 3 3experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants or a clearing of the bacterial background lawn, reduction of the titer.
The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

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.

Key result

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:

A weak bacteriotoxic effect in the standard plate test at 6000 µg/plate (TA 100). In the preincubation assay bacteriotoxicity was found depending on the strain and test conditions at doses >= 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect in the standard plate test at 6000 µg/plate. In the preincubation assay bacteriotoxicity was found at doses >= 2500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation was found

Remarks on result:

other: all strains/cell types tested

Table 1: Test results of experiment 1 (plate incorporation)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	116 ± 13	14 ± 2	30 ± 1	27 ± 5	9 ± 2
–	20	130 ± 32	13 ± 3	39 ± 3	25 ± 7	8 ± 2
–	100	153 ± 14	16 ± 5	41 ± 7	32 ± 5	8 ± 1
–	500	161 ± 23	20 ± 4	37 ± 8	22 ± 7	10 ± 2
–	2500	153 ± 13	18 ± 2	48 ± 4	25 ± 3	9 ± 3
–	5000	179 ± 3	21 ± 9	44 ± 7	27 ± 8	9 ± 2
Positive controls, –S9	Name	MNNG	MNNG	ENNG	NOPD	AAC
	Concentrations (μg/plate)	5	5	10	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	1223 ± 268	1070 ± 278	698 ± 36	982 ± 16	575 ± 6
+	0	119 ± 13	15 ± 3	46 ± 5	42 ± 4	9 ± 2
+	20	171 ± 24	16 ± 5	56 ± 5	40 ± 8	11 ± 0
+	100	143 ± 23	15 ± 5	39 ± 4	40 ± 13	10 ± 3
+	500	138 ± 22	19 ± 5	44 ± 6	43 ± 6	12 ± 3
+	2500	154 ± 10	22 ± 3	54 ± 13	40 ± 3	7 ± 1
+	5000	147 ± 17	21 ± 3	57 ± 7	33 ± 4	9 ± 3
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1297 ± 57	112 ± 3	327 ± 37	577 ± 33	214 ± 21

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

NOPD = 4-nitro-o-phenylendiamine

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

B = reduced background growth

Table 2: Test results of experiment 2 (plate incorporation)

 

With or without S9-Mix	Test substance concentration (μg/plate)
		TA 100	WP2 uvrA
–	0	117 ± 5	35 ± 2
–	2000	105 ± 5	38 ± 3
–	3000	120 ± 13	41 ± 2
–	4000	108 ± 11	38 ± 7
–	5000	90 ± 10	45 ± 4
–	6000	148 ± 20	31 ± 9
Positive controls, –S9	Name	MNNG	ENNG
	Concentrations (μg/plate)	5	10
	Mean No. of colonies/plate (average of 3 ± SD)	1307 ± 138	505 ± 6
+	0	126 ± 28	36 ± 6
+	2000	136 ± 11	32 ± 3
+	3000	142 ± 5	32 ± 4
+	4000	119 ± 19	36 ± 4
+	5000	97 ± 11	24 ± 3
+	6000	0B	0B
Positive controls, +S9	Name	2AA	2AA
	Concentrations (μg/plate)	2.5	60
	Mean No. of colonies/plate (average of 3 ± SD)	1182 ± 66	220 ± 34

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

NOPD = 4-nitro-o-phenylendiamine

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

B = reduced background growth

 

 

Table 3: Test results of experiment 3 (preincubation)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	141 ± 10	22 ± 4	33 ± 4	31 ± 3	8 ± 2
–	20	117 ± 4	18 ± 1	36 ± 2	28 ± 7	9 ± 2
–	100	113 ± 13	19 ± 1	34 ± 4	30 ± 2	8 ± 1
–	500	94 ± 8	7 ± 2	34 ± 5	16 ± 2	8 ± 1
–	2500	0B	0B	14 ± 3	0B	0B
–	5000	0B	0B	0B	0B	0B
Positive controls, –S9	Name	MNNG	MNNG	ENNG	NOPD	AAC
	Concentrations (μg/plate)	5	5	10	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	1104 ± 47	789 ± 44	521 ± 33	1087 ± 70	408 ± 42
+	0	138 ± 10	23 ± 0	41 ± 7	46 ± 4	9 ± 1
+	20	124 ± 10	20 ± 3	40 ± 3	41 ± 2	11 ± 3
+	100	103 ± 1	21 ± 3	40 ± 2	44 ± 9	13 ± 6
+	500	88 ± 8	21 ± 3	35 ± 3	36 ± 9	11 ± 5
+	2500	102 ± 5	13 ± 3	50 ± 4	30 ± 7	8 ± 2
+	5000	43 ± 9B	0B	18 ± 2	0B	7 ± 1
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	983 ± 31	121 ± 9	227 ± 14	574 ± 54	103 ± 11

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

NOPD = 4-nitro-o-phenylendiamine

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

B = reduced background growth

Conclusions:

In this study, the test substance demonstrated to be negative with or without metabolic activation under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read-across from related substance

Justification for type of information:

Data from the related substance DMAPA is used to cover this endpoint. Justification for the read-across approach is included in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains / cell types tested

Conclusions:

No reliable chromosome aberration study with the test substance is available. Data generated with the related substance DMAPA is used for endpoint coverage.
Under the conditions of the study, DMAPA did not induce noteworthy increase in the number of cells with structural chromosome aberration, both with and without metabolic activation at any harvest time.
The same is assumed to be correct for the target substance.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Dimethylaminopropylamine (DMAPA) was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 Guideline and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice.
With metabolic activation, lymphocytes cultures were exposed for 3 hours to solvent vehicle or DMAPA at 0, 350.5, 500.8, 715.4 µg/ml and then incubated for a further 17 or 41 hours.
Without metabolic activation, lymphocytes cultures were exposed for 20 or 44 hours to solvent vehicle or DMAPA at 0, 120.2, 171.8, 245.4 µg/ml.
The proportion of cells with structural aberrations in negative controls cultures fell within historical solvent control ranges. Positive controls induced statistically significant increases in the proportion of cells with structural aberrations.
Chromosome aberrations were analyzed in cells sampled 20 hours after the start of treatment at 3 consecutive dose levels. The highest concentrations chosen for analysis at this time, 245.4 and 715.4 11µg/ml, induced approximately 51% and 54% mitotic inhibition in the absence and presence of S-9 respectively. The effects of single concentrations only, 245.4 µg/ml without and 715.4 µg/ml with S-9 were investigated at the delayed harvest at which time 0% and 34% mitotic inhibition was induced.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): N, N-dimethuylaminopropylamine
- Physical state: liquid
- Analytical purity: 99.8%
- Purity test date: 21 Jan 1991
- Lot/batch No.: P91-01
- Stability under test conditions: Stable until january 1992 in the specified conditions of storage
- Storage condition of test material: Storage in the dark at room temperature

Target gene:

Chromosome defects

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: A single female donor was used in this study. The volunteer was not suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. An appropriate volume of whole blood was drawn from the peripheral circulation on the day of culture. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 ml heparinised blood into 9.0 ml Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 ug/ml gentamycin. Phytohaemagglutinin (PHA) was included at a concentration of 37.5 µl per ml of culture to stimulate the lymphocytes to divide. Cultures were rocked continuously during incubation.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9) prepared from male Sprague Dawley rats induced with Aroclor 1254 (obtained from Molecular Toxicology Incorporated, Annapolis, Maryland, USA.)

Test concentrations with justification for top dose:

Without metabolic activation: 120.2, 171.8, 245.4 µg/ml for 20 hours sampling time and 245.4 µg/ml for 44 hours sampling time.
With metabolic activation: 350.5, 500.8, 715.4 µg/ml for 20 hours sampling time and 715.4 µg/ml for 44 hours sampling time.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: Methylmethansulfonate (MMS) 50.0 µg/ml. +S9: Cyclophosphamide (CPA) 25.0 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours at 37°C
- Exposure duration: 20 and 44 hours for cultures without metabolic activation and 3 hours for cultures with metabolic activation (followed by 17 and 41 hours of additional sampling time before harvesting)
- Expression time (cells in growth medium): 20 or 44 hours
- Selection time (if incubation with a selection agent): colchicine was added 1.5 hour prior to harvest.

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 (for cultures treated with DMAPA and positive controls) and 4 (for cultures treated with negative controls)

NUMBER OF CELLS EVALUATED: One hundred metaphases from each culture were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; percentage of cell in mitosis

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: hyperploidy

Evaluation criteria:

The test chemical was to be considered as clearly positive if:
- statistically significant increases in the proportion of structurally aberrant cells (without gaps) occurred at one or more concentrations
- the proportion of aberrant cells at such data points exceeded the normal range

Statistics:

The proportion of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of cells in category 2 for each test treatment condition were compared with the proportion in negative controls by using Fisher's exact test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 1: Selection of doses

Mitotic Index (%)
Treatment (µm/ml)	20 hours				44 hours
	- S-9		+ S-9		- S-9		+ S-9
	A	B	A	B	A	B	A	B
Solvent	5.8	4.0	5.3	4.1	4.5	3.3	3.5	3.8
20.21	NM	NM	NM	NM	NM	NM	NM	NM
28.87	NM	NM	NM	NM	NM	NM	NM	NM
41.24	NM	NM	NM	NM	NM	NM	NM	NM
58.92	NS	NS	NM	NM	NM	NM	NM	NM
84.17	NS	NS	NM	NM	NM	NM	NM	NM
120.2	5.8	4.8	NS	NS	NS	NS	NM	NM
171.8	4.9	4.6	NS	NS	NS	NS	NS	NS
245.4	1.6	3.2	NS	NS	4.2	4.3	NS	NS
350.5	0.3	0.4	3.8	3.5	0.7	0.2	NS	NS
500.8	0	0	2.9	3.5	0	0	NS	NS
715.4	0	0	2.2	2.1	0	0	3.2	1.6
1022	0	0	0	0	0	0	0.6	0.4
	NS = not scored		NM = not made

Table 2: Cells with structural aberrations

20 hour sampling time, - S-9

Treatment (µg/ml)	Replicate	Cells scored	Cells with aberrations including gaps	Cells with aberrations excluding gaps	Significance §	Mitotic index (mean)
Solvent	A	100	1	0		5.8
	B	100	1	0		4.0
	Totals	200	2	0		(4.9)
120.2	A	100	0	0		5.8
	B	100	5	0		4.8
	Totals	200	5	0	NS	(5.3)
171.8	A	100	5	1		4.9
	B	100	1	0		4.6
	Totals	200	6	1	NS	(4.8)
245.4	A	100	3	2		1.6
	B	100	1	0		3.2
	Totals	200	4	2	NS	(2.4)
MMS, 50	A	25	14	13
	B	25	18	17
	Totals	50	32	30	p <0.001
	§ Statistical significance
	NS = not significant

Table 3: Cells with structural aberrations

20 hour sampling time, + S-9

Treatment (µg/ml)	Replicate	Cells scored	Cells with aberrations including gaps	Cells with aberrations excluding gaps	Significance §	Mitotic index (mean)
Solvent	A	100	5	2		5.3
	B	100	1	0		4.1
	Totals	200	6	2		(4.7)
350,5	A	100	3	1		3.8
	B	100	5	1		3.5
	Totals	200	8	2	NS	(3.7)
500,8	A	100	4	2		2.9
	B	100	5	4		3.5
	Totals	200	9	6	NS	(3.2)
715,4	A	100	8	5		2.2
	B	100	5	3		2.1
	Totals	200	13	8	p<0.05	(2.2)
CPA, 25	A	25	11	7
	B	25	8	6
	Totals	50	19	13	p <0.001
	§ Statistical significance
	NS = not significant

Table 4: Cells with structural aberrations

44 hour sampling time, - S-9

Treatment (µg/ml)	Replicate	Cells scored	Cells with aberrations including gaps	Cells with aberrations excluding gaps	Significance §	Mitotic index (mean)
Solvent	A	100	3	2		4.5
	B	100	4	4		3.3
	Totals	200	7	6		(3.9)
245.4	A	100	6	4		4.2
	B	100	2	0		4.3
	Totals	200	8	4	NS	(4.3)

Table 5: Cells with structural aberrations

44 hour sampling time, + S-9

Treatment (µg/ml)	Replicate	Cells scored	Cells with aberrations including gaps	Cells with aberrations excluding gaps	Significance §	Mitotic index (mean)
Solvent	A	100	1	1		3.5
	B	100	3	1		3.8
	Totals	200	4	2		(3.7)
715,4	A	100	5	2		3.2
	B	100	2	1		1.6
	Totals	200	7	3	NS	(2.4)

Cultures treated with DMAPA in the absence and presence of metabolic activation resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls..

Under these experimental conditions, DMAPA did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without metabolic activation at any harvest time.

Conclusions:

Under these experimental conditions, DMAPA did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without metabolic activation at any harvest time.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read-across from related substance

Justification for type of information:

Data from the related substance DMAPA is used to cover this endpoint. Justification for the read-across approach is included in section 13.

Reason / purpose for cross-reference:

read-across source

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

4-nitroquinoline 1-oxide : 0.10 µg/ml / 0.15 µg/ml (without S-9 mix); Benzo[a] pyrene: 2.00 µg/ml / 3.00 µg/ml ( with S-9 mix)

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: all strains/cells tested

Conclusions:

No reliable in vitro gene mutation study with mammalian cells with the test substance is available. Data generated with the related substance DMAPA is used for endpoint coverage.
Under the conditions of the study, DMAPA did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments, in the absence or presence of a rat liver metabolic activation system (S9).
The same is assumed to be correct for the target substance.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N-Dimethyl-1,3-propanediamine
- Analytical purity: >= 99.8 %
- Lot/batch No.: 31740675LO

Target gene:

hypoxanthine phosporibosyl transferase (HPRT)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Range finder: 31.94; 63.88; 127.8; 255.5; 511.0; 1022 µg/ml (with and without S9 mix)
Experiment 1: 30; 60; 90; 120; 150; 180; 210; 240; 270; 300 µg/ml (with and without S9 mix)
Experiment 2: 50; 100; 150; 175; 200; 210; 220; 240; 260; 275 µg/ml (without S9 mix)
50; 100; 150; 200; 220; 240; 250; 260; 275; 300 µg/ml (with S9 mix)

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

4-nitroquinoline 1-oxide : 0.10 µg/ml / 0.15 µg/ml (without S-9 mix); Benzo[a] pyrene: 2.00 µg/ml / 3.00 µg/ml ( with S-9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days

SELECTION AGENT (mutation assays): 6TG, 15 µg/ml

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
There was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
The effects described above were reproducible.
Results that only partially satisfy the assessment criteria described above will be considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

In Experiment 1, ten concentrations, ranging from 30 to 300 µg/mL, were tested in the absence and presence of S 9. Seven days after treatment, the highest two concentrations tested in the absence of S-9 (270 and 300 µg/mL) and the highest concentration tested in the presence of S 9 (300 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition 60 µg/mL in the absence of S-9 and 60, 120 and 150 µg/mL in the presence of S-9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected. The highest concentrations analysed were 240 µg/mL in the absence of S 9 and 270 µg/mL in the presence of S 9, which gave 4% and 8% RS, respectively. It may be noted that in both the absence and presence of S-9, no concentration gave 10-20% RS. However, cultures treated at 210 and 240 µg/mL gave 28% and 21% RS, respectively and therefore both concentrations were analysed.
In Experiment 2, ten concentrations, ranging from 50 to 275 µg/mL in the absence of S-9 and from 50 to 300 µg/mL in the presence of S-9, were tested. Seven days after treatment all concentrations were plated. However, following survival scoring, the highest two concentrations tested in the absence of S 9 (260 and 275 µg/mL) and the highest concentration tested in the presence of S-9 (300 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, in the presence of S-9, 220 µg/mL was not selected as there were sufficient non-toxic concentrations. The highest concentrations analysed were 240 µg/mL in the absence of S 9 and 275 µg/mL in the presence of S 9, which gave 17% and 16% RS, respectively.
Osmolality and pH
At the highest concentration tested in the cytotoxicity Range-Finder Experiment (1022 µg/mL), no marked changes in osmolality, compared to the concurrent vehicle controls, were observed but a marked increase in pH (of ≥1 unit, compared to the concurrent vehicle controls) was observed at 511 µg/mL and above in the absence and presence of S-9. Further pH measurements were therefore made in Experiments 1 and 2.
In Experiment 1, marked increases in pH (of ≥1 unit, compared to the concurrent vehicle controls) were observed at 300 µg/mL in the absence and presence of S-9.
In Experiment 2, marked increases in pH (of ≥1 unit, compared to the concurrent vehicle controls) were observed at 260 µg/mL and above in the absence of S-9.
The individual data for osmolality and pH are not reported.

Remarks on result:

other: all strains/cell types tested

Experiment 1 (3 hours treatment in the absence and presence of S-9)

Treatment (mg/mL)		-S-9				Treatment (mg/mL)		+S-9
		%RS	MF§					%RS	MF§
0		100	6.31			0		100	4.39
30		94	5.22		NS	30		96	4.55		NS
90		74	4.05		NS	90		88	5.51		NS
120		64	4.29		NS	180		75	4.67		NS
150		47	5.60		NS	210		53	5.40		NS
180		34	5.38		NS	240		21	5.22		NS
210		28	5.31		NS	270		8	5.15		NS
240		4	6.27		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		53	36.93			2		76	38.36
0.15		40	39.65			3		48	59.43

Experiment 2 (3 hours treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		%RS	MF§					%RS	MF§
0		100	3.31			0		100	2.51
50		89	2.20		NS	50		102	2.75		NS
100		85	3.55		NS	100		102	4.03		NS
150		71	3.32		NS	150		89	1.55		NS
175		60	4.83		NS	200		70	2.51		NS
200		43	3.37		NS	240		30	2.33		NS
210		34	3.74		NS	250		28	2.22		NS
220		30	2.28		NS	260		31	2.83		NS
240		17	2.51		NS	275		16	3.47		NS
Linear trend				NS		Linear trend				NS
NQO						B[a]P
0.1		61	18.78			2		47	37.83
0.15		50	35.13			3		30	71.65

§ 6 -TG resistant mutants/ 106 viable cells 7 days after treatment

%RS Percent relative survival adjusted by post treatment cell counts

NS Not significant

Conclusions:

It is concluded that N,N-Dimethyl-1,3-propanediamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments, in the absence or presence of a rat liver metabolic activation system (S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three reliable in vitro genetic toxicity studies are available.

Bacterial reverse mutation assay:

BASF (1998) performed an Ames (plate incorporation assay and preincubation test) test with S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli WP2 with and without metabolic activation in presence of the test substance.

 

Following test concentrations were applied: 0, 20, 100, 2500 and 5000 µg/plate (in triplicate) with and without metabolic activation (S9 -mix) using the standard plate incorporation assay for all strains, and 2000, 3000, 4000, 5000 and 6000 µg/plate (with and without S9 -mix) for S. typhimurium strain TA100 and E. coli WP2 uvrA. Solvent control, negative control and positive controls were run in triplicate. Following cytotoxic effects were observed in Salmonella strains: A weak bacteriotoxic effect in the standard plate test at 6000 µg/plate (TA 100). In the preincubation assay bacteriotoxicity was found depending on the strain and test conditions at doses >= 500 µg/plate. Following cytotoxic effects were observed in E. coli WP2 uvrA: toxic effect in the standard plate test at 6000 µg/plate. In the preincubation assay bacteriotoxicity was found at doses >= 2500 µg/plate. Solvent, negative and positive controls were valid.

 

Cytogenicity test (read-across):

 

A chromosome aberration test was performed with the related substance DMAPA with human lymphocytes with and without metabolic activation (Mc Enaney, 1991). Following doses were tested in duplicate: without metabolic activation: 120.2, 171.8, 245.4 µg/ml for 20 hours sampling time and 245.4 µg/ml for 44 hours sampling time; with metabolic activation: 350.5, 500.8, 715.4 µg/ml for 20 hours sampling time and 715.4 µg/ml for 44 hours sampling time. Cultures treated with DMAPA in the absence and presence of metabolic activation resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls. Under these experimental conditions, DMAPA did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without metabolic activation at any harvest time.

 

Mammalian cell gene mutation assay (read-across):

 

A mammalian cell gene mutation assay has been performed in a mouse lymphoma test with the related substance DMAPA in L5178Y cells with and without metabolic activation (Covance Laboratories Ltd). Following doses were tested in duplicate:

Range finder: 31.94; 63.88; 127.8; 255.5; 511.0; 1022 µg/ml (with and without S9 mix)

Experiment 1: 30; 60; 90; 120; 150; 180; 210; 240; 270; 300 µg/ml (with and without S9 mix)

Experiment 2: 50; 100; 150; 175; 200; 210; 220; 240; 260; 275 µg/ml (without S9 mix)

                      50; 100; 150; 200; 220; 240; 250; 260; 275; 300 µg/ml (with S9 mix)

It is concluded that DMAPA did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments, in the absence or presence of a rat liver metabolic activation system (S‑9).

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance should not be classified for mutagenicity.