Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro studies:

The test substance did not show genotoxic potential in an in vitro Mammalian Cell Gene Mutation Test, in the Ames test, in an in vitro Mammalian Chromosome Aberration Test as well as in an Unscheduled DNA Synthesis Test in Mammalian Cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
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
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

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:

- 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.

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

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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
The potential of Dimethylaminopropylamine (DMAPA) to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 1538) was evaluated in accordance with the international guidelines (OECD 471) and in compliance with the principles of GLP. DMAPA was tested in two independent experiments, with and without a metabolic activation system, according the preincubation method (60 min at 37°C). Bacterias were exposed to DMAPA at five dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 0, 50, 100, 250, 500 and 750 μg/plate. After 48 hours of incubation at 37°C, the revertant colonies were scored.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254-induced rat liver (10%)
Test concentrations with justification for top dose:
Bacterial toxicity: 100, 250, 500, 750 µg/plate (TA98) and 500, 1000, 5000, 10000 µg/plate (TA100)
Genotoxicity: 0, 50, 100, 250, 500, 750 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 : Na azide: 5 pg/plate (TA 100, TA1535), 2-nitrofluorene: 5 pg/plate (TA98, TA 1538), 9-aminoacridine: 100 pg/plate (TA 1537). + S9: 2-aminoanthracene: 5 pg/plate
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY (preliminary range-finding test)
- Test: in TA 98 and TA 100 strains, with or without S9 mix ; 4 dose-levels (three plates/dose level)
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn);

METHOD OF APPLICATION:
- Preincubation
DURATION
- Preincubation period: 60 min
- Exposure duration: 48H
NUMBER OF REPLICATIONS: triplicates
Evaluation criteria:
Reproducible increase in the number of revertant colonies compared with vehicle controls in any strain at any dose-level and/or evidence of a dose-relationship. Reference to historical data and consideration to biological relevance may also be taken into account.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
As seen in the preliminary tests, cytotoxicity was observed for the highest doses (500 µg/plate and above) with and without metabolic activation in all strains.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo study:

The test substance was not genotoxic in the Mammalian Erythrocyte Micronucleus Test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:

- Lot/batch No.: 98/390-5
- Expiration date of the lot/batch: March 15, 2000
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd.; Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 43.6 g (SD ± 2.4 g), females mean value 32.8 g (SD ± 1.8 g)
- Assigned to test groups randomly: yes
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 18 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
Duration of treatment / exposure:
24 h or 48 h
Frequency of treatment:
single application
Post exposure period:
24 h or 48 h
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
48 h preparation interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (40 mg/kg bw)
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
As estimated in the pre-experiments 100 mg 3-dimethylaminopropylamine per kg b.w. was suitable, i.e suitable to the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
A small drop of the cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was deterrnined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and biologically relevant positive response.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The mean numbers of normochromatic erythrocytes at the highest dose (100 mg/kg b.w.) of the test item was clearly increased
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 150, 250 and 500 mg/kg bw
- Clinical signs of toxicity in test animals: death (150 mg/kg bw onwards)
- Harvest times: 24 h and 48 h

Summary of Micronucleus Test Results:

 Test group  Dose: mg/kg bw  Sampling time (h)

 PCEs with micronuclei (%)

 Range*  PCE / NCE
 Vehicle  24  0.145  0 - 6  2000 / 1802
 Test item  25  24  0.055  0 - 5  2000 / 1965
 Test item  50  24  0.080  0 - 5  2000 / 1738
 Test item 100   24  0.160  0 - 8  2000 / 2673
Cyclophosphamide  40  24  1.530  12 - 42  2000 / 2180
Vehicle  0  48  0.07  0 - 5  2000 / 1794
 Test item  100  48  0.04  0 - 3  2000 / 2067

*: Number of micronucleated PCEs

The mean numbers of normochromatic erythrocytes at the highest dose (100 mg/kg b.w.) of the test item was clearly increased (2673 and 2067 NCEs per 2000 PCEs for the 24 and 48 h sampling times, respectively) as compared to the mean values of NCEs of the vehicle controls (1802 and 1794 NCEs per 2000 PCEs for the 24 and 48 h sampling times, respectively). This indicates a test item specific toxic effect in the bone marrow of the treated mice. At all other doses and sampling times no relevant changes in the NCE/PCE ratio were observed. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 3-Dimethylaminopropyl were below or near to the values of the vehicle control groups.

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies:

Several in-vitro genetic toxicity studies are available: One HPRT test in mouse lymphoma L5178Y cells (of validity 1 [Covance-8218073]),5 Ames tests (1 of validity 1 [Sanofi Recherche-1988], 3 of validity 2 [Zeiger-1987, Huntingdon-1980, and Huntsman-PH 301-TX-008-81] and 1 of validity 3 [Murphy-Corb-1983], 1 chromosome aberration test in human lymphocytes (of validity 1 [Hazleton Microtest-1991]), and 1 UDS test in rats hepatocytes (of validity 1 [Huntsman-030882]). In the HPRT test conducted according to the OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test), mouse lymphoma L5178Y cells were treated with test substance concentration (using water as vehicle) ranging from 31.94 to 1022 µg/ml (range-finding study) or 30 to 300 µg/ml (main study), with and without metabolic activation. 4-nitroquinoline 1-oxide (0.10 µg/mL / 0.15 µg/mL; without S-9 mix) and benzo[a] pyrene (2.00 µg/mL / 3.00 µg/mL; with S-9 mix) were used as positive control. For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if it induces a 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), and the effects described above were reproducible. The test substance 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). In the publication of Zeiger the test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. DMAPA doses of 0, 33, 100, 333, 1000, 1666, 3333, 6666, and 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate. A slight clearing of the background lawn was reported at this dose level when tested in the presence of hamster liver S9. A second Ames-test (Sanofi-1988) with the following Salmonella typhimurium strains: TA 1535, TA 1537, TA 98, TA 100 and TA 1538) was performed in accordance with the international guidelines (OECD 471) and in compliance with the principles of GLP. DMAPA was tested in two independent experiments, with and without a metabolic activation system, according the preincubation method (60 min at 37°C). Bacteria were exposed to DMAPA at five dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 0, 50, 100, 250, 500 and 750 μg/plate. After 48 hours of incubation at 37°C, the revertant colonies were scored. No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation on the 5 tested strains. 3 further Ames-tests confirmed these negative results. In an in vitro cytogenetics assay using human lymphocyte cultures (Hazleton-1991), the test substance was tested both in the absence and presence of metabolic activation (S9 mix), according to the OECD Guideline 473 and the EC 92/69/EEC B.10 guideline, in compliance with the principles of GLP. With metabolic activation, lymphocytes cultures were exposed for 3 hours to solvent vehicle or test substance 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 test substance at 0, 120.2, 171.8, 245.4 µg/mL. 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 11 µg/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. Cultures treated with test substance 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. The test substance was also tested negative in the UDS test (conducted similar to the OECD Guideline 482, with some restrictions).  

In vivo studies:

In a micronucleus assay with NMRI mice according to OECD TG 474 (RCC-2000), 5 male and 5 female animals per group were orally administered single doses of of  25, 50, and 100 mg/kg bw (24 h preparation interval) and 100 mg/kg bw (48 h preparation interval). The mean number of normochromatic erythrocytes (NCE) was increased after treatment with the test substance at the highest dose as compared to the mean value of NCE of the vehicle control indicating that the substance had cytotoxic properties in the bone marrow. There was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. It can be stated that during the study described and under the experimental conditions reported, the test substance in bone marrow cells of the mouse is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.