Amides, tall-oil fatty, N-[3-(dimethylamino)propyl]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 October 2007 - 26 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

Amides, C18-unsatd., N-[3-(dimethylamino)propyl]

EC Number:

800-353-8

Cas Number:

1379524-06-7

Molecular formula:

C23H46N2O, C23H44N2O, C23H42N2O C18-unsat DMAPA condensation product is including the substance with 1, 2 and 3 unsaturations in CIS position.

IUPAC Name:

Amides, C18-unsatd., N-[3-(dimethylamino)propyl]

Test material form:

other: liquid

Details on test material:

Name of test material (as cited in study report): 3-Dimethyl-aminopropyl-ölsäureamide
- CAS Number : 109-28-4
- Physical state: Reddish liquid
- Analytical purity: 78.2 area %
Composition by GC-MS :
• cis-Octadecenoic acid, 3-dimethylaminopropyl amide 78-80 %
• trans-Octadecenoic acid, 3-dimethylaminopropyl amide 0.5 - 2 %
• C16-acid, 3-dimethylaminoproyl amide, 4 isomers 8 - 12 %
• Other carbonic acids, 3-dimethylaminopropyl amide 6-8 %
• Other carbonic acids, 3-dimethylaminopropyl amide 1-2%

- Lot/batch No.: R 401/57
- Storage condition of test material: Ambient (room temperature); under light exclusion; under Nitrogen

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal Essential Medium (MEM)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (0.75 mg/mL final concentration); S9 fraction was obtained from the liver of rats treated with Phenobarbital i.p. (80 mg/kg bw) and ß-Naphthoflavone p.o.

Test concentrations with justification for top dose:

Range finding study: 0.3, 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0 and 150.0 µg/mL; with and without S9 mix (4 h exposure) and without S9 mix (24 h exposure). In earlier range finding study, 39.1 to 5000 µg/mL (with and without S9 mix) was tested and results showed strong cytotoxicity and test item precipitation observed up to 5000 µg/mL.

Based on the results of range finding study, the dose levels selected for main study as follows:

Main study:
- Experiment I* (without S9 mix; 4 h exposure and 18 h preparation interval): 0.7, 1.3, 2.5, 5.0**, 10.0** and 20.0** µg/mL
- Experiment I* (with S9 mix; 4 h exposure and 18 h preparation interval): 0.7, 1.3, 2.5, 5.0, 10.0 and 20.0 µg/mL
- Experiment I (without S9 mix; 4 h exposure and 18 h preparation interval): 1.3, 2.5, 5.0, 10.0, 20.0, 40.0**, 80.0** and 160.0** µg/mL
- Experiment I (with S9 mix; 4 h exposure and 18 h preparation interval): 1.3, 2.5, 5.0, 10.0, 20.0**, 40.0**, 80.0** and 160.0** µg/mL
- Experiment IIA (without S9 mix; 18 h exposure and 18 h preparation interval): 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 µg/mL
- Experiment IIA (without S9 mix; 28 h exposure and 28 h preparation interval): 1.25, 2.5, 5.0 and 10.0 µg/mL
- Experiment IIA (with S9 mix; 4 h exposure and 28 h preparation interval): 2.5, 5.0, 10.0, 20.0**, 40.0** and 80.0** µg/mL
- Experiment IIB (with S9 mix; 4 h exposure and 28 h preparation interval): 10.0, 15.0, 20.0, 25.0, 30.0** and 35.0** µg/mL

* repeated due to missing cytotoxicity; ** precipitation occurred 4 h after start of treatment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5 % (v/v).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol 0.5 % (v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate at 500-900 µg/mL

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol 0.5 % (v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide at 1.4-2.0 µg/mL

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
- Thawed stock cultures were propagated at 37 °C in 80 cm^2 plastic flasks. About 5 x 10^5 cells per flask were seeded into 15 mL of MEM supplemented with 10 % fetal calf serum. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

METHOD OF APPLICATION: In MEM with 10 % FCS (complete medium)

DURATION
- Exposure duration: Experiment I: 4 h (±S9); Experiment IIA: 18 h (-S9) and 28 h (-S9); Experiment IIA and IIB: 4 h (+S9)
- Fixation time (start of exposure up to harvest of cells): Experiment I: 18 h (±S9); Experiment IIA: 18 h (-S9) and 28 h (-S9); Experiment IIA and IIB: 28 h (+S9)

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of colcemid at 0.2 µg/mL culture medium, 2.5 h before the harvest.

STAIN (for cytogenetic assays): Giemsa staining

NUMBER OF REPLICATIONS: Duplicate cultures for test item, vehicle and positive control groups

NUMBER OF CELLS EVALUATED:
- Cytotoxicity of the test item was evaluated using the mitotic index (% cells in mitosis), which indicates whether an item induces mitotic inhibition. The mitotic index was determined in a sample of 1000 cells per culture of each test group.
- At least 100 metaphases/culture (with 22 ± 1 chromosomes) were scored for chromosomal aberrations. In addition the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps) and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps) and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical control data range (0.0 - 5.2 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 389 mOsm, pH 7.3 versus 407 mOsm and pH 7.4 at 150 µg/mL).

RANGE-FINDING/SCREENING STUDY:
Clear toxic effects were observed after 4 h treatment with 9.4 µg/mL and above in the absence of S9 mix and with 18.8 µg/mL and above in the presence of S9 mix. In addition, 24 h continuous treatment with 4.7 µg/mL and above in the absence of S9 mix induced strong toxic effects. Precipitation of the test item in culture medium was observed after 4 h treatment with 37.5 µg/mL and above in the absence and presence of S9 mix.

PRECIPITATION:
- Experiment I: In the absence of S9 mix, precipitation of the test item in culture medium was observed at preparation interval 18 h with 40 µg/mL & above and in the presence of S9 mix at preparation interval 18 h with 20 µg/mL.
- Experiment IIA: In the presence of S9 mix, precipitation of the test item in culture medium was observed at preparation interval 28 h with 20 µg/mL and above.
- Experiment IIB: In the presence of S9 mix, precipitation of the test item in culture medium was observed at preparation interval 28 h with 30 µg/mL and above.

CYTOTOXICITY:
- Experiment I: Cytotoxicity was observed at 20 µg/mL and above without S9 mix as well as with S9 mix at 40 µg/mL.
- Experiment IIA: Cytotoxicity was observed in the absence of S9 mix after 18 h continuous treatment with 5 µg/mL (1.7 % of control) and after 28 h continuous treatment with 2.5 µg/mL (7.3 % of control).
- Experiment IIB: Cytotoxicity was observed in the presence of S9 mix after 4 h treatment with 30 µg/mL (35.9 % of control) at preparation interval 28 h.
-In addition, reduced mitotic indices were observed after 18 h continuous treatment with 5 µg/mL (33.9 % of control) in Experiment IIA in the absence of S9 mix and after 4 h treatment with 25 µg/mL (49.1 % of control) in the presence of S9 mix at preparation interval 28 h.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with the historical data (2005 - 2006) of the laboratory.

Remarks on result:

other: strain/cell type: Chines hamster V79 cell line

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/ 2: Summary of results of the chromosome aberration study with Trennmittel ZM 121

Experiments	Preparation interval	Test item concentration (µg/mL)	Polyploid cells (%)	Cell numbers in % of control	Mitotic indices in % of control	Aberrant cells
						incl. gaps*	in % excl. gaps*	with exchanges
Exposure period 4 h without S9 mix
I	18 h	Solvent control^1	2.4	100.0	100.0	5.0	3.5	0.5
		Positive control^2	2.4	NT	96.3	10.5	10.0**	5.5
		2.5	2.5	73.0	115.0	3.0	2.5	0.0
		5.0	2.4	92.9	80.0	4.5	3.5	2.0
		10.0	1.5	73.0	65.0	4.0	2.5	0.5
Exposure period 18 h without S9 mix
IIA	18 h	Solvent control^1	2.4	100.0	100.0	2.5	2.5	0.0
		Positive control^3	2.7	NT	72.1	15.0	13.5**	5.5
		1.25	3.0	98.6	104.9	3.5	3.0	0.0
		2.5	2.3	76.3	82.5	3.5	2.5	0.5
		5.0	1.7	1.7	33.9	3.0	3.0	0.0
Exposure period 28 h without S9 mix
IIA	28 h	Solvent control^1	2.9	100.0	100.0	2.5	1.5	0.0
		Positive control^3	2.8	NT	90.0	17.5	16.5**	8.5
		1.25	3.2	86.9	110.8	2.0	1.0	0.0
		2.5	2.3	7.3	101.9	1.5	1.0	0.0
Exposure period 4 h with S9 mix
I	18 h	Solvent control^1	3.0	100.0	100.0	3.0	2.5	0.0
		Positive control^4	2.9	NT	64.0	11.5	11.0**	3.0
		2.5	3.0	110.3	98.7	2.5	2.0	0.0
		5.0	2.8	113.3	77.3	2.5	2.0	1.0
		10.0	4.1	93.6	83.6	2.0	2.0	0.5
IIA	28 h	Solvent control^1	1.7	100.0	100.0	2.0	2.0	0.0
		Positive control^5	2.1	NT	106.4	13.5	13.0**	3.5
		5.0	2.2	91.5	82.3	2.5	2.5	0.5
		10.0	2.4	91.3	97.7	3.0	2.0	0.5
		20.0^P	2.6	72.7	75.8	6.0	5.5**	1.3
IIB	28 h	Solvent control^1	1.4	100.0	100.0	2.5	2.5	0.0
		Positive control^5	1.5	NT	88.2	10.0	10.0**	2.5
		20.0	1.8	96.0	106.6	1.5	1.0	0.0
		25.0	1.5	58.6	49.1	1.0	1.0	0.0
		30.0^P	2.5	35.9	66.2	4.0	4.0	1.0

 

* Inclusive cells carrying exchanges; NT- Not tested; P-Precipitation occurred; **- Aberration frequency statistically significant higher than corresponding control values

 

1: Ethanol 0.5 % (v/v); 2: EMS 900 µg/mL; 3: EMS 500 µg/mL; 4: CPA 1.4 µg/mL; 5: CPA 2 µg/mL

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Trennmittel ZM 121 is not considered as clastogenic in Chinese hamster V79 cell lines with and without metabolic activation.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured Chinese hamster V79 cells were exposed to N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) at the following concentrations:

Range finding study: 0.3, 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0 and 150.0 µg/mL; with and without S9 mix (4 h exposure) and without S9 mix (24 h exposure). In earlier range finding study, 39.1 to 5000 µg/mL (with and without S9 mix) was tested and results showed strong cytotoxicity and test item precipitation observed up to 5000 µg/mL. Based on the results of range finding study, the dose levels selected for main study as follows:

Main study:

- Experiment I (without S9 mix; 4 h exposure + 14 h recovery period): 0.7, 1.3, 2.5, 5.0, 10.0 and 20.0 µg/mL

- Experiment I (with S9 mix; 4 h exposure + 14 h recovery period): 0.7, 1.3, 2.5, 5.0, 10.0 and 20.0 µg/mL

- Experiment I (without S9 mix; 4 h exposure + 14 h recovery period): 1.3, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 µg/mL

- Experiment I (with S9 mix; 4 h exposure + 14 h recovery period): 1.3, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0 µg/mL

- Experiment IIA (without S9 mix; 18 h exposure without recovery period): 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 µg/mL

- Experiment IIA (without S9 mix; 28 h exposure without recovery period): 1.25, 2.5, 5 and 10 µg/mL

- Experiment IIA (with S9 mix; 4 h exposure and 24 h recovery period): 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 µg/mL

- Experiment IIB (with S9 mix; 4 h exposure and 24 h recovery period): 10.0, 15.0, 20.0, 25.0, 30.0 and 35.0 µg/mL

 

Mitotic activity was arrested by addition of colcemid at 0.2 µg/mL culture medium, 2.5 h before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was 0.75 mg/mL (final concentration). S9 fraction was obtained from the liver of rats treated with Phenobarbital i.p. (80 mg/kg bw) and and ß-Naphthoflavone p.o.

In Experiment I, without S9 mix precipitation of the test item in culture medium was observed at preparation interval 18 h with 40 µg/mL & above and with S9 mix at preparation interval 18 h with 20 µg/mL. In Experiment IIA, with S9 mix precipitation of the test item in culture medium was observed at preparation interval 28 h with 20 µg/mL and above. In Experiment IIB, with S9 mix precipitation of the test item in culture medium was observed at preparation interval 28 h with 30 µg/mL and above. In Experiment I, cytotoxicity was observed at 20 µg/mL and above without S9 mix as well as with S9 mix at 40 µg/mL. In Experiment IIA, cytotoxicity was observed in the absence of S9 mix after 18 h continuous treatment with 5 µg/mL (1.7 % of control) and after 28 h continuous treatment with 2.5 µg/mL (7.3 % of control). In Experiment IIB, cytotoxicity was observed in the presence of S9 mix after 4 h treatment with 30 µg/mL (35.9 % of control) at preparation interval 28 h. In addition, reduced mitotic indices were observed after 18 h continuous treatment with 5 µg/mL (33.9 % of control) in Experiment IIA in the absence of S9 mix and after 4 h treatment with 25 µg/mL (49.1 % of control) in the presence of S9 mix at preparation interval 28 h. In Experiment I, in the absence and the presence of S9 mix and in Experiment IIA, in the absence of S9 mix no clastogenicity was observed up to the highest evaluable concentrations. In contrast, in Experiment IIA, in the presence of S9 mix the number of aberrant cells, excluding gaps was statistically significantly increased at the highest evaluated concentration (20 µg/mL); this value (5.5 % aberrant cells, excluding gaps) slightly exceeded the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps). In the confirmatory Experiment IIB this finding could not be verified. No relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test item as compared to the frequencies of the controls. The positive control substances, ethylmethane sulfonate (500 or 900 µg/mL, without S9 mix) and cyclophosphamide (1.4 or 2.0 µg/mL, with S9 mix) gave the expected statistically significant increases in the number of cells with structural chromosome aberrations, which demonstrates the sensitivity of the test system.

 

Under the test conditions, N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) is not considered as clastogenic in Chinese hamster V79 cell lines with and without metabolic activation.