Amides, C16-18, N-[3-(dimethylamino)propyl]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2012-08-15 to 2012-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(31 May 2008)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted July 21, 1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7651-02-7 (Stearic acid 3-(dimethylaminopropyl)amide)

IUPAC Name:

7651-02-7 (Stearic acid 3-(dimethylaminopropyl)amide)

Details on test material:

- Chemical name: Stearic acid 3-(dimethylaminopropyl)amide

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Horse serum: inactivated by incubation at 56°C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium
For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information given
- Periodically "cleansed" against high spontaneous background: yes, prior to experiment

Additional strain / cell type characteristics:

other: thymidine kinase deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1
Without S9-mix: 0.003, 0.01, 0.03, 0.1, 0.3, 0.6, 1, 2.5, 5, 7.5 and 10 μg/mL exposure medium.
With 8% (v/v) S9-mix: 0.1, 0.6, 1, 5, 10, 20, 30, 40, 50, 60, 70 and 85 μg/mL exposure medium.

Experiment 2
Without S9-mix: 0.01, 0.03, 0.1, 0.3, 0.6, 1, 3, 4, 5, 6.5 and 7.5 μg/mL exposure medium.
With 12% (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 20, 30, 40, 50 and 60 μg/mL exposure medium.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Experiment 1:
Without S9-mix: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2.5 and 5 μg/mL exposure medium.
With S9-mix: 0.1, 0.6, 1, 5, 10, 20, 30 and 40 μg/mL exposure medium.

Experiment 2:
Without S9-mix: 0.01, 0.03, 0.1, 0.3, 0.6, 1 and 3 μg/mL exposure medium.
With S9-mix: 0.1, 1, 3, 10, 30, 40, 50 and 60 μg/mL exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol, final concentration of the solvent in the exposure medium was 0.4% (v/v)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Remarks:

15 µg/mL (3 h) and 5 μg/mL (24 h)

Positive control substance:

methylmethanesulfonate

other: (without metabolic activation)

Controls 2

Positive controls:

yes

Remarks:

10 μg/mL

Positive control substance:

cyclophosphamide

other: (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h / 24 h (without metabolic activation); 3 h with 8 or 12 % S9 mix
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 (cloning efficiency) or 12 days (mutant frequency)
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments, 1 replicate each

NUMBER OF CELLS EVALUATED:
8E06 cells (1E06 cells/mL) for 3 hours treatment, 5E06 cells (1.25E05 cells/mL) for 24 hours treatment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (1E06) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 1E06 survivors and ≤ 170 per 1E06 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 1E06 survivors, and for CP not below 700 per 1E06 survivors.

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix > 4 µg/mL, with S9 > 50 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation in the exposure medium at concentrations of 100 μg/mL and above.
The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 1000 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
After the 3 hours treatment, severe precipitate of the test substance was observed in the exposure medium at the highest dose levels of 333 and 1000 μg/mL. Therefore, no cell count was possible at these dose levels. In the absence of S9-mix, hardly any or no cell survival was observed at all test substance concentrations tested. In the presence of S9-mix, the relative suspension growth was 39% at the test substance concentration of 33 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test substance concentrations of 100 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range except in the absence of S9-mix, in which the mutation frequency of the solvent control cultures were just below the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test:
In the presence of S9-mix, the dose levels of 50 to 85 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test substance concentration was reduced by 84% compared to the total growth of the solvent controls in the absence and presence of S9-mix.
Second mutagenicity test:
In the absence of S9-mix, the dose levels of 4 to 7.5 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 81% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 60% compared to the total growth of the solvent controls.

Any other information on results incl. tables

Experiment 1:
					Mutation frequency per 1E06 survivors
Dose (µg/mL)	RSG (%)	CE day 2 (%)	RS day 2 (%)	RTG	Total	small	large
Without metabolic activation 3 hours treatment
SC1	100	131	100	100	48	15	32
SC2		123			48	16	32
0.003	101	93	73	74	61	23	36
0.01	110	101	79	87	64	19	43
0.03	119	118	93	110	59	22	35
0.1	118	95	75	89	58	22	34
0.3	114	104	82	93	51	19	30
1	89	135	106	95	48	15	31
2.5	74	78	61	45	84	33	49
5	23	88	69	16	56	28	27
MMS	80	51	40	32	840	192	578
With 8% (v/v) metabolic activation 3 hours treatment
SC1	100	104	100	100	82	22	57
SC2		95			85	23	59
0.1	94	94	94	89	89	26	60
0.6	97	110	110	108	74	24	47
1	102	83	83	85	97	34	60
5	47	95	96	45	88	42	42
10	79	74	74	59	119	42	72
20	57	104	104	59	69	30	36
30	44	86	87	38	101	26	72
40	19	84	84	16	107	32	71
CP	52	44	44	23	1502	395	892

 

 

Experiment 2:
					Mutation frequency per 1E06 survivors
Dose (µg/ml)	RSG (%)	CE day 2 (%)	RS day 2 (%)	RTG	Total	small	large
Without metabolic activation 24 hours treatment
SC1	100	95	100	100	59	33	25
0.01	108	88	93	100	55	38	16
0.03	103	102	107	110	68	40	25
0.1	96	93	98	94	72	43	26
0.3	89	115	121	107	48	24	22
0.6	105	95	100	105	69	17	50
1	97	101	106	103	69	23	43
3	21	89	94	19	74	23	49
MMS	100	88	93	93	527	197	271
With 12% (v/v) metabolic activation 3 hours treatment
SC1	100	95	100	100	92	48	40
SC2		78			104	72	29
0.1	101	86	100	101	90	64	23
1	101	85	98	99	57	34	21
3	109	120	138	151	46	32	13
10	102	84	97	99	125	64	55
30	78	93	107	83	98	44	49
40	77	97	112	86	67	31	33
50	56	85	98	55	130	55	67
60	31	111	129	40	90	51	34
CP	58	50	58	34	1056	430	487

 

Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

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

It is concluded that Stearic acid 3-(dimethylaminopropyl)amide is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 467, adopted July 21, 1997 (thymidine kinase (TK)), L5178Y mouse lymphoma cells cultured in vitro were exposed to Stearic acid 3-(dimethylaminopropyl)amide (100% purity) in ethanol in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

First experiment

Without S9-mix, 3 h treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2.5 and 5 μg/mL

With 8% S9-mix, 3 h treatment: 0.1, 0.6, 1, 5, 10, 20, 30 and 40 μg/mL

Second experiment

Without S9-mix, 3 h treatment: 0.01, 0.03, 0.1, 0.3, 0.6, 1 and 3 μg/mL

With 12% S9-mix, 24 h treatment: 0.1, 1, 3, 10, 30, 40, 50 and 60 μg/mL

Stearic acid 3-(dimethylaminopropyl)amide was tested up to cyctotoxic concentrations.The positive controls induced the appropriate response.There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.