Amides, coco, N-[3-(dimethylamino)propyl], N-oxides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 28, 2000 to March 25, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD guideline 471 and EU guideline B14. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amides, coco, N-[3-(dimethylamino)propyl], N-oxides

EC Number:

268-938-5

EC Name:

Amides, coco, N-[3-(dimethylamino)propyl], N-oxides

Cas Number:

68155-09-9

Molecular formula:

Unspecified

IUPAC Name:

Amides, coco, N-[3-(dimethylamino)propyl], N-oxides

Details on test material:

- Purity: approximately 79%
- Lot/batch No.: 876 TK
- Physical state: off-white paste
- Storage condition of test material: room temperature, in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Stepan Company
- Batch No.of test material: 876 TK
- Date received: 10 May 1999
- Description: off-white paste

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was accurately weighed and approximately half-log dilutions prepared in dimethyl formamide. An allowance for test material purity (77%) was made prior to each days formulation.

Method

Target gene:

his and trp

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment 1:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
15, 50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A

Experiment 2:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 98 and TA 100
50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537

Experiment 3:
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537

Justification:
In order to select an appropriate dose level for use in the main study, a preliminary test was carried out to determine the toxicity of the test material.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Dimethyl formamide is an acceptable vehicle for use in the Ames assay.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

(Dimethyl formamide)

Controls 2

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA

Controls 3

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Controls 4

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA98

Controls 5

Positive controls:

yes

Remarks:

(with metabolic activation)

Positive control substance:

other: 2-aminoanthracene

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP uvrA

Controls 6

Positive controls:

yes

Remarks:

(with metabolic activation)

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Evaluation criteria:

The test material was considered positive in this test system if there is a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at and above 200 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at and above 500 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance was noted at the dose levels tested.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity study was performed with strains TA100 and WPuvrA with and without metabolic activation. Ten concentrations of the test material and a vehicle control (dimethyl formamide) were tested. The dose range of the test material was: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number, within the range of historical data, of spontaneous revertants per plate in the vehicle and untreated controls.

Any other information on results incl. tables

Experiment 1

 

Table 1: without metabolic activation

With or without S9-Mix	Test substance concentration µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type    TA100             TA1535        WP2uvrA			Frameshift type         TA98         TA1537
-	0	146 10.1 #	22 2.9	19 3.1	20 6.1	9 0.6
-	5	144 12.5	20 3.1	N/T	20 3.8	10 1.5
-	15	145 11.4	14 4.2	23 3.8	25 2.3	14 4.4
-	50	143 7.6	12 2.1	16 7.0	28 8.4	16* 3.8
-	150	112 4.0	13 2.3	24 3.1	25 4.0	18$$$ 2.3
-	500	23 3.2	3 2.0	22 9.3	6 2.0	3V 1.7
-	1500	0V 0.0	0V 0.0	20 3.5	0V 0.0	0T 0.0
-	5000	N/T	N/T	0V 0.0	N/T	N/T
Positive controls	Name	ENNG	ENNG	ENNG	4NQO	9AA
S9-Mix -	Concentration (µg/plate)	3	5	2	0.2	80
	Nº Colonies per plate	425 56.4	372 1.5	615 77.6	111 12.3	807 226.2

 

ENNG  N-ethyl-N'-nitro-N-nilrosoguanidine N/T  Not tested at this dose level

4NQO  4-Nitroquinoline-l-oxide                                   S  Sparse bacterial background lawn

9AA    9-Aminoacridine                                           T  Toxic, no bacterial background lawn

V        Very weak bacterial background lawn            $$$ p≤0.005

#         Standard deviation                                         *    p≤ 0,05         

 

 Table 2: with metabolic activation 

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type   TA100           TA1535      WP2uvrA			Frameshift type     TA98               TA1537
+	0	151 18.6#	13 2.1	22 0.0	24 3.6	7 1.0
+	5	174 51.0	14 1.5	N/T	21 3.2	12 2.0
+	15	132 13	11 5.1	22 1.2	29 2.5	9 3.1
+	50	144 11	12 2.1	19 4.5	30 8.3	13 5.3
+	150	154 14.6	14 1.7	23 1.0	27 3.6	13 4.2
+	500	38 5.3	5 4.0	27 6.1	17 7.2	4 1.7
+	1500	0V 0.0	0V 0.0	20 5.5	0V 0.0	0V 0.0
+	5000	N/T	N/T	0V 0.0	N/T	N/T
Positive controls S9-Mix +	Name Concentration µg/plate) Nº colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		2122 505.5	212 41.0	479 7.6	231 25.4	546 60.5

 BP        Benzo(a)pyrene                                        N/T   Not testedat this dose level

2AA     2-Aminoanthracene                                      S      Sparse bacterial background lawn

V          Very weak bacterial background lawn           #      Standard deviation

*           p≤ 0,05

 

 Experiment 2

 

Table 3: without metabolic activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type    TA100           TA1535     WP2uvrA			Frameshift type        TA98           TA1537
-	0	81 12.3#	19 1.7	18 6.1	15 2.3	9 5.3
-	5	89 1.0	16 3.2	N/T	14 2.1	N/T
-	15	79 15.0	15 6.2	N/T	14 1.5	9 1.2
-	50	85 3.2	22 7.0	20 9.8	16 2.3	10 3.8
-	100	N/T	N/T	N/T	N/T	13 3.2
-	150	76 9.6	15 1.2	17 3.8	15 3.1	9 4.4
-	200	N/T	N/T	N/T	N/T	6 1.5
-	500	10S 6.0	3S 1.0	16 3.8	6S 3.1	0V 0.0
-	1500	0V 0.0	0V 0.0	14 3.6	0V 0.0	N/T
-	5000	N/T	N/T	2 1.7	N/T	N/T
Positive controls S9-Mix -	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	No. colonies per plate	359 12.2	269 35.5	817 40.6	89 8.4	940 164.2

 

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine                  N/T   Not tested at this dose level

4NQO   4-Nttroquinoline-l-oxide                                   S      Sparse bacterial background lawn

9AA      9-Aminoacridine                                            V      Very weak bacterial background lawn

#          Standard deviation

 

Table 4: with metabolic activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type   TA100        TA1535       WP2uvrA			Frameshift type TA98        TA1537
+	0	93 4.0#	12 4.5	21 2.6	22 3.1	10 4.2
+	5	97 8.2	13 2.3	N/T	23 4.6	N/T
+	15	83 11.8	12 1.7	N/T	17 3.5	11 3.1
+	50	91 12.1	12 1.0	20 2.5	29 9.2	10 2.0
	100	N/T	N/T	N/T	N/T	13 1.7
+	150	82 12.7	13 2.1	20 3.5	31 12.4	17 6.6
+	200	N/T	N/T	N/T	N/T	11 4.6
+	500	30 10.8	4S 1.7	19 5.3	11S 4.2	5S 2.5
+	1500	0V 0.0	0V 0.0	13 3.1	0V 0.0	N/T
+	5000	N/T	N/T	2S 2.1	N/T	N/T
Positive controls S9-Mix +	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	No. colonies per plate	929 68.6	128 36.0	708 24.3	174 36.3	435 56.5

 

BP       Benzo(a)pyrene                      N/T   Not tested at this dose level

2AA     2-Aminoanthracene                  S      Sparse bacterial background lawn

#         Standard deviation                    V      Very weak bacterial background lawn

 

 

Experiment 3

Table 5: with and without metabolic activation

Test substance concentration µg/plate)	Number of revertants (mean number of colonies per plate)
	Frameshift type (Without metabolic activation) TA1537	Frameshift type (With metabolic activation) TA1537
0	8 3.2#	12 3.1
15	9 2.0	11 4.2
50	10 1.5	13 2.6
100	9 0.7	13 4.5
150	11 3.2	13 3.5
200	2S 1.7	14 4.0
500	0V 0.0	10S 7.8
Name	9AA	2AA
Concentration (µg/plate)	80	2
No. colonies per plate	940 87.0	399 137.2

9AA     9-Aminoacridine

2AA     2-Arninoanthracene

S         Sparse bacterial background lawn

V         Very weak bacterial background lawn

#          Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents bacteria, four strains of Salmonella typhimurium T1535, TA98, TA100, and TA1537 and E.Coli WP2 uvrA. Plate Incorporation Method was carried out at the concentrations of 5000, 1500, 500, 150, 50 and 15 µg/plate for the strains of Salmonella typhimurium in the presence and absence of metabolic activation and at the concentrations of 1500, 500, 150, 50, 15 and 5 µg/plate for the E.Coli WP2 uvrA in the presence and absence of metabolic activation.The dose range for TA1537 (with/without S9) was modified slightly, based on results observed in the Experiment 1and was15, 50, 100, 150, 200 and 500 µg/plate. Intermediate doses were included to allow for small but statistically significant increases observed in the first experiment. The test material caused a visible reduction in the growth of the bacterial lawn to all of the bacterial tester strains both with and without metabolic activation. The toxicity of the test material to the bacterial tester strains varied both between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Very small, statistically significant increases were observed to TA1537 in Experiment 1 only both with and without S9. However, these increases were not considered to be biologically significant as they were within the strains historical range and were non-reproducible over three separate experiments. According to the obtained results, the test substance was, therefore, considered to be non-mutagenic under the conditions of this test.