1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C18(unsaturated) acyl derivs., hydroxides, inner salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

2-[dimethyl({3-[(10E,13E)-nonadeca-10,13-dienamido]propyl})azaniumyl]acetate

Molecular formula:

C25H47N2O3 (if R= C18:2)

IUPAC Name:

2-[dimethyl({3-[(10E,13E)-nonadeca-10,13-dienamido]propyl})azaniumyl]acetate

Test material form:

liquid

Specific details on test material used for the study:

The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Preliminary Toxicity Test - (a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 µg/plate
Initial mutation assay - (a) 8, (b) 25, (c) 80, (d) 253 and (e) 800 µg/plate
Confirmatory mutation assay - (a) 41, (b) 86, (c) 181, (d) 380 and (e) 800 µg/plate.

Vehicle / solvent:

One hundred microliters (100 µL) of DMSO was used as the vehicle control.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

- Source of the Test System:
-Salmonella typhimurium: Health Protection Agency, National Collection of Type, Cultures (NCTC), 61, Colindale Avenue, London NW9 5EQ, Great Britain
- Escherichia coli: The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, U.K.

- Storage of Test System
Stock cultures of tester strains were stored in Oxoid nutrient broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated at 2 to 8ºC. The master plates prepared on 14 March 2016 were used in the study.

- Genotypic Characterization of Test System
The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance of strains
TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates

Evaluation criteria:

- Evaluation and Interpretation : To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Results of Preliminary Toxicity Test
Treatment (mg/plate)	TA 100 revertant colonies/plate^*
	Presence of S9			Absence of S9
	Mean	Background lawn	Precipitation	Mean	Background lawn	Precipitation
DMSO (100mL)	114	4+	Nil	112	4+	Nil
50	108	4+	Nil	107	4+	Nil
100	103	4+	Nil	101	4+	Nil
200	72	4+	Nil	70	4+	Nil
400	56	3+	Nil	51	3+	Nil
800	46	3+	Nil	43	3+	Nil
1600	37	3+	Nil	33	3+	Nil
3200	9	1+	Nil	7	1+	Nil
5000	4	0	Nil	3	0	Nil

*: Mean of two replicates

4+ - Normal

3+ - Slightly reduced   

1+ - Severely reduced

0  - Absent

Table 2: Viable Counts of the Overnight Culture of the Tester Strains
Tester Strains	Viable Counts ( x 10^9CFU/mL*)
	Initial Mutation Assay	Confirmatory Mutation Assay
TA98	1.55	1.55
TA100	1.64	1.54
TA1535	1.53	1.52
TA1537	1.55	1.52
WP2uvrA (pKM101)	1.62	1.63

* Required Cell count: 1-2x10⁹Colony Forming Units (CFU)/mL

Table 3: Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation
Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	26	2	NA	116	1	NA	14	2	NA	11	2	NA	134	3	NA
8	26	2	1.00	114	1	0.98	13	2	0.93	10	2	0.91	130	3	0.97
25	28	1	1.08	112	1	0.97	11	2	0.79	11	2	1.00	127	3	0.95
80	26	2	1.00	112	2	0.97	14	1	1.00	12	1	1.09	129	2	0.96
253	21	2	0.81	71	2	0.61	9	2	0.64	7	1	0.64	105	3	0.78
800	14	1	0.54	44	4	0.38	2	2	0.14	3	2	0.27	93	2	0.69
Positive controls	545^c	6^c	20.96^c	882^c	11^c	7.60^c	139^c	9^c	9.93^c	126^c	1^c	11.45^c	572^d	4^d	4.27^d
^aValues are means of three replicates calculated from Appendix 2 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                          ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                             SD: Standard deviation

Table 4: Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation
Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	27	1	NA	115	2	NA	13	3	NA	12	2	NA	132	3	NA
8	27	2	1.00	112	2	0.97	13	2	1.00	12	1	1.00	129	4	0.98
25	27	2	1.00	112	3	0.97	14	3	1.08	11	2	0.92	129	3	0.98
80	27	1	1.00	112	1	0.97	14	2	1.08	10	2	0.83	128	2	0.97
253	21	1	0.78	75	3	0.65	9	1	0.69	8	1	0.67	109	2	0.83
800	15	1	0.56	40	1	0.35	2	1	0.15	3	2	0.25	91	2	0.69
Positive controls	241^c	5^c	8.93^c	530^d	6^d	4.61^d	143^d	5^d	11.00^d	128^e	2^e	10.67^e	572^f	7^f	4.33^f
^aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number  ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98: 2-Nitrofluorene (2 µg/plate),             ^dTA100, TA1535: Sodium azide (1 µg/plate),            ^eTA1537: 9-Aminoacridine (50 µg/plate),      ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)             NA: Not applicable                                         SD: Standard deviation

Table 5: Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation
Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	25	2	NA	114	2	NA	14	2	NA	11	2	NA	133	4	NA
41	25	2	1.00	112	1	0.98	14	2	1.00	11	2	1.00	135	5	1.02
86	26	1	1.04	111	1	0.97	15	1	1.07	12	1	1.09	131	5	0.98
181	27	1	1.08	96	2	0.84	14	2	1.00	10	1	0.91	130	3	0.98
380	23	3	0.92	63	2	0.55	8	1	0.57	8	2	0.73	102	4	0.77
800	15	2	0.60	44	3	0.39	3	1	0.21	3	1	0.27	91	2	0.68
Positive control	557^c	17^c	22.28^c	876^c	10^c	7.68^c	134^c	6^c	9.57^c	129^c	4^c	11.73^c	580^d	6^d	4.36^d
^aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number          ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                                                                             SD: Standard deviation

Table 6: Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation
Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
Vehicle control DMSO	25	1	NA	114	2	NA	15	1	NA	12	1	NA	130	4	NA
41	26	1	1.04	111	2	0.97	15	2	1.00	11	2	0.92	131	5	1.01
86	25	1	1.00	111	1	0.97	14	2	0.93	12	1	1.00	128	3	0.98
181	26	1	1.04	96	3	0.84	15	1	1.00	10	1	0.83	127	2	0.98
380	21	1	0.84	64	3	0.56	9	2	0.60	6	1	0.50	99	3	0.76
800	16	3	0.64	43	2	0.38	2	2	0.13	3	1	0.25	90	2	0.69
Positive control	246^c	6^c	9.84^c	537^d	3^d	4.71^d	133^d	6^d	8.87^d	133^e	4^e	11.08^e	584^f	5^f	4.49^f
^aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate) ^cTA98: 2-Nitrofluorene (2 µg/plate),             ^dTA100, TA1535: Sodium azide (1 µg/plate)             ^eTA1537: 9-Aminoacridine (50 µg/plate),      ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)             NA: Not applicable                                         SD: Standard deviation

Applicant's summary and conclusion

Conclusions:

The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. No mutagenicity was found.

Executive summary:

The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. In the preliminary cytotoxicity test, the S.typhimurium TA 100 cultures were treated up to 5000 µg/plate with and without metabolic activation. Significantly reduced background lawn was observed at 400 µg/plate or higher. Two independent experiments for mutagenicity were performend, each with and without metabolic activation up to the concentration of 800µg/plate. No increase of number of reverstants were found in any of performed experiments.