4-(octadecylamino)-4-oxoisocrotonic acid

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:

25 July - 11 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(octadecylamino)-4-oxoisocrotonic acid

EC Number:

221-361-2

EC Name:

4-(octadecylamino)-4-oxoisocrotonic acid

Cas Number:

3077-27-8

Molecular formula:

C22H41NO3

IUPAC Name:

4-(octadecylamino)-4-oxoisocrotonic acid

Test material form:

solid

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from livers treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Experiment 1a, plate incorporation method
S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535: 5000, 1500, 500, 150 and 50 μg/plate with and without metabolic activation

Experiment 1b, plate incorporation method
S. typhimurium TA102: 5000 μg/plate, 1500, 500, 150, 50, 15 and 5 μg/plate with and without metabolic activation

Experiment 2, pre-incubation method
S. typhimurium TA 97a, TA 98, TA 100 and TA 1535): 5000, 2500, 1250, 625, 313, 156 and 78 μg/plate with and without metabolic activation
S. typhimurium TA 102: 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in demineralized water, dimethyl sulfoxide (DMSO), ethanol and tetrahydrofurane. Based on these results, ethanol was used as solvent in all experiments, because the test item showed in ethanol the best result for handling in the experiments. The test suspension was homogeneous and pipettable. In addition, ethanol does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino-anthracene (2-AA): +S9: 1 µg/plate (in DMSO) for TA 97a, TA 100, TA 102, TA 1535; 4-Nitro-1,2-phenylenediamine (NPD): -S9: 20 µg/plate (in DMSO) for TA 97a, TA 98, TA 102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, experiments 1a and 1b) and pre-incubation (experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments (experiments 1a/1b and 2)

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn and of the number of revertants

Evaluation criteria:

The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-/+S9: 5000 and 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in any experiment at any concentration

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : data is summarised in "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Mean Revertants Experiment 1a (all strains tested)

 

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	85	80	29	34	73	93	307	377	27	26
	sd	6.9	12.7	6.4	9.8	5.9	13.6	48.4	61.1	1.5	7.0
DMSO	Mean	98	97	35	38	78	78	320	388	22	24
	sd	19.6	4.0	5.9	6.7	5.5	3.5	31.2	28.8	3.2	4.0
Ethanol	Mean	76	83	24	26	72	72	295	313	22	22
	sd	11.4	7.0	6.4	6.0	10.6	8.6	96.4	37.2	3.8	5.9
Positive Controls*	Mean	637	781	383	86	451	1528	1411	1472	323	183
	sd	99.4	64.2	27.2	8.1	22.7	424.2	100.0	36.7	56.6	28.4
	f(I)	6.50	8.05	10.94	2.26	6.18	19.59	4.41	3.79	11.96	7.63
5000 µg/plate	Mean	99	93	22	26	76	82	79	48	20	19
	sd	18.9	15.1	3.1	5.9	4.0	18.9	12.6	14.5	2.1	1.5
	f(I)	1.30	1.12	0.92	1.00	1.06	1.14	0.27	0.15	0.91	0.86
1500 µg/plate	Mean	79	98	24	21	75	74	66	95	17	19
	sd	16.2	9.0	4.0	6.4	11.5	8.0	19.9	23.1	1.5	2.5
	f(I)	1.04	1.18	1.00	0.81	1.04	1.03	0.22	0.30	0.77	0.86
500 µg/plate	Mean	90	94	26	24	82	77	285	331	23	19
	sd	22.0	15.9	5.5	2.9	6.0	13.6	60.2	10.1	4.5	3.2
	f(I)	1.18	1.13	1.08	0.92	1.14	1.07	0.97	1.06	1.05	0.86
150 µg/plate	Mean	79	84	26	20	66	76	281	384	18	23
	sd	15.0	20.3	6.9	2.6	5.5	10.7	89.3	10.6	3.6	0.6
	f(I)	1.04	1.01	1.08	0.77	0.92	1.06	0.95	1.23	0.82	1.05
50 µg/plate	Mean	88	89	25	32	83	64	291	355	22	24
	sd	5.2	6.4	4.0	1.2	14.5	6.2	63.8	64.7	4.6	4.2
	f(I)	1.16	1.07	1.04	1.23	1.15	0.89	0.99	1.13	1.00	1.09

f(I) = increase factor

* Different positive controls were used

Table 2: Mean Revertants Experiment 1b (strain TA 102)

 

Strain		TA102
Induction		-S9	+S9
Demin. water	Mean	356	345
	sd	62.9	72.6
DMSO	Mean	257	324
	sd	26.6	20.8
Ethanol	Mean	292	258
	sd	58.9	24.0
Positive Controls*	Mean	689	1083
	sd	60.2	400.0
	f(I)	2.68	3.34
5000 µg/plate	Mean	12	4
	sd	1.0	1.5
	f(I)	0.04	0.02
1500 µg/plate	Mean	80	56
	sd	10.0	9.0
	f(I)	0.27	0.22
500 µg/plate	Mean	315	268
	sd	46.4	58.9
	f(I)	1.08	1.04
150 µg/plate	Mean	284	284
	sd	52.3	52.0
	f(I)	0.97	1.10
50 µg/plate	Mean	355	292
	sd	50.0	34.6
	f(I)	1.22	1.13
15 µg/plate	Mean	339	349
	sd	47.7	120.2
	f(I)	1.16	1.35
5 µg/plate	Mean	304	287
	sd	65.8	33.5
	f(I)	1.04	1.11

f(I) = increase factor

* Different positive controls were used

Table 3: Mean Revertants Experiment 2 (strains TA 97a, TA 98, TA 100 and TA 1535)

 

Strain		TA97a		TA98		TA100		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	64	81	24	28	51	64	22	23
	sd	4.0	17.9	4.7	2.1	4.0	6.7	4.2	6.1
DMSO	Mean	82	67	28	34	44	62	24	24
	sd	13.7	11.0	8.2	5.6	3.6	7.5	4.4	4.2
Ethanol	Mean	61	87	32	35	40	48	21	23
	sd	8.5	14.2	4.0	4.2	1.0	1.5	5.0	4.9
Positive Controls*	Mean	631	237	459	165	429	491	280	141
	sd	83.2	59.8	88.1	8.1	113.5	132.8	59.7	9.2
	f(I)	7.70	3.54	16.39	4.85	8.41	7.92	12.73	5.88
5000 µg/plate	Mean	71	80	22	29	54	64	15	17
	sd	5.7	1.2	7.2	3.0	6.1	8.5	1.7	2.6
	f(I)	1.16	0.92	0.69	0.83	1.35	1.33	0.71	0.74
2500 µg/plate	Mean	69	76	25	29	46	73	18	26
	sd	2.5	5.0	4.9	1.5	9.1	6.1	2.0	2.5
	f(I)	1.13	0.87	0.78	0.83	1.15	1.52	0.86	1.13
1250 µg/plate	Mean	86	103	21	31	52	63	18	23
	sd	14.7	19.3	2.1	10.1	7.0	14.5	2.0	5.5
	f(I)	1.41	1.18	0.66	0.89	1.30	1.31	0.86	1.00
625 µg/plate	Mean	71	85	26	28	57	68	18	28
	sd	12.7	17.2	9.6	4.7	4.6	9.0	1.5	3.5
	f(I)	1.16	0.98	0.81	0.80	1.43	1.42	0.86	1.22
313 µg/plate	Mean	66	72	28	25	42	56	17	23
	sd	0.0	4.2	5.2	4.5	7.0	5.6	2.3	2.1
	f(I)	1.08	0.83	0.88	0.71	1.05	1.17	0.81	1.00
156 µg/plate	Mean	74	95	30	35	55	69	21	26
	sd	2.9	4.0	7.2	7.9	8.7	8.1	6.6	4.0
	f(I)	1.21	1.09	0.94	1.00	1.38	1.44	1.00	1.13
78 µg/plate	Mean	72	74	27	28	37	45	16	23
	sd	4.6	6.4	8.0	9.0	7.9	11.4	3.1	2.9
	f(I)	1.18	0.85	0.84	0.80	0.93	0.94	0.76	1.00

f(I) = increase factor

* Different positive controls were used

Table 4: Mean Revertants Experiment 2 (strain TA 102)

 

Strain		TA102
Induction		-S9	+S9
Demin. water	Mean	287	283
	sd	19.7	28.1
DMSO	Mean	287	268
	sd	42.0	38.2
Ethanol	Mean	253	335
	sd	42.4	43.9
Positive Controls*	Mean	952	1325
	sd	298.2	51.4
	f(I)	3.32	4.94
500 µg/plate	Mean	320	332
	sd	39.4	35.6
	f(I)	1.26	0.99
250 µg/plate	Mean	292	292
	sd	13.9	10.6
	f(I)	1.15	0.87
125 µg/plate	Mean	301	287
	sd	58.0	26.6
	f(I)	1.19	0.86
62.5 µg/plate	Mean	271	269
	sd	24.1	64.8
	f(I)	1.07	0.80
31.3 µg/plate	Mean	276	283
	sd	52.9	42.0
	f(I)	1.09	0.84
15.6 µg/plate	Mean	289	315
	sd	42.8	77.7
	f(I)	1.14	0.94
7.8 µg/plate	Mean	280	275
	sd	17.4	15.1
	f(I)	1.11	0.82

f(I) = increase factor

* Different positive controls were used

Table 5: Historical Data of Spontaneous Revertants and Comparison with Experiments 1a, 1b and 2

 

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
H2O de-min.	Mean	92	97	15	18	93	97	279	296	17	17
	Min	60	63	6	8	62	66	85	67	6	7
	Max	144	138	37	41	141	141	425	511	31	33
	SD	19	16	6	6	15	15	65	75	6	6
	Exp 1a	85	80	29	34	73	93	307	377	27	26
	Exp 1b	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	356	345	n.r.	n.r.
	Exp 2	64	81	24	28	51	64	287	283	22	23
DMSO	Mean	91	100	15	16	90	93	279	289	17	16
	Min	58	70	7	8	60	63	79	80	8	6
	Max	135	144	46	36	136	199	393	459	33	31
	SD	18	16	7	6	16	19	61	65	6	6
	Exp 1a	98	97	35	38	78	78	320	388	22	24
	Exp 1b	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	257	324	n.r.	n.r.
	Exp 2	82	67	28	34	44	62	287	268	24	24
Ethanol	Mean	89	99	16	17	86	90	284	275	16	16
	Min	57	65	8	9	61	69	111	141	9	9
	Max	181	205	35	40	116	129	368	339	28	29
	SD	24	29	7	8	13	16	52	47	6	6
	Exp 1a	76	83	24	26	72	72	295	313	22	22
	Exp 1b	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	292	258	n.r.	n.r.
	Exp 2	61	87	32	35	40	48	253	335	21	23
Positive Controls*	Mean	552	501	394	83	513	716	1160	1237	254	115
	Min	264	241	100	39	223	273	491	408	55	45
	Max	1152	1181	793	333	984	1912	2331	6083	484	712
	SD	169	144	143	49	153	290	452	659	89	79
	Exp 1a	637	781	383	86	451	1528	1411	1472	323	183
	Exp 1b	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	689	1083	n.r.	n.r.
	Exp 2	631	237	459	165	429	491	952	1325	280	141

* Different positive controls were used

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative