(Z)-hex-3-enyl (Z)-hex-3-enoate

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:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP; minor deviation is not considered to to affect the reliability of the 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:

yes

Remarks:

The bacterial cultures were incubated in a shaking water bath for 3 hours at 37° C since the bacterial density had been reached at that incubation time.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see above.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected February 1998; signature: March 1998

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-hex-3-enyl (Z)-hex-3-enoate

EC Number:

262-797-3

EC Name:

(Z)-hex-3-enyl (Z)-hex-3-enoate

Cas Number:

61444-38-0

Molecular formula:

C12H20O2

IUPAC Name:

(Z)-hex-3-enyl (Z)-hex-3-enoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: liquid
- Storage condition of test material: at approx. 4 degrees C

Method

Target gene:

The histidine dependent strains are derived from S. typhimurium strain L T2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate (with strains TA98 and TA100)
Experiment 1: 33, 100, 333, 1000, 2500, 5000 ug/plate
Experiment 2: 33, 100, 333, 1000, 2500, 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene,

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

DURATION
- Preincubation period: 60 minutes
- Expression time (cells in growth medium): 48 h at 37 degrees C in the dark

NUMBER OF REPLICATIONS: 3 plates per dose

Evaluation criteria:

- A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
- A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.
- A biologically relevant response is described as follows:
A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no toxicity below 5000 μg/plate limit level in TA100 strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
An isolated reduction of the colony count in strain TA 98 at 100 μg/plate in the first experiment without metabolic activation was judged as an artefact since the colony count was back to normal at higher concentrations. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). However, a slight increase in revertant colony numbers close to or reaching the threshold of twice the colony count of the corresponding solvent control was observed in experiment I at 2500 and 5000 μg/plate in strain TA 100 without metabolic activation. This increase was judged as biologically irrelevant since it is, based upon a solvent control colony count close to the lower limit of the laboratory historical range. The absolute numbers of colonies remained well within the range of negative and solvent controls. Furthermore, this increase was not reproduced in the second experiment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary of revertants

+/- S9 mix	Test material concentration (μg/plate)	TA100			TA1535			TA1537			TA98			TA102
			Revertants			Revertants			Revertants			Revertants			Revertants
		Replicate	Experiment 1	Experiment 2	Replicate	Experiment 1	Experiment 2	Replicate	Experiment 1	Experiment 2	Replicate	Experiment 1	Experiment 2	Replicate	Experiment 1	Experiment 2
- S9 Mix	Negative control	1	78	85	1	9	7	1	5	9	1	16	15	1	227	267
		2	77	100	2	9	9	2	6	7	2	14	19	2	216	290
		3	83	96	3	11	15	3	4	11	3	17	12	3	220	265
		Mean	79	94	Mean	10	10	Mean	5	9	Mean	16	15	Mean	221	274
	Solvent control	1	94	84	1	7	8	1	8	8	1	19	16	1	186	229
		2	82	54	2	11	12	2	5	7	2	15	12	2	198	213
		3	73	93	3	9	8	3	6	9	3	13	18	3	180	255
		Mean	83	77	Mean	9	9	Mean	6	8	Mean	16	15	Mean	188	232
	Positive control	1	1292	715	1	213	268	1	57	67	1	96	89	1	991	904
		2	1361	820	2	367	412	2	41	65	2	99	86	2	972	867
		3	1321	931	3	509	468	3	44	56	3	92	92	3	1154	1017
		Mean	1325	822	Mean	363	383	Mean	47	63	Mean	96	89	Mean	1039	929
	33	1	59	58	1	8	9	1	3	5	1	7	11	1	192	216
		2	70	65	2	7	5	2	5	7	2	12	12	2	205	225
		3	63	55	3	6	3	3	3	7	3	4	18	3	223	268
		Mean	64	59	Mean	7	6	Mean	4	6	Mean	8	14	Mean	207	236
	100	1	68	61	1	5	8	1	6	5	1	3	13	1	184	234
		2	66	62	2	6	7	2	5	6	2	2	12	2	159	202
		3	66	69	3	9	3	3	2	4	3	2	13	3	171	214
		Mean	67	64	Mean	7	6	Mean	4	5	Mean	2	13	Mean	171	217
	333	1	105	75	1	10	9	1	4	6	1	15	11	1	177	195
		2	114	82	2	4	3	2	2	4	2	8	15	2	164	176
		3	83	77	3	5	7	3	3	3	3	5	10	3	210	237
		Mean	101	78	Mean	6	6	Mean	3	4	Mean	9	12	Mean	184	203
	1000	1	132	112	1	3	2	1	6	3	1	9	7	1	170	225
		2	133	119	2	5	2	2	7	2	2	8	7	2	185	260
		3	122	102	3	1	3	3	5	1	3	19	10	3	207	263
		Mean	129	111	Mean	3	2	Mean	6	2	Mean	12	8	Mean	187	249
	2500	1	149	84	1	6	6	1	3	5	1	9	11	1	212	262
		2	170	93	2	1	17	2	4	7	2	13	12	2	204	150
		3	167	72	3	1	5	3	2	5	3	11	25	3	214	218
		Mean	162	83	Mean	3	9	Mean	3	6	Mean	11	16	Mean	210	210
	5000	1	158	96	1	5	13	1	3	4	1	3	13	1	222	188
		2	182	94	2	2	15	2	6	12	2	3	11	2	223	193
		3	125	68	3	3	14	3	8	6	3	5	3	3	144	197
		Mean	155	86	Mean	3	14	Mean	6	7	Mean	4	9	Mean	196	193
+ S9 Mix	Negative control	1	133	144	1	4	8	1	10	8	1	14	18	1	253	170
		2	127	122	2	15	9	2	4	7	2	15	17	2	263	144
		3	126	116	3	11	7	3	4	8	3	18	21	3	250	159
		Mean	129	127	Mean	10	8	Mean	6	8	Mean	16	19	Mean	255	158
	Solvent control	1	122	104	1	9	6	1	6	14	1	17	19	1	233	147
		2	110	98	2	5	5	2	5	7	2	15	23	2	258	135
		3	127	94	3	16	9	3	9	8	3	11	23	3	230	167
		Mean	120	99	Mean	10	7	Mean	7	10	Mean	14	22	Mean	240	150
	Positive control	1	636	454	1	74	81	1	55	42	1	123	167	1	409	1255
		2	569	546	2	75	74	2	50	46	2	114	179	2	1132	1127
		3	608	622	3	86	65	3	52	49	3	100	152	3	1144	1036
		Mean	604	541	Mean	78	73	Mean	52	46	Mean	112	166	Mean	895	1139
	33	1	112	88	1	8	6	1	9	7	1	17	14	1	191	136
		2	111	110	2	5	10	2	9	10	2	15	9	2	202	157
		3	106	115	3	9	4	3	5	7	3	16	15	3	202	164
		Mean	110	104	Mean	7	7	Mean	8	8	Mean	16	13	Mean	198	152
	100	1	125	101	1	6	5	1	7	6	1	9	17	1	192	159
		2	121	113	2	4	12	2	6	6	2	10	22	2	209	154
		3	132	90	3	7	4	3	6	7	3	12	22	3	195	160
		Mean	126	101	Mean	6	7	Mean	6	6	Mean	10	20	Mean	199	158
	333	1	122	89	1	9	8	1	6	5	1	21	9	1	215	187
		2	136	95	2	4	6	2	6	6	2	15	17	2	222	205
		3	145	136	3	4	7	3	10	11	3	20	19	3	246	170
		Mean	134	107	Mean	6	7	Mean	7	7	Mean	19	15	Mean	228	187
	1000	1	120	111	1	6	5	1	11	10	1	6	11	1	179	131
		2	122	98	2	10	5	2	8	8	2	3	22	2	189	171
		3	133	100	3	3	10	3	13	7	3	14	15	3	154	169
		Mean	125	103	Mean	6	7	Mean	11	8	Mean	8	16	Mean	174	157
	2500	1	123	93	1	8	3	1	8	10	1	9	10	1	169	130
		2	126	110	2	4	10	2	10	6	2	13	16	2	182	140
		3	132	90	3	6	6	3	9	5	3	7	11	3	161	125
		Mean	127	98	Mean	6	6	Mean	9	7	Mean	10	12	Mean	171	132
	5000	1	107	87	1	7	6	1	6	3	1	5	15	1	91	102
		2	113	81	2	3	5	2	2	5	2	8	18	2	139	94
		3	105	96	3	5	3	3	9	3	3	5	6	3	141	100
		Mean	108	88	Mean	5	5	Mean	6	4	Mean	6	13	Mean	124	99

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to guideline OECD 471 and EU Method B.14 under GLP to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, TA1537 in both the presence and absence of S-9 mix. A preliminary test was performed to determine the test concentrations for main study. In the main study two experiments (one using the plate incorporation method and the other with the pre-incubation method) were performed and the test substance was evaluated at a concentration of up to 5000 µg/plate in DMSO vehicle. Positive controls appropriate for each strain, in the presence and absence of S9 -mix were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of S-9 mix.