Phenethyl cinnamate

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:

20 Mar - 27 Apr 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fuer Umwelt und Verkehr Baden-Württemberg, Stuttgart

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenethyl cinnamate

EC Number:

203-120-3

EC Name:

Phenethyl cinnamate

Cas Number:

103-53-7

Molecular formula:

C17H16O2

IUPAC Name:

phenethyl cinnamate

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Source: Dr. Bruce N. Ames, University of California, Berkeley, California, U.S.A.
- Type and identity of media: Vogel-Bonner minimal medium plates enriched with histidine (260µM) and biotin (3μM). Ampicillin (25 µg per mL medium of the plate) is added to the plates used for the strains with the R-factor.
- complete medium plates: agar, 1.5%; Difco bacto nutrient broth, 0.8%; NaCL, 0.5%
- minimal agar plates: 20 to 25 mL of 1.5 % agar in Vogel-Bonner medium E with 2 % glucose
- Properly maintained: yes
- The Vogel-Bonner minimal plates, the test compound, and the S9-mix were checked for sterility.

Additional strain / cell type characteristics:

other: All strains contain an additional mutation: rfa. except TA102, the latter contains an uvrB-mutation. Strains TA98 & TA100 were developed by transferring a resistance factor (ampicillin) carried on plasmid pKM101, to strains TA1538 & TA1535 respectively.

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate fractions (S9)

Test concentrations with justification for top dose:

0, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence of S9
0, 5, 15, 50, 150, 1500 and 5000 μg/plate in the absence of S9

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard plate-incorporation assay +/- liver homogenate activation

DURATION
- Preincubation period: at 37°C for 14-15 hours to a density of 0.5-3 x 109 cells per mL
- Exposure duration: inoculation overnight
- Expression/Selection time (cells in growth medium): 48 to 72 h at 37°C in the dark

NUMBER OF REPLICATIONS: 3 for each experimental point. The experiment was repeated in full after an intervall of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- counting # revertant colonies (his+revertants)
- normal background lawn and/or precipitates
- microscopically: microcolony growth.
- reduction # revertant colonies and/or a diminution of the background lawn was taken as indication of bacteriotoxicity
- if there was doubt about the nature of colonies (revertants) or if positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked

POSITIVE CONTROLS
In the absence of S9 mix:
- 0.7 µg/plate of sodium azide for strains TA100 and TA1535
- 2.5 µg/plate of 2-nitrofluorene for strain TA98
- 50 µg/plate of 9-aminoacridine for strain TA1537
- 0.15 µg/plate of mitomycin C for strain TA102

In the presence of SG9 mix:
- 0.8 µg/plate of 2-aminoanthracene for TA98 and TA100
- 0.9 µg/plate of 2-aminoanthracene for TA1535
- 1.0 µg/plate of 2-aminoanthracene for TA102
- 1.7 µg/plate of 2-aminoanthracene for TA1537

Statistics:

X2-test (Mohn and Ellenberger, 1977)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test compound on the plates was observed at 1500 and 5000 µg/plate.
The test compound was bacteriotoxic, but failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. Similarly, the estimation of the statistical significance of the differente between the mean number of revertants in the negative controls and the plates at each dosage level did not reveal a significant effect at any one of the test points.

Any other information on results incl. tables

Overview of individual data.
Legend:

p = precipitation

T = baceteriotoxic

Individual data experiment I, without S9

			Strain TA 98			Strain TA 100			Strain TA 102			Strain TA 1535			Strain 1537
Substance	Conc. (µg/plate)	S9	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD
control	0	-	29	35	5	65	79	11	211	260	35	12	10	2	8	11	4
			37			86			283			10			11
			39			87			285			9			15
solvent control	0	-	30	32	3	78	81	3	240	237	3	11	10	2	11	11	1
			34			83			234			9			11
			32			83			238			11			12
test item	15	-				82	80	3	229	256	20	8	10	3
						78			269			12
						79			271			10
test item	50	-	32	39	8	78	83	5	220	231	11	15	14	3	9	9	2
			36			83			228			16			11
			48			88			245			12			8
test item	150	-	37	34	5	70	72	2	196	199	15	19	15	4	7	8	3
			37			72			217			16			11
			29			73			184			11			6
test item	500	-	49	37	10	82	86	6	204	204	16	10	12	6	5	7	2
			26			84			185			6			7
			35			93			222			19			8
test item	1500 p	-	44	36	7	92	86	8	196	174 T	18	9	8	2	9	8	2
			35			90			156			8			9
			29			77			170			7			7
test item	5000 p	-	20	26	6										7	6 T	2
			28												4
			31												7
NaN3	0.7	-				430	443	15				394	471	65
						437						550
						463						468
2-NF	2.5	-	506	549	41
			602
			538
9-AA	50	-													161	180	21
															207
															171
Mitocyin C	0.15	-							918	854	46
									826
									818

Individual data experiment I, with S9

			Strain TA 98			Strain TA 100			Strain TA 102			Strain TA 1535			Strain 1537
Substance	Conc. (µg/plate)	S9	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD
control	0	+	30	27	3	122	123	13	273	320	34	8	11	3	12	10	3
			26			109			342			12			10
			26			139			344			13			8
solvent control	0	+	17	21	4	118	117	5	292	299	6	16	13	4	13	14	4
			22			112			304			9			18
			25			122			302			13			11
test item	50	+	19	22	3	87	103	13	263	292	27	14	11	3	13	13	2
			22			116			287			8			14
			25			105			325			12			12
test item	150	+	27	26	6	135	120	14	293	311	14	8	10	2	13	13	3
			19			103			319			11			11
			31			121			322			10			16
test item	500	+	22	22	5	93	97	4	290	295	7	9	9	1	14	12	4
			26			99			303			9			8
			17			99			291			8			15
test item	1500 p	+	22	19	5	109	106	3	235	259	42	7	11	4	13	13	2
			22			106			316			12			12
			13			104			225			14			15
test item	5000 p	+	16	19	3	104	100	9	175	166 T	24	10	10	4	12	9 T	3
			22			89			188			14			7
			20			107			135			7			8
2-AA	0.8	+	800	799	40	1097	1018	60
			750			954
			846			1002
2-AA	0.9	+										90	91	4
												88
												95
2-AA	1.0	+							550	592	81
									704
									552
2-AA	1.7	+													164	203	36
															249
															195

Individual data experiment II, without S9

			Strain TA 98			Strain TA 100			Strain TA 102			Strain TA 1535			Strain 1537
Substance	Conc. (µg/plate)	S9	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD
control	0	-	21	31	8	141	142	17	259	274	12	13	12	2	6	8	3
			37			161			277			11			10
			36			123			286			11			8
solvent control	0	-	33	31	4	135	118	14	235	264	23	14	13	2	9	8	2
			27			104			268			12			9
			32			114			289			14			6
test item	5	-							246	259	14
									254
									277
test item	15	-	33	33	2				247	242	6				7	8	2
			35						243						9
			32						235						7
test item	50	-	44	39	5	145	131	16	238	255	34	5	8	3	6	9	4
			34			138			226			9			13
			40			111			302			11			8
test item	150	-	32	35	7	126	121	4	233	226	13	8	8	4	12	12	3
			43			120			235			4			9
			29			118			209			12			14
test item	500	-	22	29	6	112	115	3	239	203	28	9	8	2	10	9	4
			34			115			196			9			12
			32			117			175			6			5
test item	1500 p		25	34	9	114	115	3				8	11	5	8	8	1
			44			112						16			8
			34			118						9			9
test item	5000 p	-				105	111	6				10	10	2
						118						11
						110						9
NaN3	0.7	-				417	489	61				464	486	22
						486						480
						564						514
2-NF	2.5	-	580	598	17
			596
			618
9-AA	50	-													132	121	13
															126
															105
Mitocyin C	0.15	-							696	722	76
									824
									646

Individual data experiment II, with S9

			Strain TA 98			Strain TA 100			Strain TA 102			Strain TA 1535			Strain 1537
Substance	Conc. (µg/plate)	S9	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD	#	Mean	SD
control	0	+	25	26	2	153	148	6	339	362	18	10	12	8	14	14	6
			25			141			369			22			20
			28			149			379			5			8
solvent control	0	+	21	23	3	153	133	20	318	348	22	12	12	4	12	13	2
			26			107			365			9			15
			21			139			362			16			13
test item	15	+							327	296	30				10	13	3
									258						14
									302						14
test item	50	+	19	20	2	125	124	10	318	302	13	10	9	2	17	16	2
			19			134			289			7			16
			21			113			298			10			15
test item	150	+	23	22	1	127	121	5	304	299	25	10	13	5	10	12	3
			22			117			325			11			14
			22			119			268			18			11
test item	500	+	19	16	3	111	124	11	321	309	26	13	12	2	13	14	4
			14			134			274			11			18
			16			128			331			13			12
test item	1500 p	+	17	16	2	81	99	14	288	309	28	13	14	2	9	10	2
			17			108			222			13			12
			15			109			251			15			9
test item	5000 p	+	17	13	4	100	99	2		254		8	9	2
			9			98						8
			14			98						11
2-AA	0.8	+	968	1018	40	1299	1291	34
			1064			1327
			1022			1248
2-AA	0.9	+										300	280	19
												284
												256
2-AA	1.0	+							593	570	17
									562
									556
2-AA	1.7	+													294	245	45
															188
															252

Applicant's summary and conclusion

Conclusions:

The test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system, under the experimental conditions described.

Executive summary:

In the current study the mutagenicity of the substance was studied with 5 mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) as metabolic activation system. The test item was dissolved in DMSO and tested in concentrations of 15 to 5000 µg/plate in the presence and of 5 to 5000 µg/plate in the absence of S9.

In the absence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and towards the strains TA1537 at 5000 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strains TA102 and TA1537 at 5000 µg/plate. Precipitation of the test compound an the plates was observed at 1500 and 5000 µg/plate.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls and confirmed the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.