Cinnamaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cinnamaldehyde

EC Number:

203-213-9

EC Name:

Cinnamaldehyde

Cas Number:

104-55-2

Molecular formula:

C9H8O

IUPAC Name:

3-phenylacrylaldehyde

Details on test material:

- Name of the test chemical: Cinnamaldehyde
- Molecular formula: C9H8O
- Molecular weight: 132.1612 g/mol
- Substnace type: Organic

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):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

- source of S9: Arochlor 1254 induced S9 metabolic activation system was procured from Defence Research and Development Organzaation
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix. Cofactor solution contains the following quantity of chemicals in 500 mL of RO Water.
D-glucose-6-phosphate 0.8 g
β-NADP 1.75 g
MgCl2 1.0 g
KCl 1.35 g
Na2HPO4 6.4 g
NaH2PO4.H2O 1.4 g
During the experiment, the S9 mix was prepared freshly and used.

- concentration or volume of S9 mix and S9 in the final culture medium : An appropriate quantity of S9 supernatant was mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency.

Test concentrations with justification for top dose:

0.0 (NC), 0.0(VC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.0(VC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentration 5 (T8) and 1.582 (T7) mg/plate and no reduction in colony count as well as in background lawn in treated concentrations 0.501 (T6) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation.

Based on the results of pre-experiment following doses were selected for the main study trials:
0.0 (NC), 0.0(VC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).
The concentrations used in the experiment (pre-experiment, Trial-1, Trial-2) were placed with (√10) half log interval

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	128	20	121	21
	R2	115	25	120	20
	R3	110	23	117	22
VC (0.00)	R1	120	27	126	28
	R2	125	25	121	26
	R3	118	19	118	24
T1 (0.002)	R1	111	19	109	21
	R2	101	20	111	19
	R3	102	21	108	23
T2 (0.005)	R1	99	19	107	21
	R2	115	20	119	18
	R3	108	21	109	20
T3 (0.016)	R1	121	19	113	20
	R2	106	21	119	18
	R3	118	23	120	21
T4 (0.050)	R1	113	21	115	22
	R2	105	20	113	21
	R3	121	21	119	19
T5 (0.158)	R1	105	22	114	17
	R2	115	20	116	23
	R3	120	19	120	21
T6 (0.501)	R1	104	20	108	24
	R2	118	23	121	23
	R3	106	20	118	23
T7 (1.582)	R1	113	19	117	21
	R2	108	22	110	22
	R3	118	23	121	24
T8 (5)	R1	120	25	122	25
	R2	119	23	117	25
	R3	123	22	115	23
PC	R1	1152	1024	1424	1184
	R2	1120	1008	1432	1144
	R3	1160	1016	1454	1208

NC          =    Negative control

VC          =  Vehicle Control

PC          =    Positive control

R             =    Replicate

T             =    Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
(TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	21	121	234
	R2	5	11	20	120	226
	R3	4	11	22	117	241
VC (0.00)	R1	8	17	28	126	262
	R2	7	16	26	121	264
	R3	7	17	24	118	272
T1 (0.005)	R1	5	11	21	107	248
	R2	4	10	18	119	230
	R3	6	12	20	109	229
T2 (0.016)	R1	5	13	20	113	240
	R2	4	12	18	119	239
	R3	4	10	21	120	246
T3 (0.050)	R1	6	13	22	115	246
	R2	5	12	21	113	258
	R3	7	14	19	119	266
T4 (0.158)	R1	6	13	17	114	252
	R2	6	14	23	116	266
	R3	7	11	21	120	270
T5 (0.501)	R1	7	15	24	108	264
	R2	8	16	23	121	258
	R3	6	15	23	118	260
PC	R1	174	428	1184	1424	1408
	R2	180	420	1144	1432	1320
	R3	160	390	1208	1454	1384

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	20	128	240
	R2	4	9	25	115	236
	R3	4	11	23	110	220
VC (0.00)	R1	7	16	27	120	264
	R2	8	17	25	125	272
	R3	8	16	19	118	282
T1 (0.005)	R1	5	11	19	99	234
	R2	5	12	20	115	228
	R3	4	10	21	108	235
T2 (0.016)	R1	5	12	19	121	230
	R2	5	10	21	106	244
	R3	5	10	23	118	256
T3 (0.050)	R1	6	12	21	113	240
	R2	6	13	20	105	238
	R3	7	13	21	121	226
T4 (0.158)	R1	7	13	22	105	248
	R2	5	14	20	115	256
	R3	6	15	19	120	264
T5 (0.501)	R1	7	16	20	104	274
	R2	8	15	23	118	266
	R3	7	16	20	106	260
PC	R1	176	1208	1024	1152	1888
	R2	182	1240	1008	1120	1712
	R3	166	1192	1016	1160	1704

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                                2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100,       
2- Aminoanthracene [10μg/plate]:TA 102,                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                                                                                                               

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate],       Methyl methanesulfonate [4μl/plate]: TA 102.

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	11	20	112	238
	R2	4	11	21	121	244
	R3	5	10	19	116	236
VC (0.00)	R1	7	17	26	125	264
	R2	8	16	25	127	274
	R3	8	16	26	119	286
T1 (0.005)	R1	6	12	20	115	237
	R2	5	11	21	117	243
	R3	5	10	23	121	258
T2 (0.016)	R1	5	14	22	115	246
	R2	6	11	21	117	248
	R3	4	13	20	120	242
T3 (0.050)	R1	6	14	23	125	254
	R2	6	12	24	119	258
	R3	7	11	20	115	262
T4 (0.158)	R1	7	15	21	120	268
	R2	6	14	23	122	254
	R3	7	13	25	126	270
T5 (0.501)	R1	7	17	25	125	276
	R2	7	16	25	117	280
	R3	7	14	24	123	266
PC	R1	163	336	1352	1528	1752
	R2	170	418	1384	1568	1736
	R3	158	434	1400	1464	1784

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	18	103	233
	R2	4	9	17	113	239
	R3	4	9	16	98	247
VC (0.00)	R1	8	16	25	119	268
	R2	7	15	26	123	284
	R3	7	15	25	127	274
T1 (0.005)	R1	4	10	18	104	234
	R2	5	11	17	109	246
	R3	5	12	18	110	243
T2 (0.016)	R1	5	10	19	117	252
	R2	6	12	17	119	258
	R3	6	10	20	115	262
T3 (0.050)	R1	5	11	17	109	254
	R2	4	13	17	107	248
	R3	6	15	18	119	242
T4 (0.158)	R1	7	14	22	121	256
	R2	6	16	21	118	268
	R3	5	15	23	123	262
T5 (0.501)	R1	7	15	25	125	276
	R2	7	15	24	121	280
	R3	7	14	25	119	268
PC	R1	182	1176	912	1176	1544
	R2	172	1192	834	1288	1624
	R3	186	1256	858	1328	1656

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100,        
2-Aminoanthracene [10μg/plate]:TA 102,

Sodium azide [10μg/plate]: TA 1535, TA 100                                                                                                                                    

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate [4μl/plate]: TA 102.

TABLE 4 -    MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	10.67	0.58	21.00	1.00	119.33	2.08	233.67	7.51
VC (0.00)	7.33	0.58	16.67	0.58	26.00	2.00	121.67	4.04	266.00	5.29
T1 (0.005)	5.00	1.00	11.00	1.00	19.67	1.53	111.67	6.43	235.67	10.69
T2 (0.016)	4.33	0.58	11.67	1.53	19.67	1.53	117.33	3.79	241.67	3.79
T3 (0.050)	6.00	1.00	13.00	1.00	20.67	1.53	115.67	3.06	256.67	10.07
T4 (0.158)	6.33	0.58	12.67	1.53	20.33	3.06	116.67	3.06	262.67	9.45
T5 (0.501)	7.00	1.00	15.33	0.58	23.33	0.58	115.67	6.81	260.67	3.06
PC	171.33	10.26	412.67	20.03	1178.67	32.33	1436.67	15.53	1370.67	45.49

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.33	0.58	10.00	1.00	22.67	2.52	117.67	9.29	232.00	10.58
VC (0.00)	7.67	0.58	16.33	0.58	23.67	4.16	121.00	3.61	272.67	9.02
T1 (0.005)	4.67	0.58	11.00	1.00	20.00	1.00	107.33	8.02	232.33	3.79
T2 (0.016)	5.00	0.00	10.67	1.15	21.00	2.00	115.00	7.94	243.33	13.01
T3 (0.050)	6.33	0.58	12.67	0.58	20.67	0.58	113.00	8.00	234.67	7.57
T4 (0.158)	6.00	1.00	14.00	1.00	20.33	1.53	113.33	7.64	256.00	8.00
T5 (0.501)	7.33	0.58	15.67	0.58	21.00	1.73	109.33	7.57	266.67	7.02
PC	174.67	8.08	1213.33	24.44	1016.00	8.00	1144.00	21.17	1768.00	104.00

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                            

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

 

 

 

 

 

 

TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	10.67	0.58	20.00	1.00	116.33	4.51	239.33	4.16
VC (0.00)	7.67	0.58	16.33	0.58	25.67	0.58	123.67	4.16	274.67	11.02
T1 (0.005)	5.33	0.58	11.00	1.00	21.33	1.53	117.67	3.06	246.00	10.82
T2 (0.016)	5.00	1.00	12.67	1.53	21.00	1.00	117.33	2.52	245.33	3.06
T3 (0.050)	6.33	0.58	12.33	1.53	22.33	2.08	119.67	5.03	258.00	4.00
T4 (0.158)	6.67	0.58	14.00	1.00	23.00	2.00	122.67	3.06	264.00	8.72
T5 (0.501)	7.00	0.00	15.67	1.53	24.67	0.58	121.67	4.16	274.00	7.21
PC	163.67	6.03	396.00	52.57	1378.67	24.44	1520.00	52.46	1757.33	24.44

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.33	0.58	9.33	0.58	17.00	1.00	104.67	7.64	239.67	7.02
VC (0.00)	7.33	0.58	15.33	0.58	25.33	0.58	123.00	4.00	275.33	8.08
T1 (0.005)	4.67	0.58	11.00	1.00	17.67	0.58	107.67	3.21	241.00	6.24
T2 (0.016)	5.67	0.58	10.67	1.15	18.67	1.53	117.00	2.00	257.33	5.03
T3 (0.050)	5.00	1.00	13.00	2.00	17.33	0.58	111.67	6.43	248.00	6.00
T4 (0.158)	6.00	1.00	15.00	1.00	22.00	1.00	120.67	2.52	262.00	6.00
T5 (0.501)	7.00	0.00	14.67	0.58	24.67	0.58	121.67	3.06	274.67	6.11
PC	180.00	7.21	1208.00	42.33	868.00	39.95	1264.00	78.79	1608.00	57.69

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay as per OECD guidleine no. 471 was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0(VC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0(VC),  0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of the historical data.

The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.