Cinnamaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

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.

In vitro mammalian chromosome aberration study:

 

The study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The method followed was as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 28-35 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase I and phase II. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations(0.5, 1 and 2mg/mL of culture media)based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes. Each tube according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37°C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37°C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37°C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

 

In vitro Mammalian Cell Gene Mutation Test was carried out in compliance with the OECD Guideline No. 476, adopted by the council on 29 July 2016. The test chemical was evaluated in mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells. Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of the test chemical,the CHO cells were exposed to the test item in duplicate cultures at the doses of 31.25, 15.625, 7.8125 and 3.90625 µg/mL of culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction prepared from, sodium phenobarbitone and b- naphthoflavone-induced Wistar rats was used in the study. Dimethyl sulphoxide was used as a vehicle.The target cells were exposed to the test compound for 3 hours at 37 ± 1^oC, with approximately 5% CO₂supply. The cells were sub-cultured immediately to determine cytotoxicity as relative survival (RS) and to initiate the phenotypic expression period. The culture flasks were incubated at 37 ± 1^oC with approximately 5% CO₂ supply during experimental periods. Concurrent vehicle and positive control groups were also included in the experiment, as specified by the test guideline. The cultures were sub-cultured during the expression period at suitable intervals. After phenotypic expression, the plates were incubated at 37 ± 1^oC with approximately 5% CO₂ supply for 11 days. After incubation, the medium was discarded, and the plates were stained and observed for the clones. Relative survivaland mutant frequency were calculated for all treatment, vehicle control, and positive control groups. The relative survival (RS) was used as the measure of treatment-related cytotoxicity. The RS (relative survival) for cultures treated withthe test chemical with and without metabolic activation system (S9) indicated that the test chemical induces 10 t 20% relative survival at the concentration of 31.25 µg/mL. The % relative survival (% RS) for the cloned cultures ranged from 18.0% to71.8% for cultures treated without metabolic activation system and 19.0% to 70.9% for cultures treated with metabolic activation system at test item concentrations of 31.25, 15.625, 7.8125 and 3.90625µg/mL in the treatment medium. The results of the present study indicate that there was no significant difference in the mutant frequencies of cultures treated withthe test chemical as compared to the vehicle control cultures, in the absence or presence of metabolic activation. An increase in the mutant frequency of concurrent positive controls demonstrated the sensitivity of the assay in the absence and presence of metabolic activation. Under the test conditions described in the study, it is concluded that the test chemical is non-mutagenic in 'InVitro Mammalian cell gene mutation test' using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells and hence it is not likely to classify as a "Gene mutant in vitro".

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Histidine

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

This in vitro chromosomal aberration assay was performed to assess the potential of the test chemical to induce structural / numerical chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 28-35 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254 induced S9
- source of S9 : Aroclor 1254 induced S9 metabolic activation system was procured from Defence Research and Development Research, Establishment, Nagpur.
- method of preparation of S9 mix : An appropriate quantity of S9 supernantant was thawed and mixed with S9 cofactor solution to result in final concentration in the S9 mix of approximately 1% v/v for Phase I of experiment and 2% v/v for Phase II of experiment

Cofactor solution contained following quantity of chemicals in 500 mL of RO water
D-glucose-6-phosphate 0.80g
MgCl2 1.00g
KCl 1.35 g
Na2HPO4 6.40g
M=NaH2PO4. H2O 1.40 g
NADP 1.75g

S9 mix was prepared freshly
- concentration or volume of S9 mix and S9 in the final culture medium : approximately 1% v/v for Phase I of experiment and 2% v/v for Phase II of experiment
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data.

Test concentrations with justification for top dose:

0.0 (NC), 0.0 (VC), 0.125, 0.25 or 0.5 mg/mL

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time: 21 hrs (with and without metabolic activation system- Phase I and II)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 21-25 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37°C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37°C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates were scored for cytogenetic damage on coded slides. The required number of well spread metaphases are equally divided into two replicates (150 metaphases each replicate) per concentration.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1.0 or 2.0 mg/mL of culture media) based on solubility, precipitation and pH test of the test item was tested. Cytotoxicity was determined by reduction in the mitotic index in comparison to the vehicle control.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- 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 can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37°C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was slight precipitation observed at 1 mg/mL concentration.
- 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 cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 1 (T2) and 2 (T3) mg/mL both in the presence and absence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 6.44 (T1), 4.17 (T2), 2.67 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 6.94 (T1), 4.35 (T2), 2.92 (T3) and 8.49 (PC). In the cytotoxicity experiment the highest test concentration 2 (T3) and 1 (T2) mg/ mL of treated culture media showed more than 50% reduction in the mitotic index when compared to the respective vehicle control both th the presence and in absence of metabolic activation confirms the cytotoxic effects. Hence 0.5 mg/mL was selected as highest concentration for main study considering the selection of test concentrations upto cytotoxicity. T

Hence the concentrations selected for the main study are 0.0 (NC), 0.0 (VC), 0.125, 0.25 or 0.5 mg/mL. The main study was performed in two independent phases - Phase I and Phase II

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: Please refer table remarks section

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

Remarks on result:

other: No mutagenic potential

CYTOTOXIC EXPERIMENT

Before conducting the chromosomal aberration study, the test chemical )was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 1 (T2) and 2 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%).

In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 6.44 (T1), 4.17 (T2), 2.67 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 6.94 (T1), 4.35 (T2), 2.92 (T3) and 8.49 (PC).

In the cytotoxicity experiment, the highest test concentration 2 (T3) and 1 (T2) mg/ mLof treated culture mediashowed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence these concentration were not selected for the main study.

Hence, 0.5 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in two independent phases;

PHASE I

In the experiment, the cultures were exposed to the test chemical for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) caused significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations.The observed mean mitotic indexin the absence of metabolic activation were 10.03, 9.81, 8.44, 7.65, 6.60 and 8.19 andin the presence ofmetabolic activation were 10.16, 9.89, 8.64, 7.80, 6.90 and 8.58 for0.0 (NC), 0.0 (VC) 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mLand 30 µg/mL (PC) concentrations respectively.

 

PHASE II

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were   0.0 (NC), 0.0 (VC) 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mLand 30µg/mL(PC)culture both in absence and presence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.667 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 11.333 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) caused significant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.13, 9.90, 8.74, 7.85, 6.65 and 8.29 and in the presence of metabolic activation were 10.03, 9.90, 8.79, 8.10, 7.30 and 8.68 for 0.0 (NC), 0.0 (VC) 0.125 (T1), 0.25 (T2) and 0.5 (T3) and 30 µg/mL (PC) concentrations respectively.

Note: NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; PC: Positive Control.

Conclusions:

The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The method followed was as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test.

 

Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 28-35 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase I and phase II. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations(0.5, 1 and 2mg/mL of culture media)based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes. Each tube according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37°C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37°C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37°C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis.

 

The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2020 to 25 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study contains experimental data of the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Purity:98.51%

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

Chinese Hamster Overy Cell Line (CHO-K1) cell was obtained from National Centre for Cell Sciences, Pune, India.
Mycoplasma contamination-free cell line was used for the study
In-house Passage No. 4 and 5

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9) was used as a metabolic activation system. The microsomal S9 fraction was obtained from the liver of sodium phenobarbitone and β-naphthoflavone-induced Wistar rats.
Composition of the S9 mix:
Sterile analytical grade water
0.2 M phosphate buffer having pH 7.4
0.1 MNADP
1 M glucose-6- phosphate
0.4 M MgCl2
1.65 M of KCl salt solution
Rat liver S9

Test concentrations with justification for top dose:

0 (VC), 3.90625, 7.8125, 15.625 or 31.25 μg/mL were selected as the doses for the main study based on results obtained from the preliminary cytotoxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was completely miscible in dimethyl sulphoxide at the concentration of 200 mg/mL. Hence, dimethyl sulphoxide was chosen as a vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate
- Number of independent experiments: Single experiment was conducted

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20000000
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 3 hrs

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):11 days
- Selection time (if incubation with a selective agent): 11 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 10% 6-Thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.0 to 2.2 million cells per concentration
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: % relative survival (RS)
- Any supplementary information relevant to cytotoxicity: CHO cells were seeded in duplicate at a density of 150 cells / 60 mm dish 5 mL culture medium. The plates were incubated at 37 ± 1° C, with approximately 5% CO2 supply for 9 days. After incubation, cells were stained with crystal violet. Cytotoxicity was expressed as % relative survival.
EXPRESSION OF THE MUTANT PHENOTYPE
The cells from the duplicate cultures of each test concentration, along with concurrent controls, were sub-cultured in 5 mL of culture medium, at a density of 2.0 to 2.2 million cells per concentration. Plates were incubated at 37 ± 1°C and sub-cultured at suitable intervals (Day 4 and day 7) for the remaining 11 days expression period.

PHENOTYPIC EXPRESSION OF MUTANT CELLS IN SELECTIVE MEDIUM
CHO cells were plated in triplicates (60 mm dish) at a density of about 0.2 million cells / 60mm dish in 5 mL culture medium containing 10% FBS and 6-Thioguanine, as a selective agent. For cloning efficiency determinations, 150 cells were plated in duplicate in 60 mm dish containing 5 mL of F12 nutrient mix medium with 10% FBS. For phenotypic expression, the plates were incubated at 37 ± 1°C, with approximately 5% CO2 supply for 11 days. After incubation, cells were stained by crystal violet, and the numbers of clones were counted.

Evaluation criteria:

Please refer additional information on materials and methods

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 31.25 ug/ml: 18% RS (-S9), 19% RS (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- pH check: Volumes of 50 µl were drawn from stock formulations of the test item, prepared in dimethyl sulphoxide, and having concentrations of 2000, 1000, 500 and 250 µg/mL (2, 1, 0.5 and 0.25 mg/mL), were added into 4.95 mL of F-12 nutrient mix medium in 15 mL culture tubes. The pH of these preparations was checked immediately (designated as 0 hrs) and after 3 hrs incubation at 37 ± 1°C. The pH of test concentrations 1000 to 250 µg/mL was found to be within the desired pH range of the vehicle control medium.
- Precipitation check: 50 µl of stock solutions having concentrations of 2000, 1000, 500 and 250 µg/mL (2, 1, 0.5 and 0.25 mg/mL), were added into 4.95 mL of F-12 nutrient mix medium in 15 mL culture tubes. These preparations were checked for precipitation with unaided eyes immediately (designated as 0 h) and after 3 hrs incubation at 37 ± 1°C.
Heavy precipitation was observed at 2000 µg/mL. Slight precipitation was observed at 1000 µg/mL, and no precipitation was observed at 500 and 250 µg/mL.

Preliminary cytotoxicity test:
CHO-K1 cells were exposed to test chemical at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 3.90625 µg/mL. Cytotoxicity was determined by the relative survival (RS) following a 3-hour treatment both in the presence and absence of S9 metabolic activation. Less than 10% relative survival was observed at concentrations from 1000 to 62.5 µg/mL. 10-20% relative survival was observed at concentrations of 31.25 µg/mL and more than 20% relative survival was observed at the concentration from 15.625 to 3.90625 µg/mL. The relative survival (RS) was 0.0, 0.0, 0.0, 0.5, 5.0, 19.9, 40.8, 58.6 and 71.3% in absence of S9 metabolic activation and 0.0, 0.0, 0.0, 0.3, 4.2, 18.4, 37.5, 58.4 and 69.0% in presence of S9 metabolic activation at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 3.90625 µg/mL respectively. 31.25 µg/mL was selected as the highest test concentration in presence and absence of metabolic activation system, for the definitive study.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The results of vehicle control considered acceptable as per the acceptance criteria. Concurrent positive controls produced a statistically significant increase in mutant frequency in the presence and absence of metabolic activation when compared with the concurrent vehicle control.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Cytotoxicity was determined by the relative survival (RS) following a 3-hour treatment both in the presence and absence of a metabolic activation system (S9). Less than 10% relative survival was observed at the concentrations from 1000 to 62.5 µg/mL. 10-20% relative survival was observed at concentration of 31.25 µg/mL, and more than 20% relative survival was observed at the concentration from 15.625 to 3.90625 µg/mL. The relative survival (RS) was observed to be 0.0, 0.0, 0.0, 0.5, 5.0, 19.9, 40.8, 58.6 and 71.3% in absence of metabolic activation system and 0.0, 0.0, 0.0, 0.3, 4.2, 18.4, 37.5, 58.4 and 69.0% in presence of metabolic activation system at the concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 3.90625 µg/mL respectively.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Available, refer to table 2 as specified in any other information on results including tables
- Negative (solvent/vehicle) historical control data: Available, refer to table 2 as specified in any other information on results including tables

 1. SUMMARY OF MUTANT FREQUENCY

                                                                                                

Experiments		Without S9		With S9
Treatment		3 hours		3 hours
Test Group	Test Concentration (µg/mL)	Mutant Frequency	Mutation Factor	Mutant Frequency	Mutation Factor
VC	0	10.03	1.00	13.60	1.00
F1	31.25	12.82	1.28	12.73	0.94
F2	15.625	14.36	1.43	9.93	0.73
F3	7.8125	12.45	1.24	10.80	0.79
F4	3.90625	19.65	1.96	13.51	0.99
PC	Ethylmethane Sulfonate (6 µg/mL)	64.76	6.46	-	-
PC	Benzopyrene (200 µg/mL)	-	-	72.02	5.30

 

Mutant frequencyis expressed as Mutants / 10⁶ Cells

2. SUMMARY

IN VITRO MAMMALIAN CELL GENE MUTATION TEST (CHO/HPRT)

LABORATORY HISTORICAL CONTROL DATA -VEHICLE CONTROL

Mutant Frequency / 10^6survival cells -Without S9							Mutant Frequency / 10^6survival cells- With S9
Vehicle	Mean	S.D.	2 S.D.	Lower	Upper		Mean	S.D.	2 S.D.	Lower	Upper
DMSO	10.77	3.88	7.76	3.01	18.53		10.52	4.15	8.30	2.22	18.82

 

LABORATORY HISTORICAL CONTROL DATA –POSITIVE CONTROL

	Mutant Frequency / 10^6survival cells -Without S9		Mutant Frequency / 10^6survival cells- With S9
	EMS(200 µg/mL)		Benzopyrene (6 µg/mL)
Mean	93.33		92.46
S.D.	67.70		69.85
2 S.D.	135.40		139.71
Lower range	42.06		47.25
Upper range	228.73		232.17

3. Relative Survival

  Experiment No. 1                                                              Without S9 Mix

Dose Level	Concentration  (µg/mL in   final medium)	Cloning Efficiency						% Relative Survival
		Individual Plate Count			Average colony counting	CE	Adjusted Cloning efficiency
		Plate 1	Plate 2	Plate 3
VC	0	126	112	116	118.0	0.79	0.99	100.0
F1	31.25	32	38	42	37.3	0.25	0.18	18.0
F2	15.625	66	76	86	76.0	0.51	0.47	47.2
F3	7.8125	78	88	73	79.7	0.53	0.61	61.2
F4	3.90625	114	84	64	87.3	0.58	0.71	71.8
PC	6	110	100	108	106.0	0.71	0.64	64.5

PC- Ethylmethanesulfonate (For without S9 treatment)

Key:CE - Cloning Efficiency

 

4. Relative Survival

  Experiment No. 1                                                                                   With S9 Mix

Dose Level	Concentration  (µg/mL in final medium)	Cloning Efficiency						% Relative Survival
		Individual Plate Count			Average colony counting	CE	Adjusted Cloning efficiency
		Plate 1	Plate 2	Plate 3
VC	0	122	126	114	120.7	0.80	1.01	100.0
F1	31.25	43	44	37	41.3	0.28	0.19	19.0
F2	15.625	66	70	66	67.3	0.45	0.40	40.2
F3	7.8125	80	82	94	85.3	0.57	0.63	62.2
F4	3.90625	90	78	90	86.0	0.57	0.71	70.9
PC	200	112	96	120	109.3	0.73	0.70	69.8

PC- Benzo(a)pyrene (For with S9 treatment)

Key:CE - Cloning Efficiency

5. PARALLEL CLONING EFFICIENCY

(% CEAND % RCE)

Experiment No. 1                                               Without S9 Mix
ID No. Dose Levels#	Concentration  (µg/mL in final medium)	Plate 1	Plate 2	Plate 3	Average	CE	% CE	% RCE
VC	0	118	124	100	114.0	0.76	76.00	100
F1	31.25	44	30	43	39.0	0.26	26.00	34.21
F2	15.625	60	34	47	47.0	0.31	31.33	41.23
F3	7.8125	90	76	75	80.3	0.54	53.56	70.47
F4	3.90625	66	85	78	76.3	0.51	50.89	66.96
PC	6	120	110	102	110.7	0.74	73.78	97.08

 

PC- Ethylmethanesulfonate (For without S9 treatment)

Key:CE - Cloning Efficiency, RCE - Relative Cloning Efficiency.

# Cloning Efficiency of pooled culture

  

6. PARALLEL CLONING EFFICIENCY (% CEAND % RCE)

 

Experiment No.1                                                                      With S9 Mix
ID No. Dose Levels #	Concentration  (µg/mL in final medium)	Plate 1	Plate 2	Plate 3	Average	CE	% CE	% RCE
VC	0	96	136	114	115.3	0.77	76.89	100
F1	31.25	42	21	30	31.0	0.21	20.67	26.88
F2	15.625	55	37	44	45.3	0.30	30.22	39.31
F3	7.8125	84	88	78	83.3	0.56	55.56	72.25
F4	3.90625	66	66	90	74.0	0.49	49.33	64.16
PC	200	116	86	100	100.7	0.67	67.11	87.28

 

PC- Benzo(a)pyrene (For with S9 treatment)

Key:CE - Cloning Efficiency, RCE - Relative Cloning Efficiency.

# Cloning Efficiency of pooled culture

 

 7. INDIVIDUAL MUTANT COLONY COUNT AND MUTANT FREQUENCY

Experiment No. 1                                                                                                                                                    Without S9 Mix

ID No.             Dose Levels #	Concentration  (µg/mL in final medium)	Mutant frequency					Mutation Factor
		Number of culture per 96 well plate				Mutant  frequency per 10^6survival cells
		Plate 1	Plate 2	Plate 3	Total
VC	0	8	1	7	16.00	10.03	1.00
F1	31.25	0	2	4	6.00	12.82	1.28
F2	15.625	2	3	4	9.00	14.36	1.43
F3	7.8125	1	7	6	14.00	12.45	1.24
F4	3.90625	5	12	4	21.00	19.65	1.96
PC	6	35	22	29	86.00	64.76	6.46

 

PC- Ethylmethanesulfonate (For without S9 treatment)

Key:# Mutant Frequency of pooled culture

 

8. INDIVIDUAL MUTANT COLONY COUNT AND MUTANT FREQUENCY

Experiment No. 1                                                                                                                                                    With S9 Mix

ID No.              Dose Levels #	Concentration  (µg/mL in final medium)	Mutant frequency					Mutation Factor
		Number of culture per 96 well plate				Mutant frequency per 10^6survival cells
		Plate 1	Plate 2	Plate 3	Total
VC	0	6	6	11	23	13.60	1.00
F1	31.25	1	3	1	5	12.73	0.94
F2	15.625	2	2	2	6	9.93	0.73
F3	7.8125	3	6	3	12	10.80	0.79
F4	3.90625	5	6	3	14	13.51	0.99
PC	200	32	35	20	87	72.02	5.30

 

PC- Benzo(a)pyrene (For with S9 treatment)

Key:# Mutant Frequency of pooled culture

 

Conclusions:

The registered substance was tested non-mutagenic (negative) in an in vitro gene mutation study performed according to OECD TG 476, when CHO cells were exposed up to 31.25 µg/ml of test chemical both in the presence and in the absence of S9 metabolic activation.

Executive summary:

In vitro gene mutation study in mammalian cells was performed according to OECD TG 476 to assess the ability of the registered substance to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the genome of Chinese hamster ovary (CHO) cells. The test concentrations were selected based on the solubility/precipitation and preliminary cytotoxicity tests. CHO cells were exposed to the test item in duplicate cultures at concentrations of 31.25, 15.625, 7.8125 and 3.90625 µg/mL for 3 hours, both in the absence and presence of S9 metabolic activation. Liver S9 fraction was prepared from sodium phenobarbitone and β-naphthoflavone-induced Wistar rats. Concurrent vehicle control (DMSO) and reference mutagens (Ethylmethanesulfonate (-S9); Benzo(a)pyrene (+S9)) were also included in the assay. The mutant frequency and  relative survival (RS, cytotoxicity) were calculated  in treated and control cultures. Results: In the absence of S9 metabolic activation, the RS values were 18%, 47.2%, 61.2%, and 71.8% at 31.25, 15.625, 7.8125 and 3.90625 µg/ml, respectively. In the presence of S9 metabolic activation, 19%, 40.2%, 62.2% and 70.9% RS values were observd at 31.25, 15.625, 7.8125 and 3.90625 µg/ml, respectively. There were no significant differences in the mutant frequency of treated cultures when compared to the vehicle control neither in the presence nor in the absence of S9 metabolic activation. The mutant frequency (mutant / 106 cell) was 10.03 (VC), 12.82 (31.25 µg/ml), 14.36 (at 15.625 µg/ml), 12.45 (at 7.8125 µg/ml), 19.65 (at 3.90625 µg/ml) without S9 and 13.60 (VC), 12.73 (31.25 µg/ml), 9.93 (at 15.625 µg/ml), 10.80 (at 7.8125 µg/ml) and 13.51 (at 3.90625 µg/ml) with S9 metabolic activation. Positive controls induced significant increase of mutant frequency (EMS: 64.76, Benzo(a)pyrene: 72.02) demonstrating the sensitivity of the assay. Conclusion: The registered substance did not induce gene mutation at Hprt locus in CHO cells with and without metabolic activation when it was tested up to a cytotxic concentration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A mouse micronucleus test was conducted to evaluate the mutagenic nature of the test chemical in vivo in male ddYmice. The test compound was dissolved in olive oil and given to male mice at a dose range of 0, 250, 313 or 500 mg/kg. Mice were irradiated with X-rays at 200 rad using a Hitachi X-ray machine with an adjusted Victreen dosimeter. After irradiation the flavoring was administered orally at the above-mentioned dosage. Bone marrow cells were sampled 24 h after the irradiation. In the time-course study, the test chemical at 500 mg/kg was given to mice immediately after the irradiation and bone marrow cells were sampled periodically at 0, 3, 6, 9, 12, 15, 18 hours. X-ray-induced chromosome aberrations were suppressed when the test chemical was given orally to mice after X-ray irradiation. Chromosome aberrations were monitored by the occurrence of polychromatic erythrocytes with micronuclei in bone marrow cells. The frequency of micronuclei was depressed about 55-60% without toxicity of the test compounds to the bone marrow. Based on the results of the current study, it is proven that the test chemical did not induce gene mutation in vivo in male ddY mice as observed by the mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Mutagenic effect of the test compound was evaluated in a mouse micronucleus test.

GLP compliance:

not specified

Type of assay:

other: Mouse bone marrow micronucleus test

Species:

mouse

Strain:

other: ddY mouse

Sex:

male

Details on test animals or test system and environmental conditions:

Details on test animals and env conditions
TEST ANIMALS
- Source: Japan SLC lnc. (Hamamatsu, Japan)
- Age at study initiation: 8 weeks (when purchased)
- Weight at study initiation: No data available
- Assigned to test groups randomly: [no/yes, under following basis: ] No data available
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil (Kosakai Seiyaku Co., Tokyo, Japan).
- Justification for choice of solvent/vehicle: The test chemical was soluble in oliv oil
- Concentration of test material in vehicle: 0, 250, 313 or 500 mg/kg with an administration volume of 10 ml/kg.
- Amount of vehicle (if gavage or dermal): No data available
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in olive oil at a concentration of 0, 250, 313 or 500 mg/kg

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

Duration of treatment / exposure:

24 hrs

Frequency of treatment:

No data available

Post exposure period:

0, 3, 6, 9, 12, 15, 18 hours

Remarks:

Doses / Concentrations:
0, 250, 313 or 500 mg/kg
Basis:
no data

No. of animals per sex per dose:

No data available

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive controls
No data available
- Justification for choice of positive control(s): No data available
- Route of administration: No data available
- Doses / concentrations: No data available

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No data available

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow cells were sampled 24 h after irradiation with X-rays. The cells were at a time interval of 0, 3, 6, 9, 12, 15, 18 hours.

DETAILS OF SLIDE PREPARATION: No data available

METHOD OF ANALYSIS: No data available

OTHER: Mice were irradiated with X-rays at 200 rad using a Hitachi X-ray machine with an adjusted Victreen dosimeter. After irradiation the flavoring was administered orally at 0, 250, 313 or 500 mg/kg. Bone marrow cells were sampled 24 h after the irradiation. In the time-course study, cinnamaldehyde at 500 mg/kg was given to mice immediately after the irradiation and bone marrow cells were sampled periodically. The micronucleus assay was performed according to the methods described by Schmid (1976).

Evaluation criteria:

The frequencies of polychromatic erythrocytes with micronuclei (MNPCEs) per 1000 polychromatic erythrocytes (PCEs), and PCEs per 1000 red blood cells per mouse were determined.

Statistics:

Student's t-test

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No data available
- Solubility: No data available
- Clinical signs of toxicity in test animals: No data available
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other: No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): The frequencies of polychromatic erythrocytes with micronuclei (MNPCEs) per 1000 polychromatic erythrocytes (PCEs), and PCEs per 1000 red blood cells per mouse were determined
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: Student's t-test

Conclusions:

The test chemical did not induce gene mutation in vivo in male ddY mice as observed by the mouse micronucleus test.

Executive summary:

A mouse micronucleus test was conducted to evaluate the mutagenic nature of the test chemical in vivo in male ddYmice. The test compound was dissolved in olive oil and given to male mice at a dose range of 0, 250, 313 or 500 mg/kg. Mice were irradiated with X-rays at 200 rad using a Hitachi X-ray machine with an adjusted Victreen dosimeter. After irradiation the flavoring was administered orally at the above-mentioned dosage. Bone marrow cells were sampled 24 h after the irradiation. In the time-course study, the test chemical at 500 mg/kg was given to mice immediately after the irradiation and bone marrow cells were sampled periodically at 0, 3, 6, 9, 12, 15, 18 hours. X-ray-induced chromosome aberrations were suppressed when the test chemical was given orally to mice after X-ray irradiation. Chromosome aberrations were monitored by the occurrence of polychromatic erythrocytes with micronuclei in bone marrow cells. The frequency of micronuclei was depressed about 55-60% without toxicity of the test compounds to the bone marrow. Based on the results of the current study, it is proven that the test chemical did not induce gene mutation in vivo in male ddY mice as observed by the mouse micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Experimental and reliable literature data available for the target chemical was reviewed to determine its mutagenic nature. The studies are mentioned below:

 

Gene mutation in vitro:

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.

 

In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The method followed was as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 28-35 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase I and phase II. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations(0.5, 1 and 2mg/mL of culture media)based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes. Each tube according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37°C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37°C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37°C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

In vitro Mammalian Cell Gene Mutation Test was carried out in compliance with the OECD Guideline No. 476, adopted by the council on 29 July 2016. The test chemical was evaluated in mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells. Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of the test chemical, the CHO cells were exposed to the test item in duplicate cultures at the doses of 31.25, 15.625, 7.8125 and 3.90625 µg/mL of culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction prepared from, sodium phenobarbitone and b- naphthoflavone-induced Wistar rats was used in the study. Dimethyl sulphoxide was used as a vehicle. The target cells were exposed to the test compound for 3 hours at 37 ± 1^oC, with approximately 5% CO₂ supply. The cells were sub-cultured immediately to determine cytotoxicity as relative survival (RS) and to initiate the phenotypic expression period. The culture flasks were incubated at 37 ± 1^oC with approximately 5% CO₂ supply during experimental periods. Concurrent vehicle and positive control groups were also included in the experiment, as specified by the test guideline. The cultures were sub-cultured during the expression period at suitable intervals. After phenotypic expression, the plates were incubated at 37 ± 1^oC with approximately 5% CO₂ supply for 11 days. After incubation, the medium was discarded, and the plates were stained and observed for the clones. Relative survival and mutant frequency were calculated for all treatment, vehicle control, and positive control groups. The relative survival (RS) was used as the measure of treatment-related cytotoxicity. The RS (relative survival) for cultures treated withthe test chemical with and without metabolic activation system (S9) indicated that the test chemical induces 10 to 20% relative survival at the concentration of 31.25 µg/mL. The % relative survival (% RS) for the cloned cultures ranged from 18.0% to71.8% for cultures treated without metabolic activation system and 19.0% to 70.9% for cultures treated with metabolic activation system at test item concentrations of 31.25, 15.625, 7.8125 and 3.90625 µg/mL in the treatment medium. The results of the present study indicate that there was no significant difference in the mutant frequencies of cultures treated withthe test chemical as compared to the vehicle control cultures, in the absence or presence of metabolic activation. An increase in the mutant frequency of concurrent positive controls demonstrated the sensitivity of the assay in the absence and presence of metabolic activation. Under the test conditions described in the study, it is concluded that the test chemical is non-mutagenic in 'InVitro Mammalian cell gene mutation test' using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells and hence it is not likely to classify as a "Gene mutant in vitro".

 

The experimental studies for the target chemical are further supported by available and reliable data from literature.

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 1.0, 3.3, 10.0, 33.0, 100 µg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. The test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

 

Another bacterial reverse mutation assay was also performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strarins TA100, TA1535, TA98, TA1537, TA1538 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activaton system. The test chemical was dissolved in DMSO and used at dose level of 0, 60, 120, 300 and 600 μg/plate. Concurrent solvent and positive control chemicals were also included in the study. The study for Salmonella strains without S9 was performed by the plate-incorporation method and the assay in the presence of S9 was performed by the pre-incubation method described by Yahagi et al. (1975). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. The mutagenicity assay with E. coli WP2 uerA trp was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli. Based on the results of the study, the test chemical did not induce gene mutation in Salmonella typhimurium strarins TA100, TA1535, TA98, TA1537, TA1538 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activaton system and hence it is not likely to classify as a gene mutant in vitro.

 

In another in vitro mammalian chromosome aberration test, the mutagenic effect the test chemical was evaluated in cloned Chinese hamster ovary (CHO-W-B1) cells in the absence and presence of an in vitro metabolic activation system (S9 mix). Cells were cultured in Mc-Coy’s 5a medium supplemented with 10% fetal calf serum, L-glutamine and antibiotics. Cells were exposed to the test chemical for 2 hr in the presence of S9 at dose level of 0.0, 50.2, 74.8, 100.3 µg/ mL or throughout the incubation period without S9 at concentrations of 0.0, 6.02, 7.96, 10.21 µg/ ml in trial 1 and 0.0, 6.4, 12.8, 18.3 µg/ ml in trial 2. Cells were collected by mitotic shake-off. Slides were stained with Giemsa. A statistically weak positive result was observed in the first trial without S9, however, this was due to the lack of aberrations in the solvent control and a slight increase based on only 18 cells at the highest dose. When repeating of the test at higher doses, the results showed no evidence for aberration induction. Based on the results provided, the test chemical was regarded to be not mutagenic in the absence and presence of S9 activation system when evaluated with a chromosomal aberration test and hence is not likely to classify as a gene mutant in vitro.

In an in vitro mammalian cell gene mutation assay, the mutagenic effects of the test chemical was evaluated in a Chinese hamster V79 cell line. The study was performed as a part to check the antimutagenic effects of the test chemical over the mutation generated by known mutagens. Cells were properly maintained in Dulbecco's MEM supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate. Cells were seeded at a density of 2.5 x 10⁶in 25-cm²tissue culture flasks. Approximately 18 h later, the medium was removed and the cells were treated with the test chemical for 2 -4 hrs. Then, cells were washed, trypsinized and 2.5 x 10⁵cells/dish were seeded in eight 100-mm petri dishes containing 20 ml of medium. The survival was measured by seeding 10²cells in three 60-mm petri dishes containing 5 ml of fresh medium. Cultures were maintained for 7 days expression time and then reseeded at a density of 5 × 10⁵cells/100-mm dish, taking four dishes for each condition. When the cells were attached, 6TG was added. Also 100 cells/60-mm dish containing growth medium were seeded in triplicate to test the viability. All colonies were stained and counted after 10 days. The mutation frequency was calculated as mutants/ 10⁶ viable cells. Since no mutation was noted in the test chemical treated V79 cells, it is likely that the test chemical is not mutagenic in vitro.

 

Also, mouse lymphoma assay was performed to evaluate the mutagenic and clastogenic potential of the test material. The assay was performed mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system. The test chemical was tested at concentrations of 0, 0.002, 0.005 or 0.007 µL/mL in the absence of S9 metabolic activation system and at the doses of 0, 0.001, 0.023 or 0.049 µL/mL in the presence of S9 metabolic activation system. The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2 × 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3×105/mL at 24 h intervals. They were then cloned (1×106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100×stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO 2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than∼0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from∼0.2 to 1.1 mm. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. Based on the details of the study, the test substance did not induce gene mutation in the mouse lymphoma L5178Y TK+/- 3.7.C cells at the lowest dose of 0.002 µL/mL in the absence of S9 metabolic activation system and at the dose of 0.023 µL/mL in the presence of S9 metabolic activation system and hence it not likely to be mutagenic in L5178Y TK +/- mouse lymphoma assay.

 

In another in vitro sister chromatid exchanges (SCEs) test, the genotoxic effects of the test chemical was evaluated in cloned Chinese hamster ovary (CHO-W-B1) cells in the absence and presence of an in vitro metabolic activation system (S9 mix). Cells were cultured in Mc-Coy’s 5a medium supplemented with 10% fetal calf serum, L-glutamine and antibiotics. In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with metabolic activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The results showed evidence of increased number of metaphasic sister chromatid exchanges, rendering to a weakly positive response for the test chemical. Therefore, the test chemical is regarded to be a weakly positive gene mutant as evaluated by the SCE assay.

 

DNA-repair test (Rec assay) was also performed to evaluate the mutagenic nature of the test chemical. The study was performed using Bacillus subtilis H17 Rec+ and M45 Rec- in the absence of metabolic activation system. 2 X 105 Spores of HI7 Rec- or M45 Rec+ were poured onto plates with 10 ml of molten nutrient agar to prepare spore-agar plates. Test samples were dissolved in ethanol immediately before use. Sample solutions (20 µL) at concentration of 0.2 mg/disk were pipetted onto sterile filter-paper disks (8 mm in diameter), which were carefully placed on the spore-agar plate. After incubation of the plate for 20-24 h at 37°C, the zones of killing (diameter of growth inhibition zone - diameter of disk) with both strains (Rec + and Rec ) were measured and the difference between them was taken as the rec effect. DNA-repair tests with S9 were not successful. Based on the results of the study, the test chemical tested positive in the Bacillus subtilis DNA-repair test (Rec assay) without S9.

 

Gene mutation in vivo:

Mouse micronucleus test was conducted to evaluate the mutagenic nature of the test chemical in vivo in male ddYmice. The test compound was dissolved in olive oil and given to male mice at a dose range of 0, 250, 313 or 500 mg/kg. Mice were irradiated with X-rays at 200 rad using a Hitachi X-ray machine with an adjusted Victreen dosimeter. After irradiation the flavoring was administered orally at the above-mentioned dosage. Bone marrow cells were sampled 24 h after the irradiation. In the time-course study, the test chemical at 500 mg/kg was given to mice immediately after the irradiation and bone marrow cells were sampled periodically at 0, 3, 6, 9, 12, 15, 18 hours. X-ray-induced chromosome aberrations were suppressed when the test chemical was given orally to mice after X-ray irradiation. Chromosome aberrations were monitored by the occurrence of polychromatic erythrocytes with micronuclei in bone marrow cells. The frequency of micronuclei was depressed about 55-60% without toxicity of the test compounds to the bone marrow. Based on the results of the current study, it is proven that the test chemical did not induce gene mutation in vivo in male ddY mice as observed by the mouse micronucleus test.

 

In another assay investigating induction of sex-linked recessive lethal (SLRL) mutations, the mutagenic effect of the test chemical was evaluated in Canton-S Drosophila melanogaster adult flies. The test chemical was administered to adult male Canton-S D. melanogaster flies in diet for 3 days in the dosage of 0 or 800 ppm. The results showed no mutagenic effects. Therefore, the test chemical was regarded to be not mutagenic when adult male Canton-S Drosophila melanogaster were exposed to the test chemical in diet for 3 days.

 

In the same study as mentioned above, in two assays investigating induction of sex-linked recessive lethal (SLRL) mutations and reciprocal translocations, the mutagenic effect of the test chemical was evaluated in Canton-S Drosophila melanogaster adult flies. The test chemical was administered to adult male Canton-S male D. melanogaster by injection in the dosage of 0 or 20000 ppm. The results showed mutagenic effects when the induction of SLRL mutations were investigated, whereas no mutagenic effects were observed in the reciprocal translocation test. Therefore, the mutagenic effects of the test chemical is regarded to be ambiguous when adult male Canton-S Drosophila melanogaster are exposed to the test chemical by injection.

 

Gene toxicity in vivo study was also performed on male ddY mice to evalaute the mutagenic nature of the test compound. The test chemical was dissolved in olive oil at dose level of 0, 62.5, 125, 250 or 500 mg/Kg. Olive oil was used as vehicle control and mitomycin C (MMC) was used as a positive control. Mice were killed by cervical dislocation at the appropriate time after an administration. Femoral marrow cells were flushed out with foetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 rain, and stained with Acridine Orange. The preparations were coded and analysed without any knowledge of the treatment. One thousand polychromatic erythrocytes per mouse were scored using a light microscope, with a high power objective (x 100), and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide. No increase in the number of MNPCEs was observed suggestive of negative mutagenic effect of the test chemical.

 

Based on the experimental studies conducted and the available literature data, the target chemical does not exhibit gene mutation vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the experimental studies conducted and the available literature data, the target chemical does not exhibit gene mutation vitro and in vivo. The positive effects though indicated in some of the studies is most probably due to the first pass extraction and metabolism of the target chemical by intestinal and hepatic tissues. Hence, cinnamaldehyde is regarded to be classified as "Not Classified" for Germ cell mutagenicity.