Decan-5-olide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 2020 to 29 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study contains experimental data with the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: July 21st, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decan-5-olide

EC Number:

211-889-1

EC Name:

Decan-5-olide

Cas Number:

705-86-2

Molecular formula:

C10H18O2

IUPAC Name:

6-pentyloxan-2-one

Test material form:

liquid

Remarks:

Colorless clear liquid

Details on test material:

Name of test material:
- CAS No. 705-86-2
- EC No. 211-889-1
- Molecular formula: C10H18O2
- Molecular weight: 170.25 g/mol
- Subsatnce type: Organic
- Physical state: Colorless clear liquid
- Purity: 98.4% (GC)

Specific details on test material used for the study:

Appearance: Colorless clear liquid
Purity (GC): 98.4%

Method

Target gene:

Histidine Operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced S9 was procured from Defence Research and Development Establishment, Nagpur (India)
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution, which contains 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 in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium
: 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Metabolic capability of S9 is certified.

Test concentrations with justification for top dose:

Test concentrations:
0,0 (NC), 0.0 (VC) 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate

Justification:
Test concentrations were selected based on a preliminary cytotoxicity experiment. This pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5.000 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The cytotoxicity was detected as a reduction in the number of spontaneous revertants or inhibition of the bacterial background lawn growth.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO at 50 mg/ml.

Controls

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine for strains TA1537, TA98 (without metabolic activation) 2-Aminoanthracene for strains TA 1535, TA1537, TA98, TA 100 and TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): NA
SELECTION AGENT (mutation assays): NA
SPINDLE INHIBITOR (cytogenetic assays): NA
STAIN (for cytogenetic assays): NA
NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicates in two independent experiments (Trial I-II).
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: NA
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was determined as a reduction in revertant counts and /or inhibition of the background lawn growth.

Evaluation criteria:

The substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding vehicle/(solvent) control was observed. A dose-dependent increase was considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose-dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative control and vehicle control, such an increase was not considered biologically relevant.

Statistics:

The mean values of the plates for each concentration together with standard deviation were compared with the spontaneous reversion rates of solvent treated cultures. Microsoft Office Excel-based calculation was used for descriptive statistical analysis.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertant counts was seen at 5 mg/plate with and without S9 mix in Trial I and Trial II.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data available
- Data on osmolality: No data available
- Possibility of evaporation from medium: No data available
- Water solubility: Insoluble in water, but soluble in DMSO at 50 mg/ml.
- Precipitation: No precipitation was observed in 5 mg/plate concentration.
RANGE-FINDING/SCREENING STUDIES (if applicable):
To evaluate the cytotoxicity of the substance, a pre-experiment was performed with strains TA 98 and TA 100 according to the plate incorporation methods. Bacterial cells were exposed to the concentrations of 0.0 (NC), 0.0 (VC), 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for 48 hours using triplicates.
At concentrations of 0.001-0.158 mg/plate, no reduction in colony count or clearing of the background lawn was observed. At 0.501 mg/plate, there was no decrease in revertant counts but a slight inhibition of the background lawn growth. At 1.582 mg/plate, moderate inhibition of the background lawn and no substantial decrease in the number of revertant colonies were seen. At 5 mg/plate marked reduction in the number of revertant counts and moderate inhibition of the background lawn was observed. Hence, the mutagenicity test was performed with the following test concentrations: 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate.
STUDY RESULTS
- Positive control data : Positive controls induced an unequivocal increase in revertant counts in all the five tester strains compared to respective controls used.
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: Please refer to the table.
- Negative (solvent/vehicle) historical control data: Please refer to the table.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE 1 : REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Absence (-S9) of Metabolic Activation		Presence (+S9) of Metabolic Activation
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	94	14	104	15
	R2	95	19	108	19
	R3	92	15	103	21
VC (0.00)	R1	125	26	142	31
	R2	126	28	137	28
	R3	129	31	141	33
T1 (0.001)	R1	103	23	136	19
	R2	108	26	130	25
	R3	104	21	129	21
T2 (0.005)	R1	114	18	125	19
	R2	116	24	129	19
	R3	111	20	124	23
T3 (0.015)	R1	100	25	119	27
	R2	105	26	123	29
	R3	103	21	126	30
T4 (0.050)	R1	96	29	116	32
	R2	100	24	118	28
	R3	102	26	120	25
T5 (0.158)	R1	113	19	120	29
	R2	118	26	124	27
	R3	114	23	126	24
T6 (0.501)	R1	120 ++++	20 ++++	119 ++++	20 ++++
	R2	117 ++++	22 ++++	122 ++++	25 ++++
	R3	123 ++++	24 ++++	115 ++++	21 ++++
T7 (1.582)	R1	114 +++	26 +++	125 +++	18 +++
	R2	109 +++	25 +++	120 +++	25 +++
	R3	111 +++	23 +++	122 +++	21 +++
T8 (5.000)	R1	81 +++	15 +++	93 +++	15 +++
	R2	87 +++	12 +++	97 +++	19 +++
	R3	85 +++	16 +++	100 +++	13 +++
PC	R1	908	516	1136	824
	R2	1012	492	1056	752
	R3	972	524	1128	800

NC = Negative control, VC = Vehicle Control, PC = Positive control, R = Replicate, ++++=Slight inhibition, +++ = Moderate inhibition.

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 (+S9) of Metabolic Activation
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	11	15	104	240
	R2	5	8	19	108	228
	R3	5	10	21	103	234
VC (0.00)	R1	7	14	31	142	296
	R2	6	16	28	137	285
	R3	8	13	33	141	291
T1 (0.050)	R1	5	14	32	116	281
	R2	5	14	28	118	295
	R3	7	12	25	120	269
T2 (0.158)	R1	7	12	29	120	268
	R2	6	16	27	124	257
	R3	5	13	24	126	238
T3 (0.501)	R1	6	10	20	119	235
	R2	8	10	25	122	248
	R3	5	12	21	115	252
T4 (1.582)	R1	5	14	18	125	267
	R2	5	11	25	120	284
	R3	6	12	21	122	278
T5 (5.000)	R1	4	9	15	93	251
	R2	4	8	19	97	246
	R3	5	11	13	100	239
PC	R1	152	320	824	1136	1784
	R2	180	424	752	1056	1680
	R3	168	408	800	1128	1624

Dose (mg/plate)	R	In the Absence (-S9) of Metabolic Activation
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	3	9	14	94	226
	R2	3	10	19	95	235
	R3	4	8	15	92	240
VC (0.00)	R1	5	15	26	125	286
	R2	6	17	28	126	306
	R3	6	14	31	129	291
T1 (0.050)	R1	5	14	29	96	261
	R2	4	12	24	100	255
	R3	5	12	26	102	269
T2 (0.158)	R1	5	12	19	113	251
	R2	6	11	26	118	229
	R3	4	14	23	114	243
T3 (0.501)	R1	5	14	20	120	262
	R2	5	15	22	117	248
	R3	6	12	24	123	269
T4 (1.582)	R1	5	10	26	114	224
	R2	4	11	25	109	251
	R3	4	13	23	111	238
T5 (5.000)	R1	3	8	15	81	238
	R2	5	8	12	87	252
	R3	3	10	16	85	230
PC	R1	112	1080	516	908	1608
	R2	124	1032	492	1012	1584
	R3	136	1008	524	972	1704

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

Dose (mg/plate)	R	In the Presence (+S9) of Metabolic Activation
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	10	20	109	266
	R2	5	9	24	113	281
	R3	5	11	23	110	260
VC (0.00)	R1	7	15	27	126	282
	R2	7	18	30	120	295
	R3	6	16	29	124	290
T1 (0.050)	R1	5	12	24	120	267
	R2	8	11	27	116	272
	R3	5	14	25	119	256
T2 (0.158)	R1	7	13	23	119	286
	R2	6	11	22	116	294
	R3	4	11	25	114	275
T3 (0.501)	R1	6	14	23	120	283
	R2	8	13	25	123	272
	R3	5	11	26	119	263
T4 (1.582)	R1	4	15	25	117	278
	R2	5	13	21	115	283
	R3	6	14	23	114	291
T5 (5.000)	R1	6	9	19	120	255
	R2	6	12	24	123	268
	R3	4	10	23	121	261
PC	R1	140	296	820	1624	1344
	R2	136	256	936	1528	1440
	R3	128	240	884	1496	1392

 

Dose (mg/plate)	R	In the Absence (-S9) of Metabolic Activation
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	8	16	108	256
	R2	5	8	18	105	260
	R3	4	11	21	109	271
VC (0.00)	R1	8	16	26	126	292
	R2	6	17	30	125	286
	R3	6	14	32	128	280
T1 (0.050)	R1	7	15	24	123	278
	R2	6	16	27	125	264
	R3	5	12	21	121	260
T2 (0.158)	R1	6	10	23	114	272
	R2	5	10	25	118	283
	R3	5	13	22	113	268
T3 (0.501)	R1	5	14	26	119	264
	R2	8	13	23	121	270
	R3	6	14	28	123	282
T4 (1.582)	R1	7	11	20	117	275
	R2	5	10	20	120	280
	R3	5	9	24	122	286
T5 (5.000)	R1	5	14	19	118	276
	R2	4	12	20	115	283
	R3	4	11	22	116	272
PC	R1	148	960	528	1112	1352
	R2	152	1016	620	1000	1424
	R3	160	928	592	1080	1480

TABLE 4: MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL-I)

Dose (mg/plate)	In the Presence (+S9) of Metabolic Activation
	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	9.67	1.53	18.33	3.06	105.00	2.65	234.00	6.00
VC (0.00)	7.00	1.00	14.33	1.53	30.67	2.52	140.00	2.65	290.67	5.51
T1 (0.050)	5.67	1.15	13.33	1.15	28.33	3.51	118.00	2.00	281.67	13.01
T2 (0.158)	6.00	1.00	13.67	2.08	26.67	2.52	123.33	3.06	254.33	15.18
T3 (0.501)	6.33	1.53	10.67	1.15	22.00	2.65	118.67	3.51	245.00	8.89
T4 (1.582)	5.33	0.58	12.33	1.53	21.33	3.51	122.33	2.52	276.33	8.62
T5 (5.000)	4.33	0.58	9.33	1.53	15.67	3.06	96.67	3.51	245.33	6.03
PC	166.67	14.05	384.00	56.00	792.00	36.66	1106.67	44.06	1696.00	81.19

Dose (mg/plate)	In the Absence (-S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	3.33	0.58	9.00	1.00	16.00	2.65	93.67	1.53	233.67	7.09
VC (0.00)	5.67	0.58	15.33	1.53	28.33	2.52	126.67	2.08	294.33	10.41
T1 (0.050)	4.67	0.58	12.67	1.15	26.33	2.52	99.33	3.06	261.67	7.02
T2 (0.158)	5.00	1.00	12.33	1.53	22.67	3.51	115.00	2.65	241.00	11.14
T3 (0.501)	5.33	0.58	13.67	1.53	22.00	2.00	120.00	3.00	259.67	10.69
T4 (1.582)	4.33	0.58	11.33	1.53	24.67	1.53	111.33	2.52	237.67	13.50
T5 (5.000)	3.67	1.15	8.67	1.15	14.33	2.08	84.33	3.06	240.00	11.14
PC	124.00	12.00	1040.00	36.66	510.67	16.65	964.00	52.46	1632.00	63.50

Key:-

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 5 : MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL-II)

Dose (mg/plate)	In the Presence (+S9) of Metabolic Activation
	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	10.00	1.00	22.33	2.08	110.67	2.08	269.00	10.82
VC (0.00)	6.67	0.58	16.33	1.53	28.67	1.53	123.33	3.06	289.00	6.56
T1 (0.050)	6.00	1.73	12.33	1.53	25.33	1.53	118.33	2.08	265.00	8.19
T2 (0.158)	5.67	1.53	11.67	1.15	23.33	1.53	116.33	2.52	285.00	9.54
T3 (0.501)	6.33	1.53	12.67	1.53	24.67	1.53	120.67	2.08	272.67	10.02
T4 (1.582)	5.00	1.00	14.00	1.00	23.00	2.00	115.33	1.53	284.00	6.56
T5 (5.000)	5.33	1.15	10.33	1.53	22.00	2.65	121.33	1.53	261.33	6.51
PC	134.67	6.11	264.00	28.84	880.00	58.10	1549.33	66.61	1392.00	48.00

Dose (mg/plate)	In the Absence (-S9) of Metabolic Activation
	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	9.00	1.73	18.33	2.52	107.33	2.08	262.33	7.77
VC (0.00)	6.67	1.15	15.67	1.53	29.33	3.06	126.33	1.53	286.00	6.00
T1 (0.050)	6.00	1.00	14.33	2.08	24.00	3.00	123.00	2.00	267.33	9.45
T2 (0.158)	5.33	0.58	11.00	1.73	23.33	1.53	115.00	2.65	274.33	7.77
T3 (0.501)	6.33	1.53	13.67	0.58	25.67	2.52	121.00	2.00	272.00	9.17
T4 (1.582)	5.67	1.15	10.00	1.00	21.33	2.31	119.67	2.52	280.33	5.51
T5 (5.000)	4.33	0.58	12.33	1.53	20.33	1.53	116.33	1.53	277.00	5.57
PC	153.33	6.11	968.00	44.54	580.00	47.16	1064.00	57.69	1418.67	64.17

Key:-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

 HISTORICAL CONTROL DATA

These data represent the laboratory's historical control data.

Trial I (Plate Incorporation Method)
Strains	Metabolic Activation	Treatment	Mean	SD	Max	Min
TA 1537	S9 +	Negative control	6	2	10	2
	S9 -		6	2	10	2
	S9 +	Solvent control	6	2	10	2
	S9 -		6	2	10	2
	S9 +	Positive control	168	38	245	92
	S9 -		175	43	261	89
TA 1535	S9 +	Negative control	12	3	18	7
	S9 -		12	3	18	7
	S9 +	Solvent control	13	3	18	7
	S9 -		13	3	18	7
	S9 +	Positive control	336	211	757	86
	S9 -		1200	263	1726	674
TA 98	S9 +	Negative control	24	6	36	11
	S9 -		23	6	35	11
	S9 +	Solvent control	25	6	37	13
	S9 -		23	5	33	13
	S9 +	Positive control	1099	312	1722	476
	S9 -		815	284	1383	248
TA 100	S9 +	Negative control	117	28	173	61
	S9 -		114	26	166	62
	S9 +	Solvent control	116	28	172	60
	S9 -		113	26	165	61
	S9 +	Positive control	1488	390	2268	709
	S9 -		1311	298	1906	715
TA 102	S9 +	Negative control	274	42	358	190
	S9 -		271	55	382	161
	S9 +	Solvent control	279	65	409	150
	S9 -		277	82	442	112
	S9 +	Positive control	1648	305	2258	1037
	S9 -		1896	364	2624	1168

Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

 

 

8        HISTORICAL CONTROL DATA (Contd.)

Trial II (Pre-Incubation Method)
Strains	Metabolic Activation	Treatment	Mean	SD	Max	Min
TA 1537	S9 +	Negative control	6	2	10	2
	S9 -		6	2	10	2
	S9 +	Solvent control	6	2	10	3
	S9 -		6	2	10	2
	S9 +	Positive control	170	39	249	91
	S9 -		182	43	268	96
TA 1535	S9 +	Negative control	13	3	18	7
	S9 -		12	3	18	7
	S9 +	Solvent control	13	3	18	8
	S9 -		13	3	18	7
	S9 +	Positive control	299	197	694	145
	S9 -		1244	260	1765	724
TA 98	S9 +	Negative control	24	6	35	13
	S9 -		23	5	33	13
	S9 +	Solvent control	24	5	35	14
	S9 -		23	5	32	14
	S9 +	Positive control	1269	275	1819	719
	S9 -		740	210	1160	320
TA 100	S9 +	Negative control	117	25	166	67
	S9 -		113	23	159	66
	S9 +	Solvent control	116	22	159	73
	S9 -		112	20	151	73
	S9 +	Positive control	1469	347	2163	775
	S9 -		1352	263	1878	827
TA 102	S9 +	Negative control	281	32	345	218
	S9 -		276	28	331	220
	S9 +	Solvent control	281	34	350	212
	S9 -		276	34	344	207
	S9 +	Positive control	1595	287	2168	1022
	S9 -		1753	248	2248	1258

Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

Applicant's summary and conclusion

Conclusions:

The registered substance, Decan-5-olide (CAS No. 705-86-2) tested non-mutagenic (negative) in Salmonella Typhimurium 1535, TA 1537, TA 98, TA 100 and TA 102 tester strains in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and in compliance with GLP.

Executive summary:

The potential of Decan-5-olide (CAS No. 705-86-2) to induce point mutations or frameshifts with the histidine operon was tested in Salmonella Typhimurium 1535, TA 1537, TA 98, TA 100 and TA 102 tester strains in the presence and absence of a metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. Dimethyl sulfoxide was selected as a vehicle of the test substance. Test concentrations were selected based on a preliminary cytotoxicity test. This pre-experiment was performed with strains TA 98 and TA 100 according to the plate incorporation methods. Bacterial cells were exposed to the substance at concentrations of 0.0 (NC), 0.0 (VC), 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for 48 hours using triplicates. At concentrations of 0.001-0.158 mg/plate, no reduction in colony count or clearing of the background lawn was observed. At 0.501 mg/plate, there was no decrease in revertant counts but a slight inhibition of the background lawn growth. At 1.582 mg/plate, moderate inhibition of the background lawn and no substantial decrease in the number of revertant colonies were seen. At 5 mg/plate marked reduction in the number of revertant counts and moderate inhibition of the background lawn was observed. Hence, the mutagenicity test was performed with the following test concentrations: 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate with and without S9 metabolic activation. The main test consisted of two trials.

Trial I was performed according to the plate incorporation method using five test concentrations along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA1537, TA1535, and TA102. For TA98 and TA 100 the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the required five concentrations (from 0.050 mg/plate to 5.000 mg/plate). Trial-II was performed independently with all the five tester strains and the negative, vehicle, and positive controls by preincubation method to confirm the results of Trial-I. Results: No substantial increase in the number of revertant colonies compared to the vehicle control was observed at concentrations tested in any of the five tester strains, either in the presence or absence of S9 metabolic activation in both trials. Cytotoxicity was detected at 5 mg/plate as a reduction in the number of revertant colonies. The positive controls induced unequivocal increases in revertant counts in all the five tester strains compared to vehicle controls in Trial I and Trial II. Conclusion: The test chemical did not induce gene mutations by base pair changes or frameshifts 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. The test was performed according to OECD TG 471.