1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

18 Apr 2017 to 08 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

EC Number:

262-104-4

EC Name:

1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Cas Number:

60207-90-1

Molecular formula:

C15H17Cl2N3O2

IUPAC Name:

1-{[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl}-1H-1,2,4-triazole

Method

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Experiment I: 5; 15; 50; 150; 500; 1500; and 5000 µg/plate
Experiment II: 5; 15; 50; 150; 500; 1500; and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent:
The solubility of test substance was assessed at 50 mg/mL in dimethyl sulfoxide (DMSO), in which it dissolved. DMSO was, therefore, used as the vehicle for this study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: three
- Number of independent experiments : two

METHOD OF TREATMENT:
- Cell density at seeding: 10^9/mL
- Test substance added in medium (Experiment 1) and by preincubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Experiment 1 Plate incubation method: Aliquots of 0.1 mL of the test item solutions (seven concentrations up to 5000 µg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar.
- Experiment 2 Preincubation method: As a clear negative response was obtained in the first experiment, a variation to the test procedure was used for the second experiment. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 34 to 39°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 µg/plate.
- Exposure duration: 48 hours

FOR GENE MUTATION:
- All plates were incubated at approximately 34 to 39 °C at least 48 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

Evaluation criteria:

If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed additionally since this is not required by the guideline.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: S. typhimurium

Remarks:

TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Experiment 1 (Plate Incubation Assay)
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA100, TA1537 and WP2 uvrA (pKM101) following exposure to test substance and in strains TA98 and TA1535 in the presence of metabolic activation only. Precipitate was observed on all plates containing test substance at 5000 µg/plate in the absence of S9 mix. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second experiment. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.
The revertant colony count seen in the vehicle control plates in strain WP2 uvrA (pKM101) in the presence of S9 mix were above the laboratory historical control data and therefore this was repeated.

Experiment 1 repeat (Plate Incubation Assay)
Toxicity, observed as a thin background lawn of non-revertant colonies, was obtained in strain WP2 uvrA (pKM101) following exposure to test substance. No precipitate was observed on plates containing test substance. No substantial increases in revertant colony numbers over control counts were obtained with
any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in the presence of S9 mix.

Experiment 2 (Pre-incubation Assay)
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101) following exposure to test substance. Precipitate was observed on all plates containing test substance at 5000 µg/plate in the absence of S9 mix.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. An insufficient number of non-toxic concentrations was achieved in strains TA100 and TA1537 in the absence of S9 mix therefore these were repeated with a modified concentration range.

Experiment 2 repeat (Pre-incubation Assay)
Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in strains TA100 and TA1537 following exposure to test substance in the absence of S9 mix. No precipitate was observed on plates containing test substance.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 µg/plate in the absence of S9 mix.

Please, see 'Any other information on results incl. tables'

Any other information on results incl. tables

Table 1. Results Obtained in the Absence of Metabolic Activation: Experiment 1.

Strain	Addition	Concentration per plate (µg)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	DMSO		22.7	9.3		33, 20, 15
	Test subst.	5	22	3.5	1	24, 18, 24
		15	32.7	5.5	1.4	38, 27, 33
		50	23.7	3.5	1	24, 27, 20
		150	25.3	8.1	1.1	24, 18, 34
		500	24.7	3.8	1.1	23, 29, 22
		1500	22	7	1	29, 22, 15
		5000	19	5.2	0.8	13 S P, 22 S P, 22 S P
TA100	DMSO		172	19.2		194, 163, 159
	Test subst.	5	179.7	17.4	1	160, 193, 186
		15	191	5.2	1.1	188, 188, 197
		50	182	15.6	1.1	200, 172, 174
		150	163.3	2.5	0.9	161, 166, 163
		500	164.3	11.4	1	177, 161, 155
		1500	123.3	13.6	0.7	139 T, 115 T, 116 T
		5000	49	14.8	0.3	59 P V, 56 P V, 32 P V
TA1535	DMSO		24.3	3.8		27, 26, 20
	Test subst.	5	23.3	7.1	1	22, 31, 17
		15	27.3	1.5	1.1	26, 27, 29
		50	21.7	4.7	0.9	27, 20, 18
		150	20	4.6	0.8	21, 24, 15
		500	20	3.6	0.8	21, 23, 16
		1500	18.7	8.1	0.8	26, 10, 20
		5000	14.7	5.7	0.6	10 S P, 21 S P, 13 S P
TA1537	DMSO		9.7	3.2		6, 12, 11
	Test subst.	5	11.3	4.5	1.2	7, 11, 16
		15	10.3	0.6	1.1	11, 10, 10
		50	9	2.6	0.9	6, 10, 11
		150	9.7	0.6	1	10, 9, 10
		500	8	2.6	0.8	5, 9, 10
		1500	10.7	3.2	1.1	7, 12, 13
		5000	10.7	5.1	1.1	5 S P, 12 S P, 15 S P
WP2 uvrA	DMSO		238.3	12.7		227, 236, 252
	Test subst.	5	238.3	15.3	1	225, 255, 235
		15	220	6.6	0.9	221, 226, 213
		50	230.7	18.1	1	244, 210, 238
		150	243.7	7	1	237, 243, 251
		500	233.3	10	1	241, 237, 222
		1500	222.3	4.2	0.9	221, 227, 219
		5000	238.7	6.7	1	223 S P, 24 S P 6, 237 S P
TA98	2NF	2	265	76.3	11.7	353, 218, 224
TA100	NaN3	2	775.3	87.6	4.5	804, 677, 845
TA1535	NaN3	2	796.3	66.4	32.7	769, 748, 872
TA1537	AAC	50	158	57.7	16.3	220, 148, 106
WP2 uvrA	NQO	2	2775	596	11.6	2110, 3261, 2954

2NF – 2-Nitrofluorene

NaN3 – Sodium azide

AAC – 9-Aminoacridine

NQO – 4-Nitroquinoline-1-oxide

P – Precipitate

S – Slight thinning of bacterial lawn

T – Thinning of bacterial lawn

V – Severe thinning of bacterial lawn

Table 2. Results Obtained in the Presence of Metabolic Activation: Experiment 1.

Strain	Addition	Concentration per plate (µg)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	DMSO		32.7	5.5		38, 27, 33
	Test subst.	5	29	2.6	0.9	32, 27, 28
		15	31.3	9.1	1	21, 38, 35
		50	31.3	6.8	1	39, 29, 26
		150	36.7	11.9	1.1	33, 27, 50
		500	35.3	1.5	1.1	35, 34, 37
		1500	27	2.6	0.8	24, 29, 28
		5000	10	2.6	0.3	7 T, 11 T, 12 T
TA100	DMSO		210.7	10.2		215, 218, 199
	Test subst.	5	174	3	0.8	174, 177, 171
		15	191	8	0.9	183, 199, 191
		50	161.3	9.5	0.8	161, 171, 152
		150	177.3	19.4	0.8	156, 182, 194
		500	203.3	18.4	1	183, 208, 219
		1500	99.7	28.3	0.5	115 V, 117 V, 67 V
		5000	0	0	0	0 V, 0 V, 0 V
TA1535	DMSO		14.3	2.1		16, 15, 12
	Test subst.	5	11	2	0.8	9, 11, 13
		15	12.7	3.1	0.9	10, 16, 12
		50	11	1	0.8	11, 12, 10
		150	16.7	6.7	1.2	24, 11, 15
		500	12.3	4.2	0.9	9, 11, 17
		1500	15	6.9	1	23 S, 11 S, 11 S
		5000	9	3.6	0.6	12 T, 10 T, 5 T
TA1537	DMSO		10	3		13, 10, 7
	Test subst.	5	14.7	1.5	1.5	16, 15, 13
		15	12	1	1.2	12, 13, 11
		50	16	5	1.6	11, 16, 21
		150	15	4.4	1.5	12, 20, 13
		500	12.3	4.9	1.2	9, 10, 18
		1500	5	1	0.5	6 S, 5 S, 4 S
		5000	0.3	0.6	0	0 V, 0 V, 1 V
WP2 uvrA	DMSO		300.3	18.6		302, 281, 318
	Test subst.	5	338.3	35.5	1.1	373, 302, 340
		15	332	14.1	1.1	319, 330, 347
		50	326.7	21	1.1	343, 334, 303
		150	292.7	23.1	1	290, 317, 271
		500	301.7	16.5	1	288, 320, 297
		1500	271	20	0.9	251 S, 271 S, 291 S
		5000	251	8.7	0.8	241 S, 257 S, 255 S
TA98	B[a]P	5	257	10.8	7.9	254, 269, 248
TA100	AAN	5	4488	115.3	21.3	4402, 4443, 4619
TA1535	AAN	5	360	13.5	25.1	346, 373, 361
TA1537	B[a]P	5	59.3	20.6	5.9	50, 45, 83
WP2 uvrA	AAN	10	1708.3	106.4	5.7	1588, 1747, 1790

B[a]P – Benzo[a]pyrene

AAN – 2-Aminoanthracene

S – Slight thinning of bacterial lawn

T – Thinning of bacterial lawn

V – Severe thinning of bacterial lawn

 

Table 3. Results Obtained in the Presence of Metabolic Activation: Experiment 1 repeat

Strain	Addition	Concentration per plate (µg)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
WP2 uvrA	DMSO		263	13.5		276, 264, 249
	Test subst.	5	231	42.3	0.9	192, 225, 276
		15	227.3	8.6	0.9	235, 229, 218
		50	229.3	16.3	0.9	218, 222, 248
		150	247.3	7	0.9	254, 240, 248
		500	245.3	2.9	0.9	247, 247, 242
		1500	252.7	4.7	1	251 S, 249 S, 258 S
		5000	242	23	0.9	265 S, 242 S, 219 S
	AAN	10	938.3	43.2	3.6	952, 973, 890

AAN – 2-Aminoanthracene

S – Slight thinning of bacterial lawn

Table 4. Results Obtained in the Absence of Metabolic Activation: Experiment 2.

Strain	Addition	Concentration per plate (µg)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	DMSO		20.3	2.1		22, 21, 18
	Test subst.	5	19.7	1.5	1	21, 20, 18
		15	23	1.7	1.1	24, 24, 21
		50	23	3	1.1	20, 23, 26
		150	20.3	3.5	1	20, 24, 17
		500	19	6.9	0.9	15 S, 27 S, 15 S
		1500	4.7	2.3	0.2	2 T, 6 T, 6 T
		5000	4	2.6	0.2	5 V P, 1 V P, 6 V P
TA100	DMSO		117.7	9.1		111, 128, 114
	Test subst.	5	139	17.4	1.2	127, 159, 131
		15	151	14	1.3	167, 145, 141
		50	151.7	8.1	1.3	143, 159, 153
		150	62	36.4	0.5	40 V, 42 V, 104 V
		500	0	0	0	0 A, 0 A, 0 A
		1500	0	0	0	0 A, 0 A, 0 A
		5000	0	0	0	0 A P, 0 A P, 0 A P
TA1535	DMSO		15	2.6		17, 16, 12
	Test subst.	5	21.3	11.5	1.4	10, 33, 21
		15	17.7	3.8	1.2	16, 22, 15
		50	17.3	4	1.2	13, 21, 18
		150	21.7	0.6	1.4	22, 22, 21
		500	11	4.6	0.7	10 T, 16 T, 7 T
		1500	8	2.6	0.5	7 T, 6 T, 11 T
		5000	7	5.3	0.5	1 P V, 9 P V, 11 P V
TA1537	DMSO		9.3	3.5		6, 13, 9
	Test subst.	5	11.7	3.8	1.2	9, 10, 16
		15	9.3	3.8	1	11, 12, 5
		50	8.3	2.1	0.9	10, 9, 6
		150	2.3	1.5	0.3	1 V, 2 V, 4 V
		500	0	0	0	0 A, 0 A, 0 A
		1500	0	0	0	0 A, 0 A, 0 A
		5000	0	0	0	0 A P, 0 A P, 0 A P
WP2 uvrA	DMSO		192.3	14.8		176, 205, 196
	Test subst.	5	186.7	13	1	174, 186, 200
		15	191.3	12.2	1	178, 194, 202
		50	201	36.1	1	187, 174, 242
		150	210.7	7.6	1.1	214, 216, 202
		500	201.3	7.5	1	197, 210, 197
		1500	215	36	1.1	208, 254, 183
		5000	206.7	17.9	1.1	222 S P, 187 S P, 211 S P
TA98	2NF	2	163.7	54.4	8	199, 101, 191
TA100	NaN3	2	745	247.7	6.3	908, 867, 460
TA1535	NaN3	2	677	22.5	45.1	664, 664, 703
TA1537	AAC	50	117.3	6.7	12.6	119, 110, 123
WP2 uvrA	NQO	2	2909.3	273.5	15.1	3173, 2627, 2928

2NF – 2-Nitrofluorene

NaN3 – Sodium azide

AAC – 9-Aminoacridine

NQO – 4-Nitroquinoline-1-oxide

P – Precipitate

S – Slight thinning of bacterial lawn

T – Thinning of bacterial lawn

V – Severe thinning of bacterial lawn

 

Table 5. Results Obtained in the Presence of Metabolic Activation: Experiment 2.

Strain	Addition	Concentration per plate (µg)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA98	DMSO		20.7	3.1		18, 24, 20
	Test subst.	5	22.7	4.5	1.1	23, 18, 27
		15	24	6.1	1.2	27, 17, 28
		50	21	1	1	21, 20, 22
		150	22	2	1.1	20, 22, 24
		500	21.7	5.9	1	15 T, 24 T, 26 T
		1500	18	3.6	0.9	22 T, 17 T, 15 T
		5000	8	1.7	0.4	9 V, 6 V, 9 V
TA100	DMSO		134	6.1		127, 137, 138
	Test subst.	5	134	3.6	1	131, 138, 133
		15	156	3.5	1.2	154, 160, 154
		50	150.3	8.7	1.1	143, 148, 160
		150	144	22.6	1.1	165, 120, 147
		500	93.7	15.7	0.7	76 T, 99 T, 106 T
		1500	81.7	9.9	0.6	93 V, 77 V, 75 V
		5000	9.3	4	0.1	10 V, 5 V, 13 V
TA1535	DMSO		10.7	1.5		9, 12, 11
	Test subst.	5	14.7	4.7	1.4	20, 13, 11
		15	20	7.2	1.9	26, 22, 12
		50	7.7	2.1	0.7	7, 6, 10
		150	13.7	2.1	1.3	13, 16, 12
		500	14.7	3.5	1.4	11, 18, 15
		1500	7	2	0.7	7 S, 5 S, 9 S
		5000	8.7	6.5	0.8	2 T, 15 T, 9 T
TA1537	DMSO		7.7	2.1		10, 6, 7
	Test subst.	5	10.3	5.1	1.3	6, 16, 9
		15	8.7	2.3	1.1	6, 10, 10
		50	11.3	4.7	1.5	15, 6, 13
		150	9.3	2.1	1.2	7, 10, 11
		500	6.7	5	0.9	2 T, 12 T, 6 T
		1500	3.7	3.2	0.5	0 V, 5 V, 6 V
		5000	0	0	0	0 A, 0 A, 0 A
WP2 uvrA	DMSO		268	19.3		260, 254, 290
	Test subst.	5	260	14.7	1	252, 277, 251
		15	264.7	10	1	264, 255, 275
		50	253.3	18.5	0.9	263, 232, 265
		150	255.3	4.5	1	260, 255, 251
		500	255.3	22.2	1	281, 243, 242
		1500	249	11.1	0.9	259 S, 251 S, 237 S
		5000	231.3	19.4	0.9	248 S, 210 S, 236 S
TA98	B[a]P	5	220	6.6	10.6	213, 226, 221
TA100	AAN	5	2970.3	275.6	22.2	2659, 3183, 3069
TA1535	AAN	5	260.7	17.6	24.4	263, 277, 242
TA1537	B[a]P	5	59.3	3.8	7.7	62, 55, 61
WP2 uvrA	AAN	10	1485.3	77	5.5	1404, 1495, 1557

B[a]P – Benzo[a]pyrene

AAN – 2-Aminoanthracene

S – Slight thinning of bacterial lawn

T – Thinning of bacterial lawn

V – Severe thinning of bacterial lawn

 

Table 6. Results Obtained in the Absence of Metabolic Activation: Experiment 2 repeat.

Strain	Addition	Concentration per plate (µg)	Mean revertants per plate	SD	Fold increase relative to vehicle	Individual revertant colony counts
TA100	DMSO		165.3	5.9		172, 161, 163
	Test subst.	0.5	137	10.8	0.8	128, 134, 149
		1.5	176.7	13.3	1.1	169, 169, 192
		5	158	20.7	1	152, 141, 192
		15	163.3	10.4	1	155, 175, 160
		50	153	14.5	0.9	167, 154, 138
		150	119.7	26.2	0.7	144 V, 123 V, 92 V
		500	0	0	0	0 A, 0 A, 0 A
TA1535	DMSO		11.7	2.9		10, 15, 10
	Test subst.	0.5	8	3.5	0.7	10, 4, 10
		1.5	8.3	1.2	0.7	9, 7, 9
		5	10.7	2.1	0.9	13, 9, 10
		15	11.3	4	1	15, 12, 7
		50	10	5.6	0.9	9, 5, 16
		150	1.7	0.6	0.1	2 V, 1 V, 2 V
		500	0	0	0	0 A, 0 A, 0 A
TA100	NaN3	2	773.3	69.8	4.7	851, 753, 716
TA1535	AAC	50	125.7	21.7	10.8	142, 101, 134

NaN3 – Sodium azide

AAC – 9-Aminoacridine

V – Severe thinning of bacterial lawn

A – Lawn absent

Applicant's summary and conclusion

Conclusions:

In this in vitro assessment of the mutagenic potential of the test substance performed in compliance with GLP and following the OECD 471 guideline, it was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test substance performed in compliance with GLP and following the OECD 471 guideline, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test substance diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. Two independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test substance up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance in both tests. Precipitate was observed on all plates containing the test substance at 5000 µg/plate in the absence of S9 mix in the first and second test. No evidence of mutagenic activity was seen at any concentration of the test substance in either experiment. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.