Reaction products of 2,4-bis(2-chlorophenyl)-5-(3,4-dimethoxy phenyl)-1H-imidazole and 2-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 100 w/w% (as a reaction product)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Dose Setup Test: 5, 15.8, 50, 158, 500, 1581, 5000 μg/plate
Main Test: 156, 313, 625, 1250, 2500, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The reason for this is that the substance to be tested was dissolved at less than 50 mg/mL in water and 50 mg/mL or more in DMSO.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Positive control substance:

other: AF-2 (2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide)

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Controls 4

Positive controls:

yes

Positive control substance:

other: ICR-191 (2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl)

Remarks:

TA1537 (-S9)

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA1535, WP2, TA98, and TA1537 (+S9)

Details on test system and experimental conditions:

PREPARATION METHOD OF S9
Species and system: Rat and SD system
Sex: Male
Weekly age: 7 weeks
Weight: 203.7 ± 9.7 g
Induced substance: PB and 5,6-BF
Dosage method: Intra-abdominal dosage
Dosage period and dose (g/kg weight): PB 30 mg/kg (1 time) and 60 mg/kg (3 times); 5,6-BF 80 mg/kg (1 time)

COMPOSITION OF S9 MIX
Component: Amount of 1 mL S9 mix
S9: 0.1 mL
MgCl2: 8 μmol
KCl: 33 μmol
Glucose-6-phosphoric acid: 5 μmol
NADPH: 4 μmol
Na-phosphoric acid buffer solution: 100 μmol

CONDITIONS OF PRE-CULTURE
Nutrient broth: Nutrient Broth No. 2: UNIPATH Ltd.
Pre-culture time: 8 hr
Incubator (shape and volume): L-shaped tube (volume of 27 mL)
Amount of culture solution: 10 mL
Amount of inoculated bacteria: 20 μl

NUMBER OF LIVE BACTERIA AT A TIME OF THE PRE-CULTURE END (x 1e9/mL)
TA100: 2.5 (both Dose setup and Main tests)
TA1535: 2.3 (both Dose setup and Main tests)
WP2 uvrA: 4.4 (both Dose setup and Main tests)
TA98: 2.3 (both Dose setup and Main tests)
TA1537: 2.4 Dose setup test (2.3 Main test)

GLUCOSE AGAR PLATE MEDIUM: Bacto-Agar, Difco Laboratores

TESTING CONDITIONS
Bacteria suspension: 0.1 mL
Testing substance solution: 0.1 mL
Na-phosphoric acid buffer solution (based on the direct method): 0.5 mL
S9 mix (based on the metabolic activation method): 0.5 mL
Top agar: 2
Pre-incubation Temperature: 37 ± 0.5 °C
Pre-incubation Time: 20 hr
Incubation Temperature: 37 ± 0.5 °C
Incubation Time: 48 hr

Evaluation criteria:

not reported

Statistics:

not reported

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: The precipitation of the test substance was recognized at 156 μg/plate in the absence of S9 mix and 500 μg/plate or more in the presence of S9 mix; however, since they could be identified as reverse mutation colonies, there was no trouble in counting the number of reverse mutation colonies.

Any other information on results incl. tables

Table 1, Dose Setup Test: Average Number of Reverse Mutation (number of colony/plate)

Metabolic Activation System	Dose (μg/plate)	TA100 (b)	TA1535 (b)	WP2 uvrA (c)	TA98 (c)	TA1537 (c)
-S9 mix	Negative control	121	13	26	28	8
-S9 mix	5	123	10	21	30	7
-S9 mix	15.8	131	16	26	32	11
-S9 mix	50	117	12	28	28	10
-S9 mix	158 (a)	115	11	17	24	9
-S9 mix	500 (a)	118	13	21	29	7
-S9 mix	1581 (a)	113	12	22	31	5
-S9 mix	5000 (a)	119	10	24	28	8
+S9 mix	Negative control	122	10	27	36	22
+S9 mix	5	110	10	33	43	22
+S9 mix	15.8	112	12	22	38	19
+S9 mix	50	129	11	28	35	17
+S9 mix	158	120	14	26	35	20
+S9 mix	500 (a)	132	6	24	33	16
+S9 mix	1581 (a)	123	9	26	40	16
+S9 mix	5000 (a)	129	10	26	38	14
Positive controls, Not requiring S9	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
Positive controls, Not requiring S9	Dose (μg/plate)	0.01	0.5	0.01	0.1	0.5
Positive controls, Not requiring S9	Average colony/plate	406	352	152	469	226
Positive controls, Requiring S9	Name	2AA	2AA	2AA	2AA	2AA
Positive controls, Requiring S9	Dose (μg/plate)	1	2	10	0.5	2
Positive controls, Requiring S9	Average colony/plate	1065	154	820	372	217

a: Precipitation was noted

b: Base-pair substitution type

c: Frameshift type

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl

2AA: 2-aminoanthracene

--

Table 2, Main Test: Average Number of Reverse Mutation (number of colony/plate)

Metabolic Activation System	Dose (μg/plate)	TA100 (b)	TA1535 (b)	WP2 uvrA (c)	TA98 (c)	TA1537 (c)
-S9 mix	Negative control	114	7	26	16	7
-S9 mix	156 (a)	113	10	19	23	7
-S9 mix	313 (a)	106	8	18	17	6
-S9 mix	625 (a)	117	9	18	15	6
-S9 mix	1250 (a)	108	9	18	15	9
-S9 mix	2500 (a)	112	9	16	18	10
-S9 mix	5000 (a)	112	10	15	17	7
+S9 mix	Negative control	108	9	24	28	8
+S9 mix	156	126	7	24	25	11
+S9 mix	313	107	10	19	32	11
+S9 mix	625 (a)	126	5	19	21	12
+S9 mix	1250 (a)	124	7	23	25	11
+S9 mix	2500 (a)	129	14	23	25	11
+S9 mix	5000 (a)	139	10	25	26	10
Positive controls, Not requiring S9	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
Positive controls, Not requiring S9	Dose (μg/plate)	0.01	0.5	0.01	0.1	0.5
Positive controls, Not requiring S9	Average colony/plate	466	252	138	488	289
Positive controls, Requiring S9	Name	2AA	2AA	2AA	2AA	2AA
Positive controls, Requiring S9	Dose (μg/plate)	1	2	10	0.5	2
Positive controls, Requiring S9	Average colony/plate	1154	153	839	376	216

a: Precipitation was noted

b: Base-pair substitution type

c: Frameshift type

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl

2AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Regardless of the existence of S9 mix, since the increase in the number of reverse mutation colonies was not shown for all testing strains at any set dose, it was decided that the mutagenicity was negative.

• The precipitation of the substance to be tested was recognized at 156 μg/plate in the absence of S9 mix and 500 μg/plate or more in the presence of S9 mix; however, since they could be identified as reverse mutation colonies, there was no trouble in counting the number of reverse mutation colonies.
• No growth hindrance was observed.

Executive summary:

The test substance, was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the pre-incubation method. Salmonella typhimurium strains TA98, TA 100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were tested in the presence and absence of an exogenous metabolic activation system (PB- and 5,6-BF-induced rat liver S9).

The test was performed in 2 phases. The Dose Setup Test used concentrations of 5, 15.8, 50, 158, 500, 1581, 5000 μg/plate. The Main Test used concentrations of 156, 313, 625, 1250, 2500, 5000 μg/plate. No growth hindrance was observed. Regardless of the existence of S9 mix, since the increase in the number of reverse mutation colonies was not shown for all testing strains at any set dose, it was decided that the mutagenicity was negative.