Veratrole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 October 2003 to 26 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD guideline compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: 2-anthamine was used as the sole positive control for the efficacy of S9- mix, whereas an other positive control (e.g BaP should have been use in addition to 2-anthramine)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Veratrole

EC Number:

202-045-3

EC Name:

Veratrole

Cas Number:

91-16-7

Molecular formula:

C8H10O2

IUPAC Name:

1,2-dimethoxybenzene

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Vératrole

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced with Aroclor 1254

Test concentrations with justification for top dose:

Pre-test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without metabolic activation.
Main test:   312.5, 625, 1250, 2500, 5000 µg/plate with and without metabolic activation.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO 0.1 ml/2ml overlay agar
- Justification for choice of solvent/vehicle: substance freely soluble in DMSO
-Vehicle controls tested: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
-without activation, and first experiment with activation: in agar (plate incorporation);
- with activation, second experiment: preincubation

DURATION
- Preincubation period: 60 minutes

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY - Method: other: reduction in number of revertant colonies per plate and/or a thinning of the bacterial lawn.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.

Statistics:

The number of revertants per plate was scored for each strain and concentration. The mean number of revertants, with standard deviation and ratio of mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle were determined.

The Reviewer considers the analyses used to be appropriate.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity testing with strains TA 98, TA 100 and TA 102 did not demonstrate toxicity up to 5000 µg per plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results obtained for solvent and positive controls were comparable to historic values

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed at the two highest concentrations with metabolic activation in the preincubation assay

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: Number of revertants per plate (mean of 3 plates), First experiment: Direct plate incorporation method

 

	TA 1535			TA 1537			TA 98
Conc. [unit]	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	16	15	No	8	9	No	24	35	No
312.5	12	11	No	9	6	No	28	21	No
625	11	15	No	6	11	No	21	32	No
1250	13	13	No	7	8	No	33	26	No
2500	7	11	No	6	6	No	35	25	No
5000	11	9	no	5	5	No	20	16	No
Positive control	420	168	no	371	102	no	190	2054	no

*solvent control with DMSO

S.D. Standard deviation

MA: metabolic activation

Table 7.6.1/2: Number of revertants per plate (mean of 3 plates), First experiment: Direct plate incorporation method

	TA 100					TA 102
Conc. [unit]	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)
0*	137	75	75	21	No	511	108	452	102	No
312.5	88	5	77	9	No	346	17	297	115	No
625	145	96	119	47	No	435	79	340	21	No
1250	86	8	74	20	No	321	17	201	33	No
2500	77	15	109	38	No	280	12	293	40	No
5000	89	25	65	9	no	185	22	193	13	No
Positive control	386	142	637	166	no	1202	49	4021	203	no

*solvent control with DMSO

S.D. Standard deviation

MA: metabolic activation

Table 7.6.1/3: Number of revertants per plate (mean of 3 plates), Second experiment:

Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)

	TA 1535					TA 1537					TA 98
Conc. [unit]	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)
0*	10	3	16	6	No	9	4	8	3	No	28	3	33	6	No
312.5	12	3	17	6	No	6	1	7	1	No	40	20	24	5	No
625	13	3	15	4	No	9	3	11	3	No	24	4	25	5	No
1250	9	5	13	3	no	8	3	12	4	No	34	10	30	11	No
2500	16	6	12	2	No	9	4	9	3	Yes	22	6	26	7	yes
5000	15	4	8	3	yes	5	3	1	1	yes	25	12	10	8	yes
Positive control	518	46	73	12		262	165	75	11		172	14	1009	16

*solvent control with DMSO

S.D. Standard deviation

MA: metabolic activation

Table 7.6.1/4: Number of revertants per plate (mean of 3 plates), Second experiment:

Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)

	TA 100					TA 102
Conc. [unit]	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)	- MA	S.D.	+ MA	S.D.	Cytotoxic (yes/no)
0*	88	12	101	11	No	365	38	489	47	No
312.5	125	42	120	32	No	363	27	538	134	No
625	94	7	96	18	No	362	4	504	44	No
1250	144	76	122	38	No	368	49	545	137	No
2500	125	9	89	14	yes	319	27	263	53	yes
5000	141	50	0	0	yes	230	29	28	49	yes
Positive control	604	18	492	74		1846	98	3484	350

*solvent control with DMSO

MA: metabolic activation

Applicant's summary and conclusion

Conclusions:

Negative with and without metabolic activation.
Veratrole was considered to be non-mutagenic in bacteria under the conditions of the test.

Executive summary:

The objective of this study was to evaluate the potential of the test item Veratrole to induce reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels of Vératrole to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item Veratrole was dissolved in dimethylsulfoxide (DMSO).

Five known mutagens (Sodium azide; 9 -Aminoacridine; 2 -nitrofluorene; Mitomycine C and 2 -Anthramine) were used to check the sensitivity of the test system. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

A moderate to marked toxicity was noted towards all the strains used, generally at dose-levels ≥ 2500 μg/plate, with S9 mix using the preincubation method.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under these experimental conditions, the test item Veratrole did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium with and without metabolic activation.