2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

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:

1991-07-01 to 1991-07-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with one minor deviation from current guideline. Only four strains were tested (instead of five).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No E. coli strain tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

EC Number:

258-946-7

EC Name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

Cas Number:

54060-92-3

Molecular formula:

C20H24N3O.CH3O4S C21H27N3O5S

IUPAC Name:

2‐{[2‐(4‐methoxyphenyl)‐2‐methylhydrazin‐1‐ylidene]methyl}‐1,3,3‐trimethyl‐3H‐indol‐1‐ium methyl sulfate

Details on test material:

- Name of test material (as cited in study report): C. I. Basic Yellow 28

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
The test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
0.1 mL compound, 0.1 mL bacteria, 0.5 mL S9 mix or buffer, 2.0 mL soft agar, 45 degree C in water bath, max. 30 sec, transfer to petri dish with solid agar

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First test: 5000, 1000, 200, 40 and 8 µg/plate
Repeat tests: 800, 400, 200, 100, 50 and 25 µg/plate
70, 60, 50, 40, 30, 20 and 10 µg/plate TA 100 with 10 %, 30 %, 50 % S9

Vehicle / solvent:

The solvent employed for the test item was methanol and for the positive controls DMSO.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 µg per plate, only for strain TA 1535 w/o S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Remarks:

0.2 µg per plate, only for TA 100 w/o S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

10 µg per plate, only for TA 1537 w/o S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Remarks:

0.5 µg per plate, only for strain TA 98 w/o S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

3 µg per plate, all strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C
- Incubation period: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

Statistics:

N.A.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TA 100: 1.6-fold increase at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of the test item at 8 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used to a limited extent up to 1000 µg per plate for assessment purposes.

In the first trial, none of the four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.

The negative findings for Salmonella typhimurium TA 1535, TA 1537 and TA 98 were confirmed by further repeat tests. In the TA 100 strain, a 1.6-fold increase in comparison of the negative controls was found in the second test. The findings of the second trial for Salmonella typhimurium TA 100 were confirmed by further repeat tests using 30 % and 50 % S9 mix. No relevant increase was found using 10 % S9 mix. The lowest dose at which this finding was reproducible was approximately 30 µg per plate for Salmonella typhimurium TA 100. Minimally increased mutation rates - below the threshold for positive effects ( 2 fold) - were obtained only with S9 mix containing 30 % and 50 % S9 fraction in S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of mean values without S9-mix from the pre-test
Pre-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		8		89		7		20
8		11		84		7		18
40		12		84		6		22
200		10		88		7		22
1000		1		20		4		19
5000		0		0		0		0
Na-azid		631
NF				382
4-NPDA						56		86

Table 2: Summary of mean values with S9-mix from the pre-test
Pre-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		17		117		10		34
8		16		137		8		28
40		20		145		10		39
200		8		148		12		40
1000		--		11		3		30
5000		0		0		0		0
2-AA		99		817		134		427

Table 3: Summary of mean values without S9-mix from the repeat tests
Repeat-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		15		130		11		32
25		16		141		12		30
50		16		124		10		39
100		13		131		10		41
200		12		138		14		37
400		13		100		13		29
800		5		42		4		20
Na-azid		793
NF				416
4-NPDA						58		105

Table 4: Summary of mean values with S9-mix from the repeat tests
Repeat-Test: µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		34		190		17		53
25		27		241		21		71
50		28		303		20		74
100		13		197		20		64
200		6		106		20		50
400		--		12		3		13
800		0		--		--		--
2-AA		242		1297		68		570

Table 5: Summary of mean values with different S9-mix concentrations tested in strain TA 100
µg/plate		Strain TA 100
		30 % S9		10% S9		30% S9		50% S9
0		155		150		149		177
10		204		142		160		233
20		189		169		185		199
30		237		175		201		207
40		253		173		212		243
50		261		174		246		261
60		168		174		256		250
70		162		185		209		271
2-AA		534		1406		942		613

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

No biologically relevant and dose dependent increase in the mutant count at non-bacteriotoxic concentrations in comparison to the negative controls was observed after treatment with the test item in the presence and absence of metabolic activation. Therefore, the test item is considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (98.5% purity) in methanol at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (first/pre-test) and at concentrations of 25, 50, 100, 200, 400, 800 µg/plate (repeat test) in the presence and absence of mammalian metabolic activation. 

The test item was tested up to the limit concentration (5000 µg/plate). Based on bacteriotoxic effects only the dose groups up to 1000 µg per plate were used to a limited extent for assessment purposes. The positive controls induced the appropriate responses in the corresponding strains. In the studies with metabolic activation, a slight increase (up to 1.6 fold) in the mutant count was observed. This slight increase was not dose-dependent and did not occur in all experiments. In the studies without metabolic activation no induction of mutant counts was observed. Therefore, the test item is considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test.

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.