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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-03-09 to 1989-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1984
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)
EC Number:
404-540-1
EC Name:
A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)
Cas Number:
159405-95-5
Molecular formula:
C50H56Cl4N14O6
IUPAC Name:
1'-[3-(dimethylamino)propyl]-5'-(2-{3-[2-(4-{2-[3-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium 1'-[3-(dimethylamino)propyl]-5'-(2-{4-[2-(4-{2-[4-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium tetrahydrochloride tetrachloride
Test material form:
solid

Method

Target gene:
The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella Iyphimurium bacterial strains to produce histidine-independent strains of these micro-organisms.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in the galactose metabolism; chi: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultravioletrepair B gene)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 3.3, 10, 33.3, 100, 333 µg/plate
Concentration range in the main test (without metabolic activation): 1, 3.3, 10, 33.3, 100 µg/plate
Vehicle / solvent:
Solvent: deionised (Milli-Q) water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycine
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (direct plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
The survival of the TA100 culture is determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate.
In the absence of S9-mix the survival of strain TA100 is severely reduced at test substance concentrations from 333 µg/plate upwards. Both in the absence and presence of S9-mix the survival is eliminated from 1000 µg/plate upwards. Based on these data, the test substance was tested up to a concentration of 100 µg/plate in the absence of S9-mix and up to 333 µg/plate in the presence of S9-mix.

Evaluation criteria:
A test substance was considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic)in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.
Statistics:
see "data evaluation criteria"

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
other: not tested; only 4 strains.
Cytotoxicity / choice of top concentrations:
other: not tested; only 4 strains.
Vehicle controls validity:
not applicable
Positive controls validity:
not applicable
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
range-finding test
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 333 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
not applicable
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
not determined
Remarks:
range-finding test
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1000 µg/plate
Vehicle controls validity:
not specified
Positive controls validity:
not applicable
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no effects
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity. The highest concentration of test article used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates. If no toxicity was observed, the highest dose level used in the mutagenesis assay was 5 mg/plate unless the test article exhibited limited solubility or was not uniformly dispersible in the solvent of choice.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values fell within the laboratory's background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Preliminary toxicity determination in TA 100

conc. µg/plate viable counts/plate (duplicate plates)
without S9 mix with S9-mix
control 420 465 490 500
1.0 317 385 357 365
3.3 418 350 366 348
10.0 398 377 319 345
33.3 358 318 413 372
100 298 199 318 353
333 87 98 218 225
1000 0 0 0 0
3330 0 0 0 0
5000 0 0 0 0

Experiment 1

conc. µg/plate without S9-mix
TA1535 TA1537 TA98 TA100
control 13±2 18±4 32±2 120±11
1.0 10±4 20±6 28±4 121±18
3.3 10±3 17±6 27±2 102±12
10.0 6±4 15±2 26±7 112±15
33.3 11±3 22±4 35±6 121±3
100 8±1 20±6 22±4 121±15
positive control 200±13 477±72 503±130 576±267
with S9-mix
control 8±2 11±4 26±4 114±13
3.3 6±1 15±3 22±1 124±7
10.0 7±2 15±4 23±2 11±10
33.3 8±5 10±1 26±9 123±7
100 10±2 16±6 26±3 125±3
333.3 10±3 13±3 30±7 112±3
positive control 189±20 414±14 599±48 730±84

Experiment 2

conc. µg/plate without S9-mix
TA1535 TA1537 TA98 TA100
control 8±5 13±4 28±6 105±20
1.0 12±3 15±3 31±11 109±11
3.3 12±5 15±1 36±10 114±12
10.0 9±3 15±4 30±10 110±5
33.3 10±7 16±4 22±2 108±7
100 11±2 14±1 28±4 126±8
positive control 201±6 477±72 1103±70 586±54
with S9-mix
control 9±2 7±1 34±2 122±2
3.3 15±1 13±2 38±6 135±11
10.0 9±1 8±5 26±4 126±9
33.3 14±1 8±3 35±8 125±6
100 13±3 11±1 37±5 132±3
333.3 8±4 10±2 44±3 139±12
positive control 161±34 588±76 705±29 815±7

Applicant's summary and conclusion

Conclusions:
Negative: test substance can be considered as not mutagenic under test conditions of the Ames Salmonella/microsome assay.
Executive summary:

Aim of the study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains to produce histidine-independent strains of these micro-organisms. The study followed OECD guideline 471.

The substance was tested in the Ames Salmonella/microsome plate test up to 100 µg/plate in the absence of S9-mix and up to 333 µg/plate in the presence of S9-mix (dose range as result of a preliminary range finding study).

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values fell within our laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Based on these findings, the test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535; TA1537; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, thus, be considered as not mutagenic under test conditions.