Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 March to 14 March, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
5 Salmonella typhimurium strains used; no E. coli strains used

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Cobalt, 4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate 4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate sodium complexes and Cobaltate(3-), bis[4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(3-)]-, trisodium and Cobaltate(5-), bis[4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(4-)]-, pentasodium
EC Number:
916-898-0
Molecular formula:
C64H46CoN18Na5O22S6 C64H47CoN18Na4O19S5 C64H48CoN18Na3O16S4
IUPAC Name:
Reaction mass of Cobalt, 4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate 4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate sodium complexes and Cobaltate(3-), bis[4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(3-)]-, trisodium and Cobaltate(5-), bis[4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(4-)]-, pentasodium
Test material form:
solid: particulate/powder

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G base-pair substitution Additional mutations: Δ uvr B, rfa
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C frameshift Additional mutations: Δ uvr B, rfa
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: his D frameshift Additional mutations: Δ uvr B, rfa
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D frameshift Additional mutations: Δ uvr B, rfa, pKM101
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G base-pair substitution Additional mutations: Δ uvr B, rfa, pKM101
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 Mix
Test concentrations with justification for top dose:
-Experiment concentrations: 1.0, 10.0, 100.0, 500.0, 1000.0, 2500.0, 5000.0, 10000.0 µg/plate
Doses for the actual assay were selected from a preliminary study conducted on the test material at 14 doses: 1.22, 2.44, 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.50, 625.00, 1250, 2500.00, 5000.00, 10000.00 µg per plate using the strain TA-100. In this preliminary study, the test material did not exhibit toxicity to the indicator strain at any of the doses tested, as evidenced by the appearance of the background lawn on the minimal plates.
Vehicle / solvent:
sterile deionized water
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
MICROORGANISM
The Salmonella typhimurium strains used in this assay were obtained from Dr. Bruce Ames, University of California at Berkeley.
All indicator strains were kept at 4 °C on minimal medium plates supplemented with a trace of biotin and an excess of histidine. The plates with plasmid-carrying strains contain in addition ampicillin (25 µg/ml) to ensure stable maintenance of plasmid pKM101. New stock culture plates are made as often as necessary from frozen master cultures or from single colony reisolates that were checked for their genotypic characteristics (his, rfa, uvrB, big) and for the presence of plasmid.
For each experiment, an inoculum from the stock culture plates is grown overnight at 37 °C in nutrient broth (Oxoid CM67).
MEDIA
The bacterial strains were cultured in Oxoid Media #2 (nutrient Broth). The selective medium was Vogel Bonner Medium E with 2 % glucose. The overlay agar consisted of 0.6 % purified agar with 0.05 mM histidine, 0.05 mM biotin and 0.1 M NaCl.
ACTIVATION SYSTEM
- S9 Homogenate: A 9,000 x g supernatant prepared from Sprague Dawley adult male rat liver induced by Aroclor 1254 was purchased commercially and used in this assay.
- S9 Mix composition: 4 µmol/ml of NADP (sodium salt), 5 µmol/ml D-glucose-6-phosphate, 8 µmol/ml MgCl2, 33 µmol/ml KCl, 100 µmol/ml sodium phosphate buffer pH 7.4, organ homogenate from rat liver (S9 fraction) 100 µl/ml
Rationale for test conditions:
The Salmonella typhimurium strains used are all histidine auxotrophs by virtue of mutations in the histidine operon. when these histidine-dependent cells are grown in a minimal media petri plate containing a trace of histidine, only those cells that revert to histidine independence (his+) are able to form colonies. The trace amount of histidine allows all the plated bacteria to undergo a few divisions; this growth is essential for mutagenesis to occur. The his+ revertants are easily scored as colonies against the slight background growth. The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar the mutation frequency is increased 2- to 100-fold. Cells which grow to form colonies on the minimal media petri plates are therefore assumed to have reverted, either spontaneously or by the action of a test substance to his+ genotype.
Evaluation criteria:
Because the procedures used to evaluate the mutagenicity of the test material were semiquantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria:
(1) Strains TA-1535, TA-1537 and TA-1538
If the solvent control value is within the normal range, a test material producing a positive response equal to three times the solvent control value is considered mutagenic.
(2) Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material producing a positive response equal to twice the solvent control value for TA-98 and TA-100 is considered mutagenic.
The following normal range of revertants for solvent controls are generally considered acceptable:
TA-1535: 8-30
TA-1537: 4-30
TA-1538: 10-35
TA-98: 20-75
TA-100 : 80-250
(3) Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are both derived from the same parental strain (D3052), to some extent there is a built-in redundancy in the microbial assay. In general, the two strains of a set respond to the same mutagen and such a pattern is sought. Generally, if a strain responds to a mutagen in nonactivation tests, it will do so in activation tests. Occasionally, exception to this pattern may also be seen.

The demonstration of dose-related increases in mutant counts is an important criterion in establishing mutagenicity. Since, several doses were employed in the actual assay, a dose response would normally be seen with a mutagenic test material. Additional tests may be performed at narrower dose, if the mutagenic test material fails to exhibit a dose-response in the initial assay. However, occasionally it is difficult to generate a dose-response and the test material will be evaluated based on the available data.
Statistics:
Statistical methods were not used.
Plate test data consisted of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Because the test material and the cells were incubated in the overlay for approximately 2 days and a few cell divisions occurred during the incubation period, the test is semiquantitative in nature. Although these features of the assay reduce the quantitation of result, they provide certain advantages not contained in a quantitative suspension test:
- The small number of cell divisions permits potential mutagens to act in replicating DNA, which is often more sensitive than nonreplicating DNA.
- The combined incubation of the test material and the cells in the overlay permits constant exposure of the indicator cells for approximately 2 days.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA-1535, TA-1537,TA-1538, TA-98 and TA-100 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity

Any other information on results incl. tables

The test with the strain TA-1537 was repeated because the activation system was not added to the positive control plates during the first trial. The repeat test was also negative.

Applicant's summary and conclusion

Conclusions:
The test item is not considered mutagenic in the Ames assay using five Salmonella typhimurium strains
Executive summary:

The test item was evaluated for its potential to induce mutagenic effects in an in vitro bacterial inverse mutation assay (Ames test), according to a method similar to the OECD 471 guideline. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to test item concentrations ranging from 1.0 to 10,000 µg/plate both in the presence and absence of S9 metabolic activation. The test item showed no toxicity to the TA-100 indicator strain during a preliminary test, which determined the appropriate test item concentrations.

The test item showed no genetic activity in any of the concentrations or replicates conducted, with or without metabolic activation. Therefore, the test item can be considered non-mutagenic in these test conditions. The higher concentrations used counterbalance the shortcomings that could result from the use of a test item with a low purity used suggest that the use of the experimental results for the evaluation of mutagenicity is acceptable.