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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:
2010-09-29 to 2010-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008; No. L142
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
255-527-0
EC Name:
Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Cas Number:
41741-86-0
Molecular formula:
C34H24CrN8O6.Na
IUPAC Name:
Sodium;chromium(3+);2-[(3-methyl-5-oxido-1-phenylpyrazol-4-yl)diazenyl]benzoate
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from induced rats (standard plate test); liver S9 mix from uninduced hamsters (preincubation test)
Test concentrations with justification for top dose:
1st experiment (with and without rat liver S9 mix): 0, 33, 100, 333, 1000, 2500 and 5000 μg/plate
2nd experiment (with and without hamster liver S9 mix): 0, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA (with rat liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA (with hamster liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
TA 98 (with hamster liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzidine
Remarks:
TA 98 (with hamster liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
TA 1535, TA 100 (without S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
TA 98 (without S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E. coli WP2 uvrA (without S9 mix)
Details on test system and experimental conditions:
PRELIMINARY NOTE
Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substances and are used to screen for possible mammalian mutagens and carcinogens. The principle of these assays is that mutations are detected by the reversion of mutations present in the bacterial strains. This leads to a restoration of the functional capability of the bacteria to synthesize the essential amino acid and thus to the ability to grow in the absence of the amino acid required by the parent strains. Most of the substances are not mutagenic or carcinogenic themselves, but only after metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems of the liver. Therefore, the tests are carried out not only directly, but also in the presence of an exogenous metabolic activation system. The most commonly used system is a cofactor-supplemented post mitochondrial fraction (S9) obtained from livers of rats treated with an enzyme-inducing agent. The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.

TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) are thawed at room temperature, and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. As a rule, a germ density of ≥108 bacteria/mL is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay. The Salmonella strains TA 98, TA 100 and TA 1537 were obtained from KNOLL Aktiengesellschaft, Ludwigshafen, Germany, on 30 Oct 1989. The Salmonella strain TA 1535 and the Escherichia coli strain were obtained from Merck KGaA, Darmstadt, Germany (22 Apr 2010 and 09 Sep 1991, respectively).

SALMONELLA TYPHIMURIUM
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

ESCHERICHIA COLI
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

CHECKING THE TESTER STRAINS
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA is checked for UV sensitivity. Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

STANDARD PLATE TEST
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.

• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:

- 0.1 mL test solution or vehicle (negative control)
- 0.1 mL fresh bacterial culture
- 0.5 mL rat S9 mix (with metabolic activation)
or
- 0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition of the minimal glucose agar:

- 980 mL purified water
- 20 mL Vogel-Bonner E medium
- 15 g Difco bacto agar
- 20 g D-glucose, monohydrate.

After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:

- 0.1 mL test solution or vehicle (negative control)
- 0.1 mL fresh bacterial culture
- 0.5 mL rat S9 mix (with metabolic activation)
or
- 0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:

- 300 mL solution B (agar)
- 100 mL solution A (saline solution)
- 8 mL solution C (glucose solution)
- 10 mL solution D (casein solution)

After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

PRIVAL PREINCUBATION TEST
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. and has been modified further to include reductive conditions by Prival et al.
0.1 mL test solution or vehicle (negative control), 0.1 mL bacterial suspension and 0.5 mL hamster S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 30°C for 30 minutes using a shaker. Subsequently, 2 mL soft agar which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl in purified water) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin or 0.5 mM tryptophan) is added. After mixing, the samples are poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.


SCOPE OF TESTS AND TEST CONDITIONS

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Type of test: Ames standard plate test with and without rat liver S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Type of test: Prival Preincubation test with and without hamster liver S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system. A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
For results please refer to "Any other information on results incl. tables"
Cytotoxicity / choice of top concentrations:
other: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY
A bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the Ames standard plate test depending on the strain and test conditions from about 333 μg/plate onward. In the Prival preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed depending on the strain and test conditions from about 1 000 μg/plate onward.

SOLUBILITY
Test substance precipitation was found from 1000 μg/plate onward with and without S9 mix in both experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Standard Plate incorporation assay (liver S9 mix from induced rats)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli
  TA1535 TA1537 TA98 TA100 WP2 uvr A
     
  Results without S9
Spontaneous Reversion 21 7 27 98 30
Positive control 752 414 332 762 628
33 16 6 23 101 38
100 23 8 22 95 36
333 17 6 17 92 23
1000 (P) 14 6 18 90 32
2500 (P) 10 8 17 89 23
5000 (P) 12 6 15 74 28
           
  Results with S9
Spontaneous Reversion 22 10 33 100 38
Positive control 141 190 629 829 251
33 20 10 22 95 38
100 20 10 25 100 40
333 21 8 30 99 44
1000 (P) 17 7 22 97 37
2500 (P) 16 6 23 78 27
5000 (P) 12 4 18 55 25

Table 2 Prival pre-incubation assay (liver S9 mix from uninduced hamsters)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli
  TA1535 TA1537 TA98 TA100 WP2 uvr A
     
  Results without S9
Spontaneous Reversion 18 8 37 101 34
Positive control 681 364 713 777 710
33 17 9 33 122 32
100 18 9 33 105 34
333 15 7 28 104 36
1000 (P) 12 7 22 97 20
2500 (P) 10 4 19 79 23
5000 (P) 7 2 18 48 21
  Results with S9
Spontaneous Reversion 18 10 48 128 38
Positive control 162 138 729 (2-AA) 687 236
917 (Benzid.)
861 (Congor.)
33 14 9 41 111 40
100 18 7 45 124 37
333 16 11 40 113 37
1000 (P) 19 9 26 126 32
2500 (P) 13 5 24 123 23
5000 (P) 11 3 14 80 23

P = Precipitation

2 -AA = 2 -Aminoanthracene

Benzid. = Benzidine

Congor. = Congo red

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Ames standard plate test and in the Prival preincubation test in the absence and the presence of metabolic activation.