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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:
weight of evidence
Study period:
December 1983 to January 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Ames, B.W.; Durston, W.W.; Yamasaki, E.; Lee, F.D.:
Carcinogens are mutagens: A simple test system combining liver homogenates for activation; and bacteria for detection. Proc. Nat. Aced. Sci. USA, 70, 2281 - 2285 (1973)
Ames, B.N.; McCann, J.; Yamasaki, E.:
Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Nut. Res., 31, 347 - 364 (1975)
Ames, B.N.; Lee, F.D.; Durston, V.E.:
An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Sci. USA, 70, 782 - 786 (1973)
McCann, J.; Spingarn, N.E.; Kobori, J.; Ames, B.N.:
Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Nat. Aced. Sd. USA, 72, 979 - 983,(1975)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Disperse Red 91

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
1st Experiment: 0, 20, 100, 500, 2500 and 5000 μg/plate
2nd Experiment: 0, 20, 100, 500, 2500 and 5000 μg/plate
No justification specified in the study report for dose concentrations.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene; 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
S-9 fraction
The S-9 fraction is prepared according to Ames at al. 5 male Sprague-Dawley rats (200 - 300 g) received a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil – w/v) per kg body weight before sacrifice.

During this time the animals are housed individually in Makrolon cages, type M II, in air-conditioned rooms. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).

Standardized pelleted feed and tap water from bottles were available ad libitum.

On the 5th day the rats are killed by a blow on the neck, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of 4 deg C). The livers are weighed and washed in an equivalent volume of a 150 mM ICC1 solution (1 ml per 1 g wet liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate
at 9000 g for 10 minutes at +4 deg C, 5-ml portions of the supernatant (so-called S-9 fraction) were quickly deep-frozen in dry ice and stored at -70 deg C to -80 deg C for 2 months at the most.

Preparation of S-9 fraction: December 12, 1983 (1st experiment) and January 30, 1984 (2nd experiment).

S-9 mix
The S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of 5-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are:

MgCl2 : 8mM
KCl : 33mM
glucose-6-phosphate: 5 mM
NADP: 4 mM
phosphate buffer (pH 7.4): 100 mM

The phosphate buffer is prepared by mixing an Na2HPO4 solution (25.42 g/l) with an NaH2PO4 solution (22.28 g/ll) in a ratio of about 4: 1

Bacteria
The rate of induced back mutations of several bacteria mutants
from histidine auxotrophy to histidine prototrophy
was determined. The indicator organisms TA 1535, TA 1537, TA 1538, TA 98 and TA 100 selected by Ames especially for this purpose are derivatives of Salmonella typhimurium. 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 histidinephototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537, TA 1538 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 1538 and TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to 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.

Deep-frozen (-70 deg C to -80 deg C) bacterial cultures (1 ml in 15 ml glass tubes) are thawed at room temperature, 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco bacto nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37 deg C for 16 hours. As a rule, a germ density of > 10 E+08 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.

Mutagenicity tests
The experimental procedure is based on the method of Ames et al.
Test tubes containing 2-ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mN histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto the nutrient plates within approx. 30 seconds.
Composition of the nutrient medium (minimal agar):
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation for 48 hours at 37 °C in the dark, the bacterial colonies (his+ revertants) are counted.

Titer determination
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent, only) and after adding the two highest doses of test substance. For this purpose, 0.1 ml of the overnight cultures is diluted to 106 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution for titer determination (5 m!' histidine + 5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 10-6)
0.5 ml S-9 mix
After mixing, the samples are poured onto the nutrient plates within approx. 30 seconds. After incubation for 48 hours at 37 °C in the dark, the bacterial colonies (his+ revertants) are counted.

Checking out 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).
Histidine auxotrophy is automatically checked in each experiment via, the spontaneous rate.

Controls
Negative control
Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive control
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix 10 μg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537, TA 1538 and TA 1535
without S-9 mix 5 μg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 μg 4nitro-o-phenylenediamine (dissolved in DMSO) for the strains TA 98 and TA 1538
100 μg 9-aminoacridine chloride monohydrate (dissolved in DMSO) Tor the strain TA 1537.

Tester strains, doses, number of plates
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 1538, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 μg/plate
Solvent: DMSO
Type of test, standard plate test with and without test condition: S-9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1538
Doses: 0, 20, 100, 500, 2500 and 5000 μg/plate
Solvent: DMSO
Type of test, standard plate test with and without test condition: S-9 mix
Number of plates: 3 test plates per dose or per control
Rationale for test conditions:
In accordance with experimental guidelines.
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfil the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Statistics:
Not specified

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
weak increace in MF only at precipitating concentrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
weak increace in MF only at precipitating concentrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
weak increace in MF only at precipitating concentrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutagenicity tests
Tests without S-9 mix
TA 1535; TA 100: No increase in the number of his+ revertants.
TA 1537; TA 1538; TA 98: Only a weak increase in the number of his+ revertants from 2500 μg/plate onward with a maximum increase in the mutation rate depending on the strain by a factor 2.4 - 3.7 at 5000 μg/plate.

Tests with S-9 mix
TA 1535; TA 100: No increase in the number of hjs+ revertants.
TA 1537: Slight increase in the number of revertants without any dose dependency.
TA 1538; TA 98: Weakly positive reaction was observed at 2500 μg --5000 μg/plate with an increase in the number of mutant colonies by a factor of 2.1 - 3.2.

Toxicity
No bacteriotoxic effect (reduced his background growth) was observed.

Solubility
Incomplete solubility of the test substance in DMSO from 2500 μg/plate onward.

Assessment/Discussion
According to the results of the present study, the test substance is weakly mutagenic in the Ames test at precipitating concentration in frame-shiht strains. However, the slight, dose-dependent reaction was observed only at high doses (> 2500 μg/plate) at which the dye precipitated on the plate, and therefore implies, that this effects might be caused by anthraquinonic impurities.
The test substance itself was predicted as negative in the OECD QSAR Toolbox.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the test substance is weakly mutagenic in the Ames test at precipitating concentration in frame-shiht strains. However, the slight, dose-dependent reaction was observed only at high doses (> 2500 μg/plate) at which the dye precipitated on the plate, and therefore implies, that this effects might be caused by anthraquinonic impurities.
The test substance itself was predicted as negative in the OECD QSAR Toolbox.
Executive summary:

The test substance was tested for mutagenicity in the Ames test.

 

Strains: TA 1535, TA 100, TA 1537, TA 1538, TA 98

 

Dose range: 20 μg - 5000 μg/plate

 

Test conditions: Standard plate test with and without metabolic activation (S-9, mix).

 

Mutagenicity

TA 1535; TA 100: No increase in the number of his+ revertants.

TA 1537; TA 1538; TA 98: Weakly mutagenic from dose ≥ 2500 μg/plate onward with a maximum increase in the number of revertant colonies depending on the strain and test conditions by a factor 2.4 - 3.7 at 5000 μg/plate.

 

Toxicity

No bacteriotoxic effect (reduced his background growth) was observed.

 

Solubility

Incomplete solubility of the test substance in DMSO from 2500 μg/plate onward.

 

Assessment/Discussion

According to the results of the present study, the test substance is weakly mutagenic in the Ames test under the experimental conditions chosen here. However, the slight, dose-dependent reaction was observed only at high doses (> 2500 μg/plate) at which the dye precipitated on the plate, and therefore implies, that this effects might be caused by anthraquinonic impurities.

The test substance itself was predicted as negative in the OECD QSAR Toolbox.