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Genetic toxicity: in vitro

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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:
From November 3 to 9, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. The OECD recommended combination of strains was tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
yes
Remarks:
(no detrimental impact on the outcome of the study).
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
(no detrimental impact on the outcome of the study).
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
EC Number:
224-073-5
EC Name:
Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Cas Number:
4193-55-9
Molecular formula:
C40H42N12Na2O10S2
IUPAC Name:
disodium 5-({4-anilino-6-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl}amino)-2-{2-[4-({4-[bis(2-hydroxyethyl)amino]-6-(cyclohexa-1,3-dien-1-ylamino)-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]vinyl}benzenesulfonate

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
TA 1537, genotype his C 3076; rfa-; uvrB; frame shift mutations.
TA 98, genotype his D 3052; rfa-; uvrB; R-factor; frame shift mutations.
TA 1535, genotype his G 46; rfa-; uvrB; base-pair mutations.
TA 100, genotype his G 46; rfa-; uvrB; R-factor; base-pair mutations.
WP2, genotype trp; trp; uvrA; base-pair substitutions and others
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
On the day of the experiment, the test article was dissolved in deionised water.
The solvent was chosen because of its solubility properties and its relative non- toxicity to the bacteria.
No precipitation of the test article occurred up to the highest investigated dose.
Controls
Untreated negative controls:
yes
Remarks:
current untreated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene
Remarks:
Without met. act: NaN3 for TA 1535, TA 100; 4-NOPD for TA 1537, TA 98; MMS for WP2 uvrA. With met. act: 2-M for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA.
Details on test system and experimental conditions:
PRECULTURE
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100.
This nutrient medium contains per litre:
8 9 Merck Nutrient Broth (MERCK, 0-64293 Darmstadt)
5 9 NaCI (MERCK, 0-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar: the plates with the selective agar were obtained from E. Merck, 0-64293 Darmstadt.

Overlay Agar: the overlay agar contains per litre:
for Salmonella strains:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
For Escherichia coli:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
2.5 mg Tryptophan*
* (MERCK, 0-64293 Darmstadt)
Sterilisations were performed at 121°C in an autoclave.

S9 PREPARATION
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to over- come this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm which received daily applications of 80 mg/kg b.w. Phenobarbital ip. dissolved in aqua deionised and β-Naphthotlavone orally dissolved in corn oil on three subsequent days. The livers were prepared 24 hours after the last treatment.
After decapitation of the anaesthetised animals the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at - 20 °C for up to one week before use. The protein content was determined using an analysis kit of BioRad Laboratories, D-80939 Munchen.
The protein concentration in the S9 preparation was 23.7 mg/ml.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT TOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

DOSE SELECTION
Based upon the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. Two independent experiments were performed.
According to the dose selection criteria the test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate .

TEST PERFORMANCE
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
Evaluation criteria:
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

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
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results

without S9 mix

Conc. µg/plate TA 1535 TA 1537 TA 98 TA 100 WP2 uvr A
I II I II I II I II I II
Negative control 16 12 20 9 22 15 81 110 40 34
Solvent control 14 13 16 10 19 16 99 131 47 38
Positive control* 1023 951 108 138 283 169 286 453 683 356
33 9 11 18 11 25 20 91 117 56 46
100 17 12 15 10 21 14 55 105 38 28
333 12 13 17 12 20 16 87 112 42 37
1000 14 18 14 11 19 14 46 127 40 35
2500 12 16 17 6 19 20 59 124 53 32
5000 18 18 20 10 19 15 94 127 48 31

with S9 mix

Conc. µg/plate TA 1535 TA 1537 TA 98 TA 100 WP2 uvr A
I II I II I II I II I II
Negative control 17 17 17 6 34 27 78 125 70 40
Solvent control 21 15 27 8 28 26 84 127 54 42
Positive control** 120 89 127 93 204 60 416 401 167 112
33 21 20 23 14 25 18 68 119 52 33
100 24 18 16 8 27 24 69 115 44 36
333 17 20 25 12 28 25 77 129 52 44
1000 19 15 22 13 20 20 78 115 50 38
2500 20 17 19 11 38 28 77 115 59 55
5000 19 19 17 9 40 26 81 125 56 49

*Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100

4 -nito-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate)

Methyl methane snlfonate (5 µl/plate) strain WP2 uvrA

**2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98 and TA 100

2 -aminoanthracene (10.0 µg/plate) strain WP2 11vrA

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

Method

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Results

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifcs in the genome of the strains used. Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.