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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance has no mutagenic potential in bacteria cells

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached read-across justification
Reason / purpose for cross-reference:
read-across source
Target gene:
Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution
Metabolic activation system:
S9-Mix from rat and hamster liver
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The results lead to the conclusion that the substance is not mutagenic in the absence and presence of metabolic activation using the standard Ames Test procedure (plate incorporation test) and the preincubation test as described.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: 21-July-1997 and U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, Aug-1998andEC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14andJapanese Substance Control Law (JSCL) Test Guideline III.l Gene Mutation Test with bacteria.The study is based on the Principles of Good Laboratory Practice (GLP).


 The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the substance was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000 μg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies. The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled. The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.


The substance did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate. In both studies toxicity was not observed either with or without metabolic activation.


 In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence or presence of the rat liver activation system (10 % (v/v)). Also in the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.


Summarizing, it can be stated that the substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2003 to 28 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
incorporating Privall Mitchell preincubation test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
JSCL TG III.1 Gene mutation test with bacteria
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine dependent
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: tryptophan dependent
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from rat and hamster liver
Test concentrations with justification for top dose:
50, 160, 500, 1600, 5000 µg/plate
Vehicle / solvent:
deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
w/o S9 TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
w/o S9 TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
w/o S9 TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
w/o S9 WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with rat S9 10%: TA98, TA100, TA1535, TA1537, WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
with hamster S9 30% TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with hamster S9 30%: TA100, TA1535, TA1537, WP2uvrA
Details on test system and experimental conditions:
Test groups
plate incorporation test:
with metabolic activation (10 % rat liver): 50,160, 500, 1600 and 5000 μg/plate
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate

preincubation test:
with metabolic activation (30 % hamster liver): 50, 160, 500, 1600 and 5000 μg/plate
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate

Control groups
negative controls:
a: untreated controls
b: solvent controls (0 μg/plate)

positive controls:
- without metabolic activation: sodium-azide for strain TA 100 and TA 1535, 9-aminoacridine for strain TA 1537, 2-nitrofluorene for strain TA 98, 4-nitroquinoline-N-oxide for strain WP2uvrA
- with metabolic activation (10 % rat liver): 2-aminoanthracene for all strains
- with metabolic activation (30 % syrian golden hamster liver): 2-aminoanthracene for strain TA 100, TA 1535, TA 1537 and WP2uvrA, congo red for strain TA 98

Formulation of test compound: dissolved in deionised water at appropriate concentrations immediately before use.

Formulation of reference compounds
Sodium-azide dissolved in deionised water final concentration: 1.0μg/plate for strain TA 1535, 2.0μg/plate for strain TA 100
9-aminoacridine dissolved in DMSO final concentration: 50.0μg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5μg/plate for strain AT 98
4-nitroquinoline-N-oxide dissolved in DMSO final concentrations: 2.0μg/plate (plate inc.), 0.5μg/plate (preinc.) for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations: (10% (v/v) rat liver S9-mix): 1.5μg/plate for strains TA 98, TA 100, TA 1535 and TA 1537, 20.0μg/plate for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations (30% (v/v) hamster liver S9-mix): 1.0μg/plate for strain TA 100, TA 1535 and TA 1537, 30.0μg/plate for strain WP2uvrA
Congo red dissolved in deionised water final concentration: 250μg/plate for strain TA98
The frozen stock solutions of each compounds were diluted progressively up to the final concentration on the day of treatment.

Source of bacteria: stock cultures in the bank of “Genetic Toxicology”, Aventis Pharma Germany, ProTox prepared from the original bacteria strains.

Test organism: Salmonella typhimurium strains – TA 98 hisD3052 rfa uvrB +R, TA 100 hisG46 rfa uvrB +R, TA 1535 hisG46 rfa uvrB, TA 1537 hisC3076 rfa uvrB and Escherichia coli WP2uvrA pKM101

Experimental conditions in vitro: approx. 37°C in an incubator.

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction of Spraque Dawley rat liver induced with Aroclor 1254 was obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, (828) 264-9099. The protein content for every batch was guaranteed by a Quality Control & Production Certificate by the supplier. Also for every batch of S9 an independent validation was performed in the laboratory with a minimum of two different mutagens, e.g. 2-aminoanthracene and benzo(a)pyrene, to confirm metabolic activation by microsomal enzymes.
The S9 fraction of Syrian golden hamster liver was prepared by the department conducting the study according to Prival et. al (1982). Male Syrian golden hamsters (7-8 weeks old), were supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany. Liver preparations were performed from the liver of non pretreated Syrian hamsters. The livers were removed from 10 male Syrian hamsters (7-8 weeks old) using cold sterile solutions at approx. 0 to 4 °C and glassware, and were then pooled and washed in approx. 150 mM KC1 (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KC1. The homogenate was centrifuged at approx. 9000g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g. 2-aminoanthracene and congo red, to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed at room temperature immediately before each test. One volume of Moltox. S9 fraction (batch no. 1530 for the plate incorporation test, protein concentration 36.1 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the rat liver S9-mix were:

8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
4mM NADP
100 mM phosphate buffer pH 7.4

According to the modification proposed by Prival (8) the test substance and the tester strains were
preincubated for 20 to 30 minutes with 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction (batch no. 2002/1 for the preincubation, protein concentration
45 g/l) were mixed with 7 volumes of the S9 cofactor solution.

This preparation is termed S9-mix. The hamster liver S9-mix consists of:

8 mM MgCl2
33 mM KC1
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4mM NADP+
2 mM NADH
2 mM FMN (Riboflavine-5’-phosphate-sodium-salt)
100 mM phosphate buffer pH 7.4

Bacteria
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. coli was obtained from E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored in liquid nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeability, ampicillin resistance, tetracyclin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.

ASSAY PROCEDURE
An independent mutation test was performed using the plate incorporation method. When results were negative or equivocal, a second test was conducted. This included a preincubation step if the first test was clearly negative. Preincubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in deionised water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approx. 30 °C.

After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose).
After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
STERILITY CHECKS AND CONTROL PLATES
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.
The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled.
The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.

SOLUBILITY AND TOXICITY
The substance was dissolved in deionized water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were used in all experiments.
The substance did not precipitate on the plates up to the highest investigated dose of 5000μg/plate.

The substance proved to be not toxic to the bacterial strains.

MUTAGENICITY
The number of colonies per plate with each strain as well as mean values of 3 plates were given.

Plate incorporation test:
The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.
Preincubation test:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increase in the number of revertant colonies under the experimental conditions described.
Conclusions:
The results lead to the conclusion that the substance is not mutagenic in the absence and presence of metabolic activation using the standard Ames Test procedure (plate incorporation test) and the preincubation test as described.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: 21-July-1997 and U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, Aug-1998andEC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14andJapanese Substance Control Law (JSCL) Test Guideline III.l Gene Mutation Test with bacteria.The study is based on the Principles of Good Laboratory Practice (GLP).


 The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the substance was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000 μg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies. The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled. The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.


The substance did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate. In both studies toxicity was not observed either with or without metabolic activation.


 In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence or presence of the rat liver activation system (10 % (v/v)). Also in the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.


Summarizing, it can be stated that the substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.

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:
1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
1. Ames, B.N., Durston, W.E., Yarnasaki, E. and Lee, F.D. Proc. Nat. Acad. Sc!. USA (1973), 70, 2281.
2. Ames, B.N., McCann, J. and Yarnasaki, E., Mutation Res. (1975),31, 347.
3. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. Proc. Nat. Acad. Sci. USA (1975), 21, 5135.
4. Garner, R.C., Miller, E.C. and Miller,~J.A., Cancer Res. (1972),33, 2058.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift
TA1538 hisD3052 Frameshift

A!l five strains are defective in DNA repair capacity ( uvr-) and have a defective lipopolysaccharide barrier on the cell wall (rfa-). These two properties confer extra sensitivity to DNA damage and also allow greater permeability of large molecules into the cell. Strains TA 98 and TA 100 also contain a resistance transfer factor (plasmid PKM 101). This factor, which confers resistance to ampicillin, enhances the operation of an error prone repair system.

Escherichia coli:
WP2uvrA trpE Base pair substitution
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Dose levels:
Dose range finding test: 5000, 500, 50, 5 µg/plate.
Mutation test: (a) 5000, 2500, 1250, 625, 312.5 µg/plate.
(b) 10000, 5000, 2500 µg/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
w/o S9 in TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine TA98, TA1537, TA538
Remarks:
w/o S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
w/o S9 E. coli
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with rat S9: TA98, TA100, TA1535, WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Neutral red - TA1537
Remarks:
with rat S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
with rat S9: TA1538
Evaluation criteria:
A compound will be deemed to provide evidence of mutagenic potential if the highest number of revertant colonies is:
(a) at least 2.5 times the control level, and
(b) a dose-related response is obtained
Statistics:
No data
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The results lead to the conclusion that Reactive Red 230 is not mutagenic in the absence and presence of metabolic activation
Executive summary:

49/81 A did not show any toxicity towards the six bacterial tester strains in the preliminary test, therefore 5000 µg/plate was chosen as the top dose level for the mutation test.

With tester strain E. coli WP2 uvrA both in the presence and absence of liver microsomal fraction a slight increase in revertant colony numbers was observed at the 5000 µg/plate dose level only. As this increase was very small and restricted to one dose level it was not thought to be significant. However, a repeat test was performed in order to clarify the situation with dose levels up to 10000 µg/plate. In the repeat test 49/81A did not produce any substantial increases in revertant colony numbers.

No substantial increases in the revertant colony numbers of any of the remaining five strains were observed following treatment with 49/81 A at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix).

It is hence concluded that no evidence of mutagenic potential of 49/81A was obtained in this bacterial test system at the dose levels tested.

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:
October 07th, 1992 to October 23rd 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift
TA1538 hisD3052 Frameshift
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from rat and hamster liver
Test concentrations with justification for top dose:
4 to 5000 µg/plate
Vehicle / solvent:
At the day of the experiment the test substance was dissolved in aqua bidest at appropriate concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
w/o S9; TA100, TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
w/o S9; TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
w/o S9; TA1538, TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with rat S9 10%: TA98, TA100, TA1535, TA1537, TA1538;
with rat S9 30%: TA98, TA100, TA1535, TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
with hamster S9 30%: TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzidine
Remarks:
with hamster S9 30%: TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with hamster S9 30%: TA100, TA1535, TA1537
Details on test system and experimental conditions:
Two independent experiments for each of the two protocols (Ames, Prival) were performed.
a) - with 10 %rat liver S-9 Mix or buffer and the strains TA 100. TA 1535, TA 1537, TA 1538 and TA 98
- with 30 %rat liver S-9 Mix and the stains TA 100. TA 1535, TA 1537 and TA 98
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 %NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at approximatly 45°C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml 10 %or 30 %rat liver S-9 Mix or buffer

After mixing. the liquid is poured into a petridish with minimal agar 1.5 % agar, Vogel-Bonner E medium with 2 %glucose). After incubation for approximatly 48 hours at approx. 37 oC in the dark. colonies (his+ revertants) are counted.
b) with 30 %Syrian golden hamster S-9 Mix and preincubation
0.1 ml test solution. 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at approx. 30°C for the durati on of 30 minutes. Subsequently, 2 ml 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 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation for approx. 48 hours at approx. 37°C in the dark, colonies (his+ revertants) are counted.
Evaluation criteria:
A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test article produces at least a 2-fold incr€ase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.
Statistics:
No data
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
up to 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

The test compound was tested at doses of 4 to 10 000 microgram/plate and proved to be not toxic to the bacterial strains.
For mutagenicity testing 5 000 microgram/plate was chosen as the highest dose in the main experiments.

Ames-Test:
The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat S-9 Mix. No dose dependent effect was obtained.

Prival-Test:
In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.
It is concluded that Reaktiv-Rot F-67787 is not mutagenic in the absence and presence of rat S-9 Mix (10 %) using the standard Ames Test procedure. Also in the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) and preincubati on the test compound did not induce a significant increase in the number of revertant colonies.
Conclusions:
The test substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.
Executive summary:

Reaktiv-Rot F-67787 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 microgram/plate to 5 000 microgram/plate was used.


Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.


Toxicity: The test compound proved to be not toxic to the bacterial strains. 5 000 microgram/plate was chosen as top dose level for the mutagenicity study.


a) Ames Test:


Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 %), treatment of the cells with Reaktiv-Rot F-67787 did not result in relevant increases in the number of revertant colonies.


b) Prival Test:


In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival Reaktiv-Rot F-67787 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.


Summarizing, it can be stated that Reaktiv-Rot F-67787 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Two separate bacterial reverse mutation tests, showed that the test substance does not have any mutagenic properties in different Salmonella and E. Coli strains.


 


In the first study, the test substance was tested in in strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of Salmonella typhimuriums and in the strain Escherichia coli WP2 uvrA in the absence and presence of a rat liver metabolic activation system. The test substance did not show any toxicity towards the six bacterial tester strains in the preliminary test, therefore 5000 µg/plate was chosen as the top dose level for the mutation test.


With tester strain E. coli WP2 uvrA both in the presence and absence of liver microsomal fraction a slight increase in revertant colony numbers was observed at the 5000 µg/plate dose level only. As this increase was very small and restricted to one dose level it was not thought to be significant. However, a repeat test was performed in order to clarify the situation with dose levels up to 10000 µg/plate. In the repeat test, the test substance did not produce any substantial increases in revertant colony numbers. No substantial increases in the revertant colony numbers of any of the remaining five strains were observed at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix). It was hence concluded that no evidence of mutagenic potential of the test substance was obtained in this bacterial test system at the dose levels tested.


 


The second study was conducted with the test substance in strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of Salmonella typhimurium int the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 microgram/plate to 5 000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was hence chosen as top dose level for the mutagenicity study.


In the standard plate test, the test substance did not show a dose dependent increase in the number of revertants in any of the bacterial strains in the absence or presence of the  rat liver activation system (10 %). Also in the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival, the test substance did not induce a significant increase in the number of revertant colonies, with any of the tester strains.


 


As in the above mentioned bacteria reverse mutation tests, E. coli has not been tested in the Prival modification, a study with a structural analogue with the comparable physico-chemical properties and idential mechanistic properties was added to the assessment to prove that the substance has not mutagenic properties. This study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: 21-July-1997 and U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, Aug-1998andEC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14andJapanese Substance Control Law (JSCL) Test Guideline III.l Gene Mutation Test with bacteria.The study is based on the Principles of Good Laboratory Practice (GLP). The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the substance was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000 μg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies. The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled. The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled. The substance did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate. In both studies toxicity was not observed either with or without metabolic activation.


In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence or presence of the rat liver activation system (10 % (v/v)). Also in the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.


 


It is hence concluded that Reactive Red 230  is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.

Justification for classification or non-classification

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008).