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Genetic toxicity: in vitro

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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 the 15th of December, 1998 to the 7th of January, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From the 15th of December, 1998 to the 7th of January, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Justification for Read Across is detailed in the section summary and it is further detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
. This study, modified according to Prival and Mitchell, was especially suited to detect possible mutagenic activity of azo dyes. Prival, M,J. and Mitchell, V.D.: Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9, Mutation Files. Q, 103-116 (1982).13
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: histidine- requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
E.Coli: base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. Tryptopham requirement, UV-sensitivity and the number of spontaneous revertants.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Source Dr, Bruce N. Ames, University of California at Berkeley, U.S.A. (Salmonella typhimurium strains)
TA100 received 18-02-1993, used batch: TA1 00.061098
TA98 received 21-02-1991, used batch: TA98.150698
TA1535 received 14-12-1994, used batch: TA1535.150698
TA1537 received 14-12-1994, used batch: TA1537.150698

Mutation type
TA1537 hisC3076 Frameshift
TA98 hlsD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*R-factor = plasmid pKM101 (increases error-prone DNA repair)
Additional strain / cell type characteristics:
other: rfa: deep rough (defective lipopolysaccharide cellooat) gal: mutation in the galactose metabolism chl: mutation in nitrate reductase bio: defective biotin synthesis uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Prof. Dr. B.A, Bridges, University of Sussex, Brighton, U.K, 7 (Escherichia coll strain)
WP2uvrA received 23-10-1987, used batch: EC.050598
Metabolic activation:
with and without
Metabolic activation system:
S9-mix: S9-mix was prepared immediately before use and kept on ice.
Test concentrations with justification for top dose:
Dose range finding test Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay.
The highest concentration of HWL-158 used in the subsequent mutation assay was 5 mg/plate.
3, 333, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
Miili-Q water (Miliipore Corp., Bediord, Mass, USA
Positive controls:
yes
Remarks:
TA1535, TA1537, TA98, TA100. Solvent saline and DMSO
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: Daunomycine
Remarks:
without metabolic activation
Positive controls:
yes
Remarks:
TA1535, TA1537, TA98, TA100. Solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Positive controls:
yes
Remarks:
WP2uvrA, solvent DMSO
Positive control substance:
4-nitroquinoline-N-oxide
other: N-oxide
Remarks:
without metabolic activation
Positive controls:
yes
Remarks:
WP2uvrA, solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
The Salmonella typhimurium and E. Coli strains were regularly checked.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

CELL CULTURE
Preparation of Bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (C-37°C, 150 spm). until the cultures reached an optical density of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilization of the Escherichia coli strain
WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-I-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris- HCL, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates
Agar plates (diameter 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code L28) in Vogei-Bonner Medium E,10 g glucose. N.B. The agar plates for the test with the Salmonell typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar
Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autociaved for 20 min at 121°C.
Environmental condition
All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.

TREATMENT OF THE TEST SUBSTANCE
The test substance was dissolved in Miili-Q water (Miliipore Corp., Bediord, Mass, USA). The stock solution was treated with ultra-sonication until the test substance had completely dissolved.
The stock solution was filter (0.22 µm)—sterilized. Test substance concentrations were prepared directly prior to use.

MUTATION ASSAY
At least five different doses (increasing with approximately halt-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C.
The following solutions were preincubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (1O*cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q water.
After the preincubation period the solutions were added to 3 ml molten top agar: The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h.
After this period revertant colonies (histidine independent tor Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Evaluation criteria:
Colony counting
The revenant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

A Salmonella typhimurium reverse mutation assay and/or Escherichia colI reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) lt induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
Precipitate
The test substance did not precipitate in the top agar. Precipitation on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.
Toxicity
To determine the toxicity of the substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revenant colonies were observed.
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.
Number of revertants
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-told, increase in the number of revertants in two independently repeated experiments.
Conclusions:
negative with metabolic activation
negative without metabolic activation
Not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The similar substance 01 was tested in the Salmonella typhimurium reverse mutation assay with tour histidine- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptopham requiring strain of Escherichia coli WP2uvrA in two independent experiments, modified according to Prival and Mitchell.

The similar substance 01 substance was tested up to concentrations of 5000 pg/plate in the absence and presence of S9 mix.

The test item did not precipitate on the plates at this dose level.

The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

The substance did not induce a dose-related increase in the number of revertant (His*) colonies in each of the tour tester strains (TA1535, TA1537, TA98 and TA100) and in the number of reveitant (Trpt) colonies in tester strain WP2uvrA both in the absence and presence of S9- metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that the similar substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
modified according to Prival and Mitchell
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
This study, modified according to Prival and Mitchell, was especially suited to detect possible mutagenic activity of azo dyes. Prival, M,J. and Mitchell, V.D.: Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9, Mutation Files. Q, 103-116 (1982).13
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Description Brown powder
Test substance storage At room temperature in the dark
Stability under storage conditions Not indicated
Expiry date 29 August 2002 (allocated by NOTOX, 1 year after)

Method

Target gene:
S. typhimurium: histidine- requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
E.Coli: base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. Tryptopham requirement, UV-sensitivity and the number of spontaneous revertants.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Source Dr, Bruce N. Ames, University of California at Berkeley, U.S.A. (Salmonella typhimurium strains)
TA100 received 18-02-1993, used batch: TA1 00.061098
TA98 received 21-02-1991, used batch: TA98.150698
TA1535 received 14-12-1994, used batch: TA1535.150698
TA1537 received 14-12-1994, used batch: TA1537.150698

Mutation type
TA1537 hisC3076 Frameshift
TA98 hlsD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*R-factor = plasmid pKM101 (increases error-prone DNA repair)
Additional strain / cell type characteristics:
other: rfa: deep rough (defective lipopolysaccharide cellooat) gal: mutation in the galactose metabolism chl: mutation in nitrate reductase bio: defective biotin synthesis uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Prof. Dr. B.A, Bridges, University of Sussex, Brighton, U.K, 7 (Escherichia coll strain)
WP2uvrA received 23-10-1987, used batch: EC.050598
Metabolic activation:
with and without
Metabolic activation system:
S9-mix: S9-mix was prepared immediately before use and kept on ice.
Test concentrations with justification for top dose:
Dose range finding test Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay.
The highest concentration of HWL-158 used in the subsequent mutation assay was 5 mg/plate.
3, 333, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
Miili-Q water (Miliipore Corp., Bediord, Mass, USA
Controlsopen allclose all
Positive controls:
yes
Remarks:
TA1535, TA1537, TA98, TA100. Solvent saline and DMSO
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: Daunomycine
Remarks:
without metabolic activation
Positive controls:
yes
Remarks:
TA1535, TA1537, TA98, TA100. Solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Positive controls:
yes
Remarks:
WP2uvrA, solvent DMSO
Positive control substance:
4-nitroquinoline-N-oxide
other: N-oxide
Remarks:
without metabolic activation
Positive controls:
yes
Remarks:
WP2uvrA, solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
The Salmonella typhimurium and E. Coli strains were regularly checked.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

CELL CULTURE
Preparation of Bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (C-37°C, 150 spm). until the cultures reached an optical density of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilization of the Escherichia coli strain
WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-I-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris- HCL, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates
Agar plates (diameter 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code L28) in Vogei-Bonner Medium E,10 g glucose. N.B. The agar plates for the test with the Salmonell typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar
Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autociaved for 20 min at 121°C.
Environmental condition
All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.

TREATMENT OF THE TEST SUBSTANCE
The test substance was dissolved in Miili-Q water (Miliipore Corp., Bediord, Mass, USA). The stock solution was treated with ultra-sonication until the test substance had completely dissolved.
The stock solution was filter (0.22 µm)—sterilized. Test substance concentrations were prepared directly prior to use.

MUTATION ASSAY
At least five different doses (increasing with approximately halt-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C.
The following solutions were preincubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (1O*cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q water.
After the preincubation period the solutions were added to 3 ml molten top agar: The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h.
After this period revertant colonies (histidine independent tor Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Evaluation criteria:
Colony counting
The revenant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

A Salmonella typhimurium reverse mutation assay and/or Escherichia colI reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) lt induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
Precipitate
The test substance did not precipitate in the top agar. Precipitation on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.
Toxicity
To determine the toxicity of the substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revenant colonies were observed.
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.
Number of revertants
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-told, increase in the number of revertants in two independently repeated experiments.

Applicant's summary and conclusion

Conclusions:
negative with metabolic activation
negative without metabolic activation
Not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The similar substance was tested in the Salmonella typhimurium reverse mutation assay with tour histidine- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptopham requiring strain of Escherichia coli WP2uvrA in two independent experiments, modified according to Prival and Mitchell.

The similar substance 01 substance was tested up to concentrations of 5000 pg/plate in the absence and presence of S9 mix.

The test item did not precipitate on the plates at this dose level.

The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

The substance did not induce a dose-related increase in the number of revertant (His*) colonies in each of the tour tester strains (TA1535, TA1537, TA98 and TA100) and in the number of reveitant (Trpt) colonies in tester strain WP2uvrA both in the absence and presence of S9- metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that the similar substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.