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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:
13 May 2003 to 17 July 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to recent EU test guidance in compliance with GLP and reported with a GLP certificate. This study is read across to the structural analogue Reactive Yellow BZK5934 which is considered to be structurally equivalent to the substance to be registered, in that this substance is the vinyl form of the substance to be registered. This alteration to the structure is not considered to adversely affect the toxicity or intrinsic effects overall between the molecules. Details of structure are detailed below under "illustrations”.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
III.l Gene Mutation Test with bacteria
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: Structural Analogue 01

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate and a hamster liver homogenate
Test concentrations with justification for top dose:
The doses ranged from 50 to 5000 µg/plate.
Vehicle / solvent:
Suspended in deionized water at appropriate concentrations immediately before use.
Controls
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
solvent controls (0 µg/plate)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
other: 2-aminoanthracene
Details on test system and experimental conditions:
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 raM KC1 (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g 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 S9 fraction (Moltox batch no. 1455 for the plate incorporation test, protein concentration 39.3 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:
8mM MgCl2
33 mM KCl
5mM glucose-6-phosphate
4 mM 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. 2001/3 for the preincubation, protein concentration 41.1 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
4 mM NADP+
2mM NADH
2mM 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 /litre) 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 liquide 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. All criteria for a valid assay were fulfilled.

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 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/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 suspension (suspended in deionized 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.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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.
In the prival modification in the presence of S9-mix the number of revertant colonies of the solvent control and the negative control with the strain TA 1537 (and of the positive control with the strains TA 1535 and WP2uvrA) were slightly above the historical control data range, but the criteria for the positive response were fulfilled.

SOLUBILITY AND TOXICITY
Reaktiv Gelb BZK 5934 was suspended in deionized water and a stock suspension of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/plate were used in all experiments.
Reaktiv Gelb BZK 5934 did not precipitate on the plates up to the highest investigated dose of 5000 ug/plate.
Reaktiv Gelb BZK 5934 proved to be not toxic to the bacterial strains.

MUTAGENICITY
In both independent mutation tests Reaktiv Gelb BZK 5934 was tested for mutagenicity with the same concentrations as described. 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 absence and 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.

All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The results lead to the conclusion that the test 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 according to Prival as described.
Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted, U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, EC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14 and Japanese 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 compound was suspended in deionized water, and each bacterial strain was exposed to 5 dose levels in both tests. The doses ranged from 50 to 5000 µg/plate. No precipitation of the test compound was observed with these doses.

 

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.

 

In the prival test in the presence of S9-mix the number of revertant colonies of the solvent control and the negative control with the strain TA 1537 (and of the positive control with the strains TA 1535 and WP2uvrA) were slightly above the historical control data range, but the criteria for the positive response were fulfilled.

 

Toxicity: In the mutagenicity experiments toxicity was not observed with and without metabolic activation.

 

Plate incorporation test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells did not result in relevant increases in the number of revertant colonies.

 

Preincubation test:

Mutagenicity: In the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the test 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 test substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.