Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic or cytogenic as derived from studies with close structural analogues

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

see attached justification in section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
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
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
Executive summary:

The results lead to the conclusion that the test substance, by analogy of read across, 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

see attached justification in section 13
Reason / purpose:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Positive controls validity:
valid
Additional information on results:
Solubility and preliminary toxicity testing
The test item was dissolved in cell culture medium. Evaluation of the solubility of that solution in cell culture medium showed that 5000 µg/mL was the highest practicable concentration and produced no precipitate.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 100 µg/mL.
After treatment with the test item in the presence of S9-mix moderate toxicity was observed at the concentrations of 4000 µg/mL, reaching 66.1% and 5000 µg/mL reaching 70.9 % of the corresponding solvent control value.
Following treatment in the absence of S9 metabolic activation, high toxicity was observed at 2000 µg/mL and above. Survival declined in a dose-related manner reaching 4.1 % of the solvent control value at the highest dose level.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Mutagenicity test
In the main experiment cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced to 13.5 % of the solvent control value without S9-mix at the concentration of 2000 µg/mL and 87.4 % in the presence of S9-mix at the highest concentration tested, 5000 µg/mL.
In the main and in the repeat experiment the mitotic index was slightly reduced (indication of toxicity) after treatment with the highest dose level at both fixation intervals in the absence of a metabolic activation.
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
The test compound was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments without metabolic activation and two independent experiments with metabolic activation. No relevant reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.
Conclusions:
The Structural Analogue was not mutagenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described
Executive summary:

This study is read across to the Structural Analogue 02, which is considered to be structurally equivalent to the substance to be registered.

The substance is not considered to induce chromosomal aberrations under the conditions of the test.

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”.
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
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.
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
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'.
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.

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:
25 March 1998 to 03 April 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
In addition to the standard plate incorporation test (Ames Test) a modified protocol using preincubation with hamster S9 supplemented with flavine mononucleotide was used. This protocol has been proposed by Prival for assessing the mutagenic activity of azo dyes.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Reverse mutation to histidine prototrophy using histidine auxotrophic mutants.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from induced rat liver and uninduced hamster liver
Test concentrations with justification for top dose:
a: without metabolic activation: 4, 20, 100, 500, 2500, 5000 ug/plate
b: with metabolic activation (10 % rat liver): 4, 20, 100, 500, 2500, 5000 ug/plate
c: with metabolic activation (30 % Syrian golden hamster liver and preincubation): 4, 20, 100, 500, 2500, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: double-distilled water
- Justification for choice of solvent/vehicle: Standard laboratory reagent with history of use.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
a: untreated control b: solvent controls (0 ug/plate)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below
Remarks:
5 substances used for +ve control
Details on test system and experimental conditions:
Positive controls:

a: without metabolic activation:
sodium-azide for strain TA 100 and TA 1535
9-aminoacridine for strain TA 1537
2-nitrofluorene for strain TA 98

b: with metabolic activation (10 % rat liver):

2-aminoanthracene for all tester strains

c: with metabolic activation (30 % Syrian golden hamster liver and preincubation):

2-aminoanthracene for strain TA 100, TA 1535 andTA 1537
Congo red for strain TA 98

Formulation of test compound:

Dissolved in double-distilled water at appropriate concentrations immediately before use

Formulation of reference compounds:

sodium-azide dissolved in double-distilled water final concentration: 1.0 ug/plate for strain TA100 and TA 1535

9-aminoacridine dissolved in DMSO final concentration: 50.0 ug/plate for strain TA 1537

2-nitrofluorene dissolved in DMSO final concentration: 2.5 ug/plate for strain TA 98

2-aminoanthracene dissolved in DMSO final concentrations (10 % rat liver S9-mix (v/v)): 0.5 ug/plate for strains TA 100 and TA 98 1.0 ug/plate for strains TA 1535 and TA 1537

Final concentrations (30 % Syrian golden hamster liver S9-mix (v/v)): 1.0 ug/plate for strain TA 100 0.5 ug/plate for strain TA 1535 2.5 ug/plate for strains TA 1537

Congo red dissolved in DMSO final concentration: 500.0 ug/plate for strain TA 98

The frozen stock solutions of each compound were diluted progressively up to the final concentration on the day of treatment.

Source of biological material:

Stock cultures in the bank of "Genetic Toxicology", Hoechst Marion Roussel Deutschland GmbH, Lead Optimization, Department of oxicology/Pathology prepared from the original bacterial 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

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

Preparation and storage of a liver homogenate fractions (S9)

The S9 fraction was prepared by the department conducting the study according to Ames et. al (1975) and Prival et. al (1982). Male Sprague Dawley rats (200-300 g) and male Syrian golden hamster (7-8 weeks old), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany. Liver preparations were performed from the liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrian hamsters. Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 Sprague Dawley rats or from 10 male Syrian hamsters (7-8 weeks old) at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (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 for not longer than six months. 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 dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix

Sufficient S9 fraction was thawed immediately at room temperature before each test. One volume of S9 fraction (batch no. 97/16 for both experiments, protein concentration 25.6 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 S9-mix of the rat liver were:

246.0 mg MgCI2 8 mM
162.65 mg KCI 33 mM
170.1 mg glucose-6-phosphoric acid
disodium salt dihydrate = 5 mM
333.36 mg NADPH 4 mM
213.76 mg NADH 4 mM

Na2HPO4/NaH2PO4 - phosphate buffer pH 7.4 = 100

According to the modification proposed by Prival (5) using 30 minutes preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction (batch no. 97/7 for both experiments, protein concentration 30.0 g/l) was mixed with 7 volumes of the S9 cofactor solution. This preparation is termed S9-mix. The S9-mix consists of:

8 mM MgCI2
33 mM KCI
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..
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 at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled as described below.

Assay procedure
Two independent mutation tests for each of the two protocols (Ames, Prival) were 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 first mutation experiment also assessed the toxicity of the test substance in order to select a suitable range of dose levels for the second mutation test. A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawns were used as toxicity indicators. Thinning of the bacterial lawns was evaluated microscopically.

Parallel to the second mutation experiment, precise toxicity testing was performed as follows using dose levels selected on the basis of toxicity results in the first test: 0.1 ml of the different concentrations of the test compound were thoroughly mixed with 0.1 ml of 106 dilution of the overnight culture of TA 100 (designated TA 100 D for the Ames Test and TA 100 HD for the Prival modification) and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

a)- with 10 % (v/v) rat liver S9-mix or buffer and the Salmonella strains TA 98, TA 100, TA 1535 and TA 1537
For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml 10 % (v/v) rat liver S9-mix (if required) or 1/3 nutrient broth-buffer mix 0.1 ml of an overnight nutrient broth culture of the bacterial tester strain 0.1 ml test compound solution (dissolved in double-distilled water)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal (histidine deficient) 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+ revertants) were counted with an automatic colony counter (Artec counter Model 880).

b)- with 30 % (v/v) Syrian golden hamster S9-mix and preincubation and the Salmonella strains TA 98, TA 100, TA 1535 and TA 1537
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S9-mix were incubated at approx. 30 °C for approx. 30 minutes. Subsequently, 2 ml of soft agar containing of 100 ml agar (0.6 % (w/v) agar + 0.5 % (w/v) NaCI) and 10 ml amino-acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) was added. After mixing, the samples were poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approximately 30 seconds. After incubation for 48 hours at 37 °C in the dark, colonies (his* revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter. A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.
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 induced increases in the mutation frequency which were both statistically 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:
a) 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
b) 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.

The test results must be reproducible.
Statistics:
Comparison with the testing laboratorie's historical range.
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
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.

Solubility and toxicity

The test compound was dissolved in double-distilled water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provides a final concentration of 5000 ug/plate. Further dilutions of 2500, 500, 100, 20 and 4 ug/plate were used in all experiments.
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 ug/plate.
The test compound proved to be not toxic to the bacterial strains.

In the toxicity test with a dilution of tester strain TA 100 (designated TA 100 D and TA 100 HD), which was performed in parallel with the second mutation experiment, no toxicity was found either in the absence or in the presence of metabolic activation up to the highest investigated dose of 5000 ug/plate.

Mutagenicity

In both independent mutation tests Reaktivgelb FD 08064 was tested for mutagenicity with the same concentrations as described in section 6.2. The number of colonies per plate with each strain as well as mean values of 3 plates are given.

Ames 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 in the presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.

Prival 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.

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'. Remarks: Both Ames and Privall tests

The results lead to the conclusion that Reaktivgelb FD 08064 is not mutagenic in the absence and presence of rat S9-mix (10 % (v/v)) using the standard Ames Test procedure. Also in the presence of hamster liver S9-mix (30 % (v/v)) and preincubation the test compound did not induce a significant increase in the number of revertant colonies.

Conclusions:
The results lead to the conclusion thatthe test substance is not mutagenic in the absence and presence of rat S9-mix (10 % (v/v)) using the standard Ames Test procedure. Also in the presence of hamster liver S9-mix (30 % (v/v)) and preincubation the test compound did not induce a significant increase in the number of revertant colonies.
Executive summary:

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.

Two independent 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 a rat liver homogenate or a hamster liver homogenate. For all studies, the compound was dissolved in double-distilled water, and each bacterial strain was exposed to 6 dose levels. Doses for all studies ranged from 4 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: In the mutagenicity experiments toxicity was not observed either with or without metabolic activation up to the highest investigated dose of 5000 µg/plate.

A toxicity test using histidine-enriched agar plates and a dilution of the tester strain TA 100 was performed in parallel with the second mutation experiment. The test compound proved to be not toxic to the bacterial strain.

Ames 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 with The test substance did not result in relevant increases in the number of revertant colonies.

Prival Test :

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 March 1998 to 08 June 1998
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 F08064, which is considered to be structurally equivalent to the substance to be registered, in that only the halogen ion differs structurally (fluorine instead of chlorine). Reactive Yellow F08064 also contains a form where hydroxyl group replaces the chlorine. Neither of these differing analogues are considered to adversely affect the toxicity or intrinsic effects overall between the molecules. Details of structure are detailed below under "illustrations”.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromatid breakage and/or reunion between chromatids.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of the laboratories "Genetic Toxicology" section, thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.

Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CO2 in 175 cm2 plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Sampling time 20 h without S9-mix with S9-mix

Solvent control: 0.00 μg/ml 0.0 μg/ml
Positive control: EMS 500.00 μg/ml CPA 3.5 μg/ml
Test group 1: 156.25 μg/ml # 1250.0 μg/ml
Test group 2: 312.50 μg/ml 2500.0 μg/ml
Test group 3: 625.00 μg/ml 5000.0 μg/ml
Test group 4: 1250.00 μg/ml
Test group 5: 2000.00μg/ml *

Sampling time 28 h without S9-mix with S9-mix

Solvent control: 0.0 μg/ml 0.0 μg/ml
Test group 3: 625.0 μg/ml # 5000.0 μg/ml
Test group 4: 1250.0 μg/ml
Test group 5: 2000.0 μg/ml *

* not evaluated because of high toxicity
# not used because higher concentrations were evaluated
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
- Justification for choice of solvent/vehicle: Historical usage within the laboratory.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: EMS & CPA
Details on test system and experimental conditions:
Reference Compounds
Without metabolic activation: Ethyl methane sulfonate EMS
With metabolic activation: Cyclophosphamide
Control Groups: Cultures treated with Solvent

Dosing levels:

Sampling time 20 h without S9-mix with S9-mix

Solvent control: 0.00 μg/ml 0.0 μg/ml
Positive control: EMS 500.00 μg/ml CPA 3.5 μg/ml
Test group 1: 156.25 μg/ml # 1250.0 μg/ml
Test group 2: 312.50 μg/ml 2500.0 μg/ml
Test group 3: 625.00 μg/ml 5000.0 μg/ml
Test group 4: 1250.00 μg/ml
Test group 5: 2000.00μg/ml *

Sampling time 28 h without S9-mix with S9-mix

Solvent control: 0.0 μg/ml 0.0 μg/ml
Test group 3: 625.0 μg/ml # 5000.0 μg/ml
Test group 4: 1250.0 μg/ml
Test group 5: 2000.0 μg/ml *

* not evaluated because of high toxicity
# not used because higher concentrations were evaluated

Formulation of test compound: dissolved in cell culture medium at appropriate concentrations immediately before use

Formulation of reference compounds (only at the sampling time 20 h):

EMS dissolved in cell culture medium on the day of treatment final concentration: 0.5 mg/ml

CPA dissolved in cell culture medium on the day of treatment final concentration: 3.5 ug/ml

Source of biological material: cell bank of "Genetic Toxicology", HMR Germany

Test organism: cell line V79 of Chinese hamster lung fibroblasts

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks

Preparation and storage of a liver homogenate fraction (S9)

The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (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, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. 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 dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 97/16 for all mutation experiments, protein concentration 25.6 g/l;) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different cofactors of the S9-mix were:
8 mM MgCI2 33 mM KCI 5 mM glucose-6-phosphate 5 mM NADP 100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C02 in 175 cm2 plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the cytogenetic assay. Cell cultures were subjected to the same treatment conditions as in the main experiment. Cytotoxic effects were determined by photometric measurement of V79 cell cultures grown in microwell plates and stained with crystal violet. The relative cell density in the microwell plates was nearly the same as in the Quadriperm® dishes.
The test included the following treatments:
Solvent control : the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 ug/ml. Treatments were performed both in the presence and absence of S9 metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection

The concentrations for the mutagenicity assay were based on the results of the toxicity experiment.
For non-toxic, freely soluble test compounds, the top dose is 10 mM or 5000 ug/ml according to international testing guidelines.
For relatively insoluble test compounds, that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium after the end of the treatment period. In the case of toxic effects, the highest dose level should reduce the survival rate to approximately 20 - 50 % and/or the mitotic index to approximately 50 % compared with the corresponding solvent control.
For toxic compounds additional concentrations may be included in the treatment series.
According to the criteria described above, three adequately spaced dose levels extend¬ing over at least one decadic logarithm were evaluated at the 20 h fixation interval. At the 28 h fixation interval one appropriate dose level was selected to evaluate the metaphases. In case of clear positive results at the 20 h fixation interval, an evaluation of the 28 h fixation interval is not necessary to perform. For each experimental point two cultures were used.

Mutagenicity test
Two independent experiments were conducted in both the presence and the absence of S9-mix. Cultured cells were seeded on to slides (2 per dose level and sampling time) then treated for either 20 hours (without S9-mix) or for 3 hours (with S9-mix), colcemide was then added to arrest cell division and the chromosomes were stained and examined. In both assays, cells were sampled 20 and 28 hours after the start of treatment. For both assays, all dose levels were evaluated for chromosome aberrations at 20 hours but a single appropriate dose level was evaluated at 28 hours only if there were negative or equivocal results at 20 hours.

Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCI. Any effects on the osmolality during the study were described in the study report.

Two-day old, exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension (culture) was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. Two slides were placed in Quadriperm® dishes which were then seeded with cells to yield 2-3 x 104 cells/slide (20 h culture) or 1-2 x 104 cells/slide (28 h culture).Thus for each dose level and sampling time, one culture and two slides were used. The Quadriperm® dishes contained 5 ml MEM with approx. 10 % (v/v) FCS.

After 48 h, the medium was replaced with one containing approx. 10 % (v/v) FCS and the test compound, or positive control, or solvent or buffer (without S9-mix), or 5 % (v/v) FCS and the test compound, or positive control, solvent or S9-mix. In those cultures with metabolic activation, the medium was replaced after 3 h with normal medium following two rinses with this. Without S9-mix the cells were exposed to the treatment medium for 20 h.
18.5 h and 26.5 h after the start of the treatment, colcemide was added (approx. 0.05 ug/ml/culture medium) to the cultures to arrest mitosis and 1.5 h later (20 h and 28 h after the start of treatment) metaphase spreads were prepared as follows.
The cultures were made hypotonic by adding about 4 ml of approx. 0.075 M potassium chloride solution at around 37 °C. The cells were then incubated for 20 minutes at approx. 37 °C. The next step was the addition of 1.5 ml fixative.
Then the liquid was replaced by 5 ml fixative (methanol: glacial acetic acid, 3:1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and airdried for 24 hours. The chromosomes were stained as follows:

1. staining for 10 minutes in approx. 2 % (w/v) orcein solution
2. rinsing 3 times in distilled water
3. rinsing twice in acetone
4. brief rinsing in acetone/xylene
5. 2 minutes in acetone/xylene
6. 5 minutes in xylene
7. 10 minutes in xylene
8. embedding in Entellan® or Eukitt®

Two slides were prepared from each experimental group.

Solvent and the positive controls were prepared 20 h after treatment in the same way. Two independent experiments with and without metabolic activation were performed.

Analysis of metaphases
The slides were coded and 25-100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified as shown in chapter 9.1. Only metaphases with 22 +/-1 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: gap, isogap, break, isobreak, fragment, isofragment, minute, isominute, deletion, isodeletion, exchanges including intrachanges, dicentrics, pulverization and ring formation. Metaphases with 5 or more aberrations were classified separately as multiple aberrations. The number of chromosomes in these metaphases and pulverised metaphases often can not be evaluated. Furthermore the rate of polyploid metaphases was determined in 1000 cells of each cell culture. Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.

After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.
Evaluation criteria:
Criteria for a valid assay

The assay was considered valid if the following criteria are met:
- the solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range


Criteria for a positive response

The evaluation of the results was performed as follows:

The test compound is classified as mutagenic if it induces a reproducible statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls. The test compound is classified as mutagenic if there is a reproducible concentration-related increase in the aberration rate (without gaps). The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation.
Statistics:
The Biometry of the results was performed with a one-sided Fisher - Exact test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and preliminary toxicity testing
The test item was dissolved in cell culture medium. Evaluation of the solubility of that solution in cell culture medium showed that 5000 µg/mL was the highest practicable concentration and produced no precipitate.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 100 µg/mL.
After treatment with the test item in the presence of S9-mix moderate toxicity was observed at the concentrations of 4000 µg/mL, reaching 66.1% and 5000 µg/mL reaching 70.9 % of the corresponding solvent control value.
Following treatment in the absence of S9 metabolic activation, high toxicity was observed at 2000 µg/mL and above. Survival declined in a dose-related manner reaching 4.1 % of the solvent control value at the highest dose level.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Mutagenicity test
In the main experiment cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced to 13.5 % of the solvent control value without S9-mix at the concentration of 2000 µg/mL and 87.4 % in the presence of S9-mix at the highest concentration tested, 5000 µg/mL.
In the main and in the repeat experiment the mitotic index was slightly reduced (indication of toxicity) after treatment with the highest dose level at both fixation intervals in the absence of a metabolic activation.
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
The test compound was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments without metabolic activation and two independent experiments with metabolic activation. No relevant reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tabulated data is enclosed below under attachments.

Conclusions:
The test substance was not mutagenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described
Executive summary:

In this study the potential of the test substance to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment two cell cultures were used.

The test compound was dissolved in cell culture medium and tested at the following concentrations:

without S9-mix:

20 h: 312.5, 625.0 and 1250.0 µg/mL

28 h: 1250.0 µg/mL

 

with S9-mix:

20 h: 1250.0, 2500.0 and 5000.0 µg/mL

28 h: 5000.0 µg/mL

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced a moderate lowering of the mitotic index in the absence of a metabolic activation at both fixation intervals. Up to the highest investigated dose the test compound induced no significant increase in the number of chromosome aberrations. Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

In conclusion, the test substance does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described. The test substance is therefore considered to be non-mutagenic in this chromosome aberration assay.

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

Genetic toxicity in vivo

Description of key information

The test substance is not clatogenic or aneugenic as derived from studies with close structural analogues

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 2002 to 30 April 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Species of animals: rat
Strain of animals: Hsd:Sprague Dawley
Origin (supplier) of animals: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMn04 and cage numbering
Body weight at start of study: male animals
mean = 189.8 g (= 100 %)
min = 178 g (-6.2%)
max = 202 g (+6.4%)
n = 15
female animals
mean = 152.7 g (= 100 %)
min = 148 g (-3.1%)
max = 162 g (+6.1%)
n = 15
Age at start of study: male animals approximately 6 weeks, female animals approximately 6 weeks
Randomisation procedure: Randomisation schemes 2003.0198 and 2003.0199
Animal maintenance: five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned room (room number 011)
Room temperature: 22 °C ± 3 °C (except short lasting deviations due to disturbances of air condition)
Relative humidity: 50 % ± 20 % (except short lasting deviations due to disturbances of air condition)
Lighting times: 12 hours light / dark cycle
Acclimatisation: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest
Water:tap water in plastic bottles, ad libitum
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used:Deionised water
- Justification for choice of solvent/vehicle: Historical use within the laboratory.
- Concentration of test material in vehicle: 200 mg/ml
Details on exposure:
Frequency of administrations: two doses separated by an interval of 24 hours (except positive control with only one dose).
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Two doses
Post exposure period:
No post exposure period
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group Dose Concentration Volume No. of animals Animal No. Killing time
(mg/kg bwt.) (mg/ml) (ml/kg bwt.) and sex (hours p.a.)
1 0 0 10 5 males 1 – 5 24
5 females 6 -10
2 2000 200 10 5 males 11 – 15 24
5 females 16 – 20
3* 40 4.0 10 5 males 21 – 25 24
5 females 26 – 30

* = positive control: Endoxan® (batch no. 9M642A) containing cyclophosphamide, dissolved in distilled water
Hours p.a. = hours after administration
Control animals:
yes, concurrent vehicle
Positive control(s):
Name: cyclophosphamide
Synonyms: Endoxan®
CAS-Register number: 6055-19-2
Batch number: 2K645B
Certificate of analysis: certified by the supplier, Quality Control, Dr. Hofmann dated 10-Dec-2002
Tissues and cell types examined:
chromosomal damage (clastogenicity) in a rat bone marrow
Details of tissue and slide preparation:
PREPARATION AND STAINING

Extraction of the bone marrow
Animals were killed by carbon dioxide asphyxiation 24 hours after second dosing. One femoral bone was removed and the bone freed of muscle tissue. The proximal end of the femoral bone was opened, the bone marrow flushed into a centrifuge tube containing about 3 ml of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Subsequently the slides were stained as follows:
Staining procedure
5 minutes in methanol
5 minutes in May-Grunwald's solution
brief rinsing twice in distilled water
10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
rinsing in distilled water drying
coating with Entellan®
Evaluation criteria:
SCORING AND EVALUATION OF DATA

Scoring
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micro-nuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychro-matic erythrocytes to 200 normochromatic erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.

Criteria for assay validity
An one-sided Wilcoxon-Test was performed to check the validity of the study. The study was considered as valid if the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p<0.05).

Data analysis
Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

CRITERIA FOR A POSITIVE RESPONSE
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered as positive if there is a significant or dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant or dose-related increase in the number of micronucleated polychromatic erythrocytes is considered as non-clastogenic in this system.
Statistics:
As detailed above under "Evaluation Criteria".
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Rats were treated twice at an interval of 24 hours with 2000 mg/kg body weight to study the induction of micronuclei in bone marrow cells.

All animals survived after treatment. No signs of toxicity were observed in the main study.

The dissection of the animals revealed no test substance related macroscopic findings.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.

A very slight increase of micronucleated polychromatic erythrocytes (mean value) was observed in the animals treated with 2000 mg/kg of the test compound. However, the incidence of micronucleated polychromatic erythrocytes in this dose group was within the historical range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7-4.9). Moreover, the increase was not statistically significant. It was therefore considered as incidental finding without biological relevance.

The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

SUMMARY TABLES AND STATISTICS

 

Sex

Dose

mg/kg b.w.

Killing time

Number of animals

Poly / animal counted

Poly/Ery

 

Mean

Poly/Ery SD Mean

Poly with MN

 

Mean

Poly with MN [%]

Mean

Poly with MN SD

Mean

Male

0 – Control

24 h

5

2000

0.48

0.08

2.4

0.12

0.08

Male

2000

24 h

5

2000

0.48

0.04

2.6

0.18

0.04

Male

40 – Endoxan

24 h

5

2000

0.43

0.05

28.2*

1.41

0.70

Female

0 – Control

24 h

5

2000

0.50

0.04

2.4

0.12

0.08

Female

2000

24 h

5

2000

0.47

0.04

3.4

0.17

0.10

Female

40 – Endoxan

24 h

5

2000

0.40

0.05

18.8*

0.94

0.24

 

Sex

Dose

mg/kg b.w.

Killing time

Number of animals

Poly / animal counted

Poly/Ery

 

Mean

Poly/Ery SD Mean

Poly with MN

 

Mean

Poly with MN [%]

Mean

Poly with MN SD

Mean

Mut. I.

Pooled

0 – Control

24 h

10

2000

0.49

0.06

2.40

0.1

0.07

1.0

Pooled

2000

24 h

10

2000

0.48

0.04

3.50

0.2

0.07

1.5

Pooled

40 – Endoxan

24 h

10

2000

0.42

0.05

23.5*

1.2

0.55

9.8

 

Mut. I. = Mutagenic Index

Control = Vehicle (deionised water)

* = Significantly different from control (p < 0.05)

 

A cross comparison of individual data and pooled data may show discrepancies since the values are rounded.

Conclusions:
The results lead to the conclusion that the test item did not cause a substantial increase in micronucleated polychromatic erythrocytes and is not clastogenic in the micronucleus test in vivo under the conditions described in this report.
Executive summary:

The test compound was suspended in deionised water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on the results of a previous dose range finding assay. Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei in all dose groups was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test item and differed less than 20% from the control value.

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test item is not clastogenic in the micronucleus test in vivo.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to EU, OECD & US EPA test guidance in compliance with GLP. This study is read across to a structural analogue 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”.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see justification under section 13
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Rats were treated twice at an interval of 24 hours with 2000 mg/kg body weight to study the induction of micronuclei in bone marrow cells.

All animals survived after treatment. No signs of toxicity were observed in the main study.

The dissection of the animals revealed no test substance related macroscopic findings.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.

A very slight increase of micronucleated polychromatic erythrocytes (mean value) was observed in the animals treated with 2000 mg/kg of the test compound. However, the incidence of micronucleated polychromatic erythrocytes in this dose group was within the historical range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7-4.9). Moreover, the increase was not statistically significant. It was therefore considered as incidental finding without biological relevance.

The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

SUMMARY TABLES AND STATISTICS

 

Sex

Dose

mg/kg b.w.

Killing time

Number of animals

Poly / animal counted

Poly/Ery

 

Mean

Poly/Ery SD Mean

Poly with MN

 

Mean

Poly with MN [%]

Mean

Poly with MN SD

Mean

Male

0 – Control

24 h

5

2000

0.48

0.08

2.4

0.12

0.08

Male

2000

24 h

5

2000

0.48

0.04

2.6

0.18

0.04

Male

40 – Endoxan

24 h

5

2000

0.43

0.05

28.2*

1.41

0.70

Female

0 – Control

24 h

5

2000

0.50

0.04

2.4

0.12

0.08

Female

2000

24 h

5

2000

0.47

0.04

3.4

0.17

0.10

Female

40 – Endoxan

24 h

5

2000

0.40

0.05

18.8*

0.94

0.24

 

Sex

Dose

mg/kg b.w.

Killing time

Number of animals

Poly / animal counted

Poly/Ery

 

Mean

Poly/Ery SD Mean

Poly with MN

 

Mean

Poly with MN [%]

Mean

Poly with MN SD

Mean

Mut. I.

Pooled

0 – Control

24 h

10

2000

0.49

0.06

2.40

0.1

0.07

1.0

Pooled

2000

24 h

10

2000

0.48

0.04

3.50

0.2

0.07

1.5

Pooled

40 – Endoxan

24 h

10

2000

0.42

0.05

23.5*

1.2

0.55

9.8

 

Mut. I. = Mutagenic Index

Control = Vehicle (deionised water)

* = Significantly different from control (p < 0.05)

 

A cross comparison of individual data and pooled data may show discrepancies since the values are rounded.

Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that the test item did not cause a substantial increase in micronucleated polychromatic erythrocytes and is not clastogenic in the micronucleus test in vivo under the conditions described in this report.
Executive summary:

This study is read across to a structural analogue, which is considered to be structurally equivalent to the substance to be registered. The micronucleus test was carried out with Structural Analogue 01 (see Jusification for Read-Across). The test compound was suspended in deionised water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on the results of a previous dose range finding assay. Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei in all dose groups was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test item and differed less than 20% from the control value.

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test item is not clastogenic in the micronucleus test in vivo.

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

Additional information

The substance, by analogue of read across, was negative in the following tests:

1) Ames and Prival Test - S. typhimurium and E.coli - Structural Analogue 01 and 02

2)  Chromosome Aberration – V79 Chinese Hamster lung cells - Structural Analogue 02

3) Mammalian Erythrocyte Micronuclues Test In Vivo - Structural Analogue 01

 

Two separate bacterial reverse mutation tests showed that the substance does not have any mutagenic properties in different Salmonella and E.Coli strains. In addition, no genotoxic properties have been seen in the in vivo mouse micronucleus test, nor in the chromosome aberration assay.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.

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