Reactive Red F03-0318

Genetic toxicity: in vitro

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:

11 February 2011 to 15 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study conducted to EU, US & OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: M.J. PRIVAL and V.D. MITCHELL: Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S9. Mutation Research 97: 103-116, 1982

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

Name: Reactive Red F03-0318

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions.
The Salmonella typhimurium strains are constructed to differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
The Escherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp- to trp+ reversions.
The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat or hamster liver post-mitochondrial fraction (S9 fraction) - phenobarbital/B-naphthoflavone-induced

Test concentrations with justification for top dose:

Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

Vehicle / solvent:

Based on the results of the Solubility Test, the test item was dissolved in Distilled water. Distilled water was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory.

Distilled water:
Supplier: TEVA Hungary Co.
Batch No.: 7530810 / 8490910*
Expiry date: August 2013 / September 2013
Grade: Aqua destillata pro injectione

*Note: Lot 7530810 was used in the preliminary experiment; lot 8490910 was used in the main tests

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD), Sodium azide (SAZ), 9-aminoacridine (9AA), Methyl-methanesulfonate (MMS), 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Formulation
The behaviour of the test item solutions with the solution of top agar and phosphate buffer was examined in a preliminary solubility test. Distilled water was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

POSITIVE AND NEGATIVE CONTROLS
Positive Controls
Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.

Negative Controls (Solvent Controls)
In the study three vehicle control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals. The following chemicals were used for vehicle (solvent) control groups:

Distilled water:
Supplier: TEVA Hungary Co.
Batch No.: 7530810 / 8490910*
Expiry date: August 2013 / September 2013
Grade: Aqua destillata pro injectione

*Note: Lot 7530810 was used in the preliminary experiment; lot 8490910 was used in the main tests

Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: BCBC5888V / BCBD8784V*
Expiry date: May 2016 / December 2016
Grade: puriss p.a., ACS reagent
Purity: ≥99.9%

*Note: Lot BCBC5888V was used in the preliminary experiments and Initial Mutation Test; lot BCBD8784V was used in the Confirmatory Mutation Test.

BACTERIAL STRAINS

Date of arrival and origin:

Salmonella typhimurium TA98 16 November 2010, MOLTOX - Molecular Toxico-logy Inc., Boone, North Carolina, USA
Salmonella typhimurium TA100 16 November 2010, MOLTOX - Molecular Toxico-logy Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1535 16 November 2010, MOLTOX - Molecular Toxico-logy Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1537 16 November 2010, MOLTOX - Molecular Toxico-logy Inc., Boone, North Carolina, USA
Escherichia coli WP2 uvrA 16 November 2010, MOLTOX - Molecular Toxico-logy Inc., Boone, North Carolina, USA

Genotypes

In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.

Storage

The strains are stored at -80 +/- 10ºC in the Culture Collection of the Microbiological Laboratory of the LAB Research Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. [1] and Maron and Ames. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of LAB Research Ltd.

Spontaneous Reversion of Tester Strains

Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli WP2 uvrA strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 1999 to 2010 as follows: Salmonella typhimurium TA98: 9-54, TA100: 58-211, TA1535: 2-31, TA1537: 1-24, Escherichia coli WP2 uvrA: 9-86.

Procedure for Growing Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum were used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37*C in a Gyrotory water bath shaker.

Viability of the Testing Cultures

The viability of each testing culture was determined by plating 0.1 mL of the 105, 106, 107 and 108 dilutions of cultures on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting.

MEDIA

The supplier, batch number and expiry date of the used chemicals described in sections 5.4.1.-5.4.5. are summarized in Table 6 (Section 5.9. Chemicals Used in the Experiments). The composition of media refers to 1000 mL.

The Typical Composition (g/1000 mL) of Minimal Glucose Agar

Glucose 20.0g
Magnesium sulfate 0.2g
Citric acid 2.0g
di-Potassium hydrogenphosphate 10.0g
Sodium ammonium hydrogenphosphate 3.5g
Agar agar 15.0g
Distilled water q.s. ad 1000 mL

Minimal glucose agar plates [MERCKOPLATE] (Batch Number: 04329, Expiry date: 29 March 2011 used in the Preliminary Range Finding Test; Batch Number: 04414, Expiry date: 15 June 2011 used in the Initial Mutation Test; and Batch Number: 04416, Expiry date: 16 June 2011 used in the Confirmatory Mutation Test) were provided by Merck. Certificates of Analysis were obtained from the Supplier.

Nutrient Broth No.2

Nutrient Broth No.2. 25.0g
Distilled water q.s. ad 1000mL
Sterilization was performed at 121°C in an autoclave.
Note: Due to technical reason, the Nutrient Broth No.2 was prepared in the preliminary experiment in the following way: 10.0 g of LAB Lemco Powder, 10.0 g of Peptone, 5.0 g of Sodium chloride dissolved in Distilled water (final volume 1000.0 mL).

Nutrient Agar:
Nutrient Agar 20.0g
Distilled water q.s. ad 1000mL
Sterilization was performed at 121°C in an autoclave.

Top Agar for Salmonella typhimurium Strains:
Agar solution:
Agar Bacteriological 4.0g
NaCl 5.0g
Distilled water q.s. ad 1000mL
Sterilization was performed at 121°C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.3) 122.2mg
L-Histidine x•HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1000mL
Sterilization was performed by filtration using a 0.22 um membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100mL
Agar solution 900mL

Top Agar for Escherichia coli Strain:
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2000mg
Distilled water q.s. ad 1000mL

Sterilization was performed by filtration using a 0.22 um membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50mL
Tryptophan solution (2 mg/mL) 2.5mL
Agar solution 947.5mL

METABOLIC ACTIVATION SYSTEM

Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction.

The rat liver post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory in the LAB Research Ltd according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

For azo-dyes and diazo-compounds the modified protocol proposed by Prival and Mitchell is referred to in the OECD guideline No. 471.

This modified protocol differs from the standard plate incorporation assay in five ways:
1. uninduced hamster liver S9 instead of induced rat liver S9 is used
2. the hamster liver S9 mix contains 30% hamster liver extract
3. flavine mononucleotide is added to the S9 mix
4. exogeneous glucose 6-phosphate dehydrogenase, NADH, and four times the standard amount of glucose 6-phosphate is added to the S9 mix
5. a 30 minutes pre-incubation step is used before addition of top agar.

These modifications are needed in order to test the mutagenic potential under conditions in which reduction of the compound to its constituent aromatic amines occurs. The hamster liver post-mitochondrial fraction (S9 fraction) was obtained from Trinova Biochem GmbH.

Rat Liver S9 fraction

Induction of Rat Liver Enzymes

Male Wistar rats (275-319 g, animals were 8 weeks old at the initiation of E10891; 257-305 g, animals were 7-8 weeks old at the initiation of E11041) were treated with phenobarbital (PB) and -naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation dates of the induction of liver enzymes for preparation S9 used in this study were 20 September 2010 (E10891) and 07 March 2011 (E11041).

Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1-3 mL portions, frozen quickly and stored at -80  10ºC.

Sterility of the preparation was checked.

The protein concentration was determined by colorimetric test by chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of LAB Research Ltd. The protein concentration of the S9 fraction used was determined to be 34.2 g/L (E10891) and 35.3 g/L (E11041). The dates of preparation of S9 fraction for this study were 23 September 2010 (LAB code: E10891) and 10 March 2011 (LAB code: E11041)*.

*Note: Lot E10891 was used in the preliminary experiment, lot E11041 was used in the Initial Mutation Test.

The biological activity in the Salmonella assay of each batch of S9 was characterized beside the 2-Aminoanthracene with another mutagen, Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. Each batch of S9 used in this study was found active under the test conditions.

The Rat Liver S9 Mix
Salt solution for S9 mix:

NADP Na 7.66g
D-glucose-6 phosphate Na 3.53g
MgCl2 x 6 H2O 4.07g
KCl 6.15g
Distilled water q.s. ad 1000.0 mL

The solution was sterilized by filtration through a 0.22 um membrane filter.

The complete S9 mix was freshly prepared containing components as follows:

Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500.0mL
Rat liver homogenate (S9) 100.0mL
Salt solution for S9 mix 400.0 mL

Prior to addition to the culture medium the S9 mix was kept in an ice bath.

Hamster Liver S9 fraction
Hamster Liver Homogenate S9 Fraction

The S9 fraction of Syrian golden hamster liver was provided by Trinova Biochem GmbH (Kerkrader Straße 10; D-35394 Gießen, Germany); Manufacturer: MOLTOX-Molecular Toxicology Inc. (P.O. Box 1189; Boone, North Carolina, 28607 USA). Certificate of Analysis is obtained from the supplier, stored in the Microbiological Laboratory.

Name: Hamster Liver Post Mitochondrial Supernatant (S9)
Supplier: Trinova Biochem GmbH
Manufacturer: MOLTOX Molecular Toxicology Inc.
Batch Number: 2696 /15-104/
Manufacturing date: 23 December 2010
Expiry date: 23 December 2012
Protein content: 42.5 mg/mL
Storage conditions: -80 ± 10oC

The Hamster liver S9 Mix

The preparation of the S9 Mix was performed according to Prival and Mitchell.

Salt solution for S9 mix:

β-NADP Na 15.31 g
NADH Na2 7.63 g
FMN (Riboflavine-5’-phosphate-sodium salt) 4.78 g
D-glucose-6 phosphate Na 28.20 g
MgCl2 x 6 H2O 8.13 g
KCl 12.31 g
Distilled water ad 1000.0mL

The solution was sterilized by filtration through a 0.22 *m membrane filter.

The complete S9 Mix will be freshly prepared as follows:

Ice cold 0.2 M Sodium phosphate-buffer, pH 7.4 500.0 mL
Hamster liver homogenate (S9) 300.0 mL
Salt solution for S9 mix 200.0 mL
D-glucose-6 phosphate dehydrogenase 2800 U

Prior to addition to the culture medium the S9 mix was kept in an ice bath.


0.2 M Sodium Phosphate Buffer, pH 7.4

Solution A:
Na2HPO4 x 12H2O 71.63g
Distilled water q.s. ad 1000mL

Sterilization was performed at 121°C in an autoclave.

Solution B:
NaH2PO4 24.0g
Distilled water q.s. ad 1000mL

Sterilization was performed at 121°C in an autoclave.

Sodium phosphate buffer pH 7.4:
Solution A 880mL
Solution B 120mL

DESCRIPTION OF THE TEST PROCEDURE

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used.

In the Confirmatory Mutation Test, a modified pre-incubation procedure proposed by Prival and Mitchell for azo-dyes and diazo-compounds was used to confirm the negative result of the Initial Mutation Test.

Concentrations

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test the same concentrations were used.

Preliminary Solubility Test

The solubility of the test item was examined in the Preliminary Solubility using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test item was insoluble in DMSO and DMF, but it was soluble in Distilled water at 100 mg/mL concentration. However, the resulting formulation was very thick, not suitable for the test. Therefore, it was further diluted to 50 mg/mL concentration. The formulation at this concentration had a proper density, thus Distilled water was chosen for solvent of the study. The obtained stock solution (100 µL) with the solution of top agar (5.4.4.) and phosphate buffer (5.5.3.) was examined in a test tube without test bacterium suspension.

Concentration Range Finding Test (Informatory Toxicity Test)

Based on the solubility test, 50 mg/mL stock solution was prepared in Distilled water, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)

Based on the results of the preliminary tests, 50 mg/mL stock solution was prepared from the test item with Distilled water, which was diluted by serial dilutions in six steps to obtain seven dosing solutions for lower doses. The maximum test concentration was 5000 μg test item/plate. Examined Reactive Red F03-0318 concentrations were: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

Control Groups Used in the Tests

Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Experimental Method
Procedure for Exposure in the Initial Mutation Test

A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.

Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
top agar 2000µL
solvent or test item solution (or reference controls) 100 (50)µL*
overnight culture of test strain 100µL
phosphate buffer (pH 7.4) or S9 mix 500µL

*Note: Treatment volume was 100 µL for test item solutions and solvent control, and 50 µL for positive reference controls and their solvent controls.

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test

A modified pre-incubation procedure was performed as a Confirmatory Mutation Test since the result of the Initial Mutation Test was negative.

Before the overlaying, the test item solution (or solvent or reference control), the bacterial culture (Section 5.3.6.) and the hamster liver S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its solvent). The tubes were gently mixed and incubated for 30 min at 30°C in a shaking incubator.

After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Evaluation criteria:

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions at least two times higher than the reversion rate of the solvent control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains
- the number of reversions at least three times higher than the reversion rate of the solvent control in Salmonella typhimurium TA1535 and TA1537 bacterial strains

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated / negative / positive control and test item treated plates were determined by manual counting. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test results

Test results 1

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each samples (including the controls) were tested in triplicate.

In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.

The observed numbers of revertant colonies compared to the solvent control were mostly in the normal range in both tester strains with and without metabolic activation. Minor differences were observed in some cases, but they were considered as biological variability of the test.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. Each test was performed in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (solvent) and positive controls. In the main test each sample (including the controls) were tested in triplicate.

The examined test item concentrations in the main tests were: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

Note: In the Confirmatory Mutation Test, in Salmonella typhimurium TA98 tester strain with metabolic activation plates of the lowest concentration were dried out, they could not be evaluated. No data are reported for this concentration, but this fact has no impact on the results of the study.

In the Initial Mutation Test and Confirmatory Mutation Tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.

Using the plate incorporation method, the highest revertant rate was observed in the Initial Mutation Test in Salmonella typhimurium TA98 tester strain without metabolic activation at the concentration of 1581 μg/plate. The mutation factor value was 1.39. Higher revertant counts compared to the solvent control were detected for other tested concentrations using this strain. However, no dose-dependence was observed, the observed mutation factor values were well below the biologically relevant threshold value and the mean numbers of revertant colonies were within the historical control range in all cases.

Using the pre-incubation method (Prival modification), the highest revertant rate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 tester strain with metabolic activation at the concentration of 500 μg/plate. The mutation factor value was 2.53. Higher revertant counts compared to the solvent control were detected in the whole tested concentration range in this experiment. However, no dose-response was observed; the observed mutation factor values did not reach the biologically relevant threshold value and the mean numbers of revertant colonies were within the historical control range in all cases.

Sporadically, higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test at some concentrations. However, the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

VALIDITY OF THE TESTS
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated and solvent control plates were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table of the Confirmatory Mutation Test

 

Concentrations (ug/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	25.0	38.0	200.3	119.3	18.0	13.3	8.3	8.7	30.7	36.3
	MF	0.93	0.83	1.08	0.86	1.20	0.80	0.86	0.81	1.07	0.92
DMSO control	Mean	26.3	41.3	--	135.0	--	17.0	5.3	12.3	--	50.3
	MF	0.98	0.90	--	0.98	--	1.02	0.55	1.16	--	1.28
Distilled water control (50 µL)	Mean	--	--	197.7	--	18.0	--	--	--	29.0	--
	MF	--	--	1.07	--	1.20	--	--	--	1.01	--
Distilled water control (100 µL)	Mean	27.0	46.0	185.3	138.0	15.0	16.7	9.7	10.7	28.7	39.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	21.0	36.3	198.0	228.7	12.3	15.7	7.3	18.7	31.3	36.7
	MF	0.78	0.79	1.07	1.66	0.82	0.94	0.76	1.75	1.09	0.93
1581	Mean	30.7	34.7	223.0	199.0	11.7	16.0	7.3	16.3	34.0	40.7
	MF	1.14	0.75	1.20	1.44	0.78	0.96	0.76	1.53	1.19	1.03
500	Mean	30.3	35.3	214.3	173.0	12.3	15.7	9.0	27.0	34.3	42.0
	MF	1.12	0.77	1.16	1.25	0.82	0.94	0.93	2.53	1.20	1.07
158.1	Mean	23.0	40.0	230.0	168.3	9.0	16.3	5.3	19.7	34.7	44.0
	MF	0.85	0.87	1.24	1.22	0.60	0.98	0.55	1.84	1.21	1.12
50	Mean	25.3	44.3	216.3	155.3	18.0	16.3	5.7	16.3	34.0	45.3
	MF	0.94	0.96	1.17	1.13	1.20	0.98	0.59	1.53	1.19	1.15
15.81	Mean	30.3	41.3	236.0	136.7	14.0	12.0	8.0	21.7	38.0	39.7
	MF	1.12	0.90	1.27	0.99	0.93	0.72	0.83	2.03	1.33	1.01
5	Mean	26.3	n.d.	232.3	146.7	12.0	15.0	6.0	19.0	41.3	44.3
	MF	0.98	n.d.	1.25	1.06	0.80	0.90	0.62	1.78	1.44	1.13
NPD (4mg)	Mean	577.3	--	--	--	--	--	--	--	--	--
	MF	21.92	--	--	--	--	--	--	--	--	--
2AA (2mg)	Mean	--	1898.7	--	1530.0	--	224.7	--	185.7	--	--
	MF	--	45.94	--	11.33	--	13.22	--	15.05	--	--
2AA (50mg)	Mean	--	--	--	--	--	--	--	--	--	328.7
	MF	--	--	--	--	--	--	--	--	--	6.53
SAZ (2mg)	Mean	--	--	1402.7	--	1429.3	--	--	--	--	--
	MF	--	--	7.10	--	79.41	--	--	--	--	--
9AA (50mg)	Mean	--	--	--	--	--	--	1187.0	--	--	--
	MF	--	--	--	--	--	--	222.56	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1076.0	--
	MF	--	--	--	--	--	--	--	--	37.10	--

n.d.: no data

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test item Reactive Red F03-0318 had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item Reactive Red F03-0318 was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats (Preliminary Range Finding Test and Initial Mutation Test) or from the livers of uninduced hamsters (Confirmatory Mutation Test).

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method, Prival modification).

Based on the results of the Solubility Test, the test item was dissolved inDistilled water. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate wereexamined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were:5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect.

In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normal biological variability of the test system.

The mean values of revertant colonies of the solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Reactive Red F03-0318 had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.