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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 November 2010 to 14 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, OECD & US EPA 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F08-0146

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- -> 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- -> trp+ reversions. The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
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:
Formulation of the Test Item for the Main Experiments
No. of concentration Concentration
of the test item Concentration (µg/plate)
1 50 mg/mL 5000
2 15.81 mg/mL 1581
3 5 mg/mL 500
4 1.581 mg/mL 158.1
5 0.5 mg/mL 50
6 0.1581 mg/mL 15.81
7 0.05 mg/mL 5
Vehicle / solvent:
Distilled water was used as solvent to prepare the stock solution of the test material.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water & DMSO
True negative controls:
not specified
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, negative (solvent) and untreated controls were included in the experiments. Routine safety precautions (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.

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:

Distilled water:
Supplier: TEVA Hungary Co.
Batch No.: 3590210*
Expiry date: February 2013
Grade: Aqua destillata pro injectione

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

*: used in the Preliminary Range Finding Test and Initial Mutation Test
**: used in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test

Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: BCBB7249*
Expiry date: January 2016
Grade: puriss p.a., ACS reagent
Purity: ≥99.9%

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

*: used in the Preliminary Range Finding Test , Initial Mutation Test and Confirmatory Mutation Test
**: used in the Complementary Confirmatory Mutation Test


BACTERIAL STRAINS

Origin
Date of arrival and origin:
Salmonella typhimurium TA98: 04 December 2008, BioReliance Corporation, Rockville, Maryland, USA
Salmonella typhimurium TA100: 04 December 2008, BioReliance Corporation, Rockville, Maryland, USA
Salmonella typhimurium TA1535: 04 December 2008, BioReliance Corporation, Rockville, Maryland, USA
Salmonella typhimurium TA1537: 04 December 2008, BioReliance Corporation, Rockville, Maryland, USA
Escherichia coli WP2 uvrA: 09 December 2008, 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 ± 10°C 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. and Maron and Ames.

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 (in Salmonella strains) or tryptophan (in 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 2009 (as guide) as follows: Salmonella typhimurium TA98: 9-54, TA100: 58-211, TA1535: 2-31, TA1537: 1-24, Escherichia coli WP2 uvrA: 9-66.

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 (Section 5.4.2.) 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 composition of media refers to 1000 mL.

The Typical Composition (g/1000 mL) of Minimal Glucose Agar:
Glucose: 20.0g
Magnesium sulphate: 0.2g
Citric acid: 2.0g
di-Potassium hydrogenphosphate: 10.0g
Sodium ammonium hydrogenphosphate: 3.5g
Agar agar: 15.0g
Distilled water: 1000mL

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:
uninduced hamster liver S9 instead of induced rat liver S9 is used
the hamster liver S9 mix contains 30% hamster liver extract
flavine mononucleotide is added to the S9 mix
exogeneous glucose 6-phosphate dehydrogenase, NADH, and four times the standard amount of glucose 6-phosphate is added to the S9 mix
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 initiation) were treated with Phenobarbital (PB) and beta-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used in the preparation of S9 fraction used in this study was 20 September 2010.

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 ± 10°C.

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. The date of preparation of S9 fraction for this study (LAB code: E10891): 23 September 2010.

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. The 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: 1000.0mL

The solution was sterilized by filtration through a 0.22 *m 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: 400.0mL

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).

Name: Post Mitochondrial Supernatant (S9)
Supplier: Trinova Biochem GmbH
Manufacturer: MOLTOX Molecular Toxicology Inc.
Species (Strain): Syrian Hamster (LVG)
Batch Number: 2380 (15-104)
Manufacturing date: 19 February 2009
Expiry date: 19 February 2011
Protein content: 35.9 mg/mL
Storage conditions: -80 ± 10°C

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

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

The complete S9 Mix will be freshly prepared as follows:

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

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

Description of the Test Procedure
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary 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, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test the same concentrations were used.

Preliminary Solubility Test
The solubility of the test item was examined in the Preliminary Solubility test using Distilled water and Dimethyl sulfoxide (DMSO). The test item was insoluble in DMSO, but it was soluble in Distilled water at 100 mg/mL concentration. However, the resulted 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 and phosphate buffer 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 F08-0146 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
The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, Prival and Mitchell, OECD Guideline No. 471 1997, Commission Regulation (EC) No 440/2008 and EPA Guidelines, OPPTS 870.5100, 1998 and 1996.

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 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:
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.

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:
- in Salmonella typhimurium TA100 strain: the number of reversions is at least twice as high as the reversion rate of the solvent control
- in Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains: the number of reversions is at least three times higher than the reversion rate of the solvent control

According to the guidelines [5][6][7], 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:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
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.

Higher numbers of revertant colonies compared to the solvent control were detected in Salmonella typhimurium TA98 strain without metabolic activation 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. Furthermore, higher revertant counts compared to the Distilled water control were observed for untreated control (MF: 1.24) and DMSO control (MF: 1.22) also in this experiment. Thus, the observed values were considered as reflecting the biological variability of the test.
Lower numbers of revertant colonies compared to the solvent control were detected in some sporadic cases, but the numbers of revertant colonies were within the historical control range in all cases.

Initial And Confirmatory Mutation Tests
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the pre-incubation method (Prival modification) was used. The Initial Mutation Test, Confirmatory Mutation Test and Complementary 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.

In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, 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 TA1535 tester strain without metabolic activation at the concentration of 5000 μg/plate. The mutation factor value was 1.81. However, the observed mutation factor value was below the biologically relevant threshold value and the mean number of revertant colonies was within the historical control range. Furthermore, higher revertant counts compared to the Distilled water control were observed for untreated control (MF: 1.37) and distilled water (50 μL) control (MF: 1.37) also in this experiment.

Using the pre-incubation method, the highest revertant rate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 tester strain with metabolic activation at the concentration of 5000 μg/plate. The mutation factor value was 1.69. Higher revertant counts compared to the solvent control were detected for other tested concentration using this strain. However, 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. Furthermore, higher revertant counts compared to the Distilled water control were observed for untreated control (MF: 1.23) and DMSO control (MF: 1.23) also in this experiment.
Sporadically, higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test, Confirmatory Mutation Test and Complementary 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, Confirmatory Mutation Test and Complementary 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 and Complementary Confirmatory Mutation Test

Concentrations (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimurium tester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

21.0

43.7

139.7

138.3

13.0

10.7

17.7

19.7

30.0

49.0

MF

1.07

1.19

0.90

0.84

1.18

1.23

0.98

1.20

0.71

0.82

DMSO control (50μL)

Mean

18.7

42.3

-

133.7

-

10.7

18.7

16.0

-

54.3

MF

0.95

1.15

-

0.81

-

1.23

1.04

0.98

-

0.91

Distilled water control (50μL)

Mean

-

-

141.3

-

9.3

-

-

-

39.3

-

MF

-

-

0.91

-

0.85

-

-

-

0.93

-

Distilled water control (100μL)

Mean

19.7

36.7

154.7

164.3

11.0

8.7

18.0

16.3

42.3

60.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

15.0

19.3

113.3

182.7

10.0

14.7

25.7

15.3

34.0

57.3

MF

0.76

0.53

0.73

1.11

0.91

1.69

1.43

0.94

0.80

0.96

1581

Mean

15.3

41.7

113.7

161.7

11.0

12.7

24.3

21.0

33.3

62.7

MF

0.78

1.14

0.73

0.98

1.00

1.46

1.35

1.29

0.79

1.04

500

Mean

18.3

38.3

117.3

165.0

11.7

10.0

20.7

16.7

34.0

55.7

MF

0.93

1.05

0.76

1.00

1.06

1.15

1.15

1.02

0.80

0.93

158.1

Mean

15.0

34.3

126.7

145.3

9.3

8.3

23.3

19.0

37.0

49.7

MF

0.76

0.94

0.82

0.88

0.85

0.96

1.30

1.16

0.87

0.83

50

Mean

15.7

41.0

141.3

149.3

7.7

7.0

23.7

17.7

39.7

50.3

MF

0.80

1.12

0.91

0.91

0.70

0.81

1.31

1.08

0.94

0.84

15.81

Mean

17.7

43.0

167.7

152.7

8.0

10.3

20.0

19.3

41.0

45.0

MF

0.90

1.17

1.08

0.93

0.73

1.19

1.11

1.18

0.97

0.75

5

Mean

15.0

34.7

145.0

159.7

9.0

6.0

16.7

18.7

30.7

42.7

MF

0.76

0.95

0.94

0.97

0.82

0.69

0.93

1.14

0.72

0.71

NPD (4μg)

Mean

239.0

--

--

--

--

--

--

--

--

--

MF

12.80

--

--

--

--

--

--

--

--

--

2AA (2μg)

Mean

--

3084.0

--

3340.0

--

177.0

--

249.0

--

--

MF

--

72.85

--

24.99

--

16.59

--

15.56

--

--

2AA (50μg)

Mean

--

--

--

--

--

--

--

--

--

267.7

MF

--

--

--

--

--

--

--

--

--

4.93

SAZ (2μg)

Mean

--

--

1076.0

--

934.7

--

--

--

--

--

MF

--

--

7.61

--

100.14

--

--

--

--

--

9AA (50μg)

Mean

--

--

--

--

--

--

1882.7

--

--

--

MF

--

--

--

--

--

--

100.86

--

--

--

MMS (2μL)

Mean

--

--

--

--

--

--

--

--

942.7

--

MF

--

--

--

--

--

--

--

--

23.97

--

 

Applicant's summary and conclusion

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

In conclusion, the test item Reactive Red F08-0146 had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.
Executive summary:

This study has been performed in accordance with the study plan agreed upon by the Sponsor, the OECD Guidelines for Testing of Chemicals (No. 471, 21 July 1997), Commission Regulation (EC) No.440/2008, B.13/14., EPA Health Effects Test Guidelines, OPPTS 870.5100 (EPA 712-C-98-247, 1998 and EPA 712-C-96-247, 1996) and the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

The test item Reactive Red F08-0146 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 WP2 uvrA) 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 (Complementary 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), a Confirmatory Mutation Test and Complementary Confirmatory Mutation Test (Pre-Incubation Method, Prival modification).

Based on the results of the Solubility Test, the test item was dissolved in Distilled water. 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 three independently performed main experiments (Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test) were:5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

In the Initial Mutation Test, Confirmatory Mutation Test and Complementary 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 F08-0146 had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.