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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2011 to 18 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Substance name: Reactive Yellow F01-0555

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 / 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 applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver post-mitochondrial fraction (S9 fraction)
uninduced hamster liver post-mitochondrial fraction (S9 fraction)
Test concentrations with justification for top dose:
No. of concentration Concentration of the test item Concentration (µg/plate)
1 100 mg/mL 5000
2 31.62 mg/mL 1581
3 10 mg/mL 500
4 3.162 mg/mL 158.1
5 1 mg/mL 50
6 0.3162 mg/mL 15.81
Vehicle / solvent:
Vehicle: Distilled water

Justification for choice of vehicle:
The solubility of the test item was examined in Distilled water and Dimethyl sulfoxide (DMSO). The test item was soluble in both solvents at 100 mg/mL concentration. Due to the better biocompatibility to the test system, Distilled water was chosen for solvent of the study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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:
The bacteria used in this assay did not possess the mammalian enzyme system that is known to convert promutagens into mutagenic metabolites. In order to overcome this major drawback, an exogenous metabolic activation system is added in the form of a mammalian microsomal enzyme activation mixture (liver extract, S9 fraction). The activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The liver extract was obtained from rats, which were pre-treated with phenobarbital and beta-naphthoflavone, two inducers of several drug-metabolizing enzymes.

In addition to the standard Ames test, a modified protocol using pre-incubation with hamster S9 supplemented with flavine mononucleotide was used as proposed by Prival for assessing the mutagenicity of azo-dyes.

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 (100 mg/mL) 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 and negative (solvent) 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 two 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:

Dimethyl sulfoxide (DMSO):

Supplier: Sigma-Aldrich Co.
Batch No.: BCBD8784V
Expiry date: December 2016
Grade: puriss p.a., ACS reagent
Purity: ≥99.9%

Distilled water:

Supplier: TEVA Hungary Co.
Batch No.: 0110111
Expiry date: January 2014
Grade: Aqua destillata pro injectione

BACTERIAL STRAINS

Origin

Date of arrival and origin:

Salmonella typhimurium TA98 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA100 04 December 2008, BioReliance Corporation, Rockville, Maryland, USA
Salmonella typhimurium TA1535 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1537 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Escherichia coli WP2 uvrA 16 November 2010, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

The true copies of original certificates and other documents of the strains are collected and stored in the Microbiological Laboratory of the CiToxLAB Hungary Ltd.

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 CiToxLAB Hungary 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.

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 CiToxLAB Hungary 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 degrees 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 (Section 5.4.3.) plates. The viable cell number of the cultures was determined by manual counting.

MEDIA

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

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

Minimal glucose agar plates [MERCKOPLATE] (Batch Number: 14518, Expiry date: 15 September 2011) were provided by Merck. Certificates of Analysis were obtained from the Supplier.

Nutrient Broth No.2

Nutrient Broth No.2. 25.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

Nutrient Agar

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

Top Agar for Salmonella typhimurium Strains

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

Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.3) 122.2 mg
L-Histidine x HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1000 mL
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) 100 mL
Agar solution 900 mL

Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2000 mg
Distilled water q.s. ad 1000 mL

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

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

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 CiToxLAB Hungary 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 (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 date of the induction of liver enzymes for preparation S9 used in this study was 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 CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 35.3 g/L. The date of preparation of S9 fraction for this study was 10 March 2011 (CiToxLAB code: E11041).

The biological activity in the Salmonella assay of the batch of S9 used in the study 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 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
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 7.4 500.0 mL
Rat liver homogenate (S9) 100.0 mL
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.
Catalogue Number: 15-104
Batch Number: 2696
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 1000.0 mL

The solution was sterilized by filtration through a 0.22 um 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 3000 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 71.63 g
Distilled water q.s. ad 1000 mL

Sterilization was performed at 121°C in an autoclave.

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

Sterilization was performed at 121°C in an autoclave.

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





Evaluation criteria:
The colony numbers on the untreated /solvent/ 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 Excel TM 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 occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred 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 produced 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:
None

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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. Higher numbers of revertant colonies compared to the solvent control were observed in some cases, but they were well below the relevant threshold value and within the historical control range. Furthermore, no dose-response was observed, therefore, these values were considered as biological variability of the test.

In Salmonella typhimurium TA100 strain, the numbers of revertant colonies on the untreated and negative (solvent) control plates were out of the historical range. However, as the aim of the Preliminary Range Finding test was to get information about toxicity and collect data for dose selection, and the results fulfilled these criteria, the preliminary experiment was considered to be acceptable.

Any other information on results incl. tables

INITIAL AND CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method (Prival modification) 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 uvr A 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; and 15.81 /plate.

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 50 μg/plate. The mutation factor value was 1.97. 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 did not reach 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 without metabolic activation at the concentration of 1581 μg/plate. The mutation factor value was 1.79. Higher revertant counts compared to the solvent control were detected at other tested concentrations in this experiment. However, no dose-response was observed; the observed mutation factor values were below the biologically relevant threshold value and the mean numbers of revertant colonies were within the historical control range. Furthermore, higher number of revertant colonies compared to the Distilled water control was observed for DMSO control (MF: 1.57) also in this experiment

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.

Summary Table of the Confirmatory Mutation Test (Pre-Incubation Method)

 

Concentrations(ug/plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvr A

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

20.3

37.7

133.0

144.3

9.7

11.0

3.7

8.3

29.0

44.0

MF

0.98

1.07

1.60

1.18

1.32

1.22

0.79

1.19

1.19

1.27

DMSO
control

Mean

18.0

33.7

--

115.3

--

12.7

7.3

12.0

--

33.7

MF

0.87

0.95

--

0.94

--

1.41

1.57

1.71

--

0.97

Distilled water control

Mean

20.7

35.3

83.3

122.3

7.3

9.0

4.7

7.0

24.3

34.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

22.0

36.3

79.3

113.7

9.0

12.0

6.0

11.3

26.7

46.0

MF

1.06

1.03

0.95

0.93

1.23

1.33

1.29

1.62

1.10

1.33

1581

Mean

19.7

45.0

87.0

126.0

9.3

9.7

8.3

10.3

19.3

45.0

MF

0.95

1.27

1.04

1.03

1.27

1.07

1.79

1.48

0.79

1.30

500

Mean

19.3

38.7

82.0

125.3

10.0

7.3

5.7

9.7

31.3

43.7

MF

0.94

1.09

0.98

1.02

1.36

0.81

1.21

1.38

1.29

1.26

158.1

Mean

24.0

42.0

73.0

142.3

9.3

6.3

4.0

7.7

30.3

45.7

MF

1.16

1.19

0.88

1.16

1.27

0.70

0.86

1.10

1.25

1.32

50

Mean

22.0

39.0

75.3

117.3

8.3

7.3

6.7

10.7

25.7

42.0

MF

1.06

1.10

0.90

0.96

1.14

0.81

1.43

1.52

1.05

1.21

15.81

Mean

20.3

43.3

86.0

139.3

9.7

8.3

4.0

6.0

19.3

37.0

MF

0.98

1.23

1.03

1.14

1.32

0.93

0.86

0.86

0.79

1.07

NPD (4ug)

Mean

293.7

--

--

--

--

--

--

--

--

--

MF

16.31

--

--

--

--

--

--

--

--

--

2AA (2ug)

Mean

--

2301.3

--

2388.0

--

210.7

--

200.7

--

--

MF

--

68.36

--

20.71

--

16.63

--

16.72

--

--

2AA (50ug)

Mean

--

--

--

--

--

--

--

--

--

353.3

MF

--

--

--

--

--

--

--

--

--

10.50

SAZ (2ug)

Mean

--

--

1880.0

--

1480.0

--

--

--

--

--

MF

--

--

22.56

--

201.82

--

--

--

--

--

9AA (50ug)

Mean

--

--

--

--

--

--

486.0

--

--

--

MF

--

--

--

--

--

--

66.27

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1202.7

--

MF

--

--

--

--

--

--

--

--

49.42

--

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.

Applicant's summary and conclusion

Conclusions:
The test item Reactive Yellow F01-0555 was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay including the Prival modification.
In this test, the test item Reactive Yellow F01-0555 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 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 uvr A) 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 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 two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were:5000; 1581; 500; 158.1; 50 and 15.81 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 Yellow F01-0555 had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.