Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not show any mutagenic effects in the bacterial reverse mutation assay including the Prival modification and in the mouse lymphoma assay.

Link to relevant study records

Referenceopen allclose all

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
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
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 / 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.
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
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.

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.

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2019 to February 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence.
In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains.
The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities.
The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers: 3512 and 3488
Test concentrations with justification for top dose:
Since the test item does not consist of a single active ingredient, the concentration of 5.00 mg/mL was selected as the maximum dose level to be tested as indicated in the Study Protocol. The test item was found to be soluble in complete medium (RPMI 5%).
A cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, where the test item was assayed at a maximum dose level of 5.00mg/mL and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195mg/mL.

Based on the results obtained in the preliminary trial, three independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (+S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.
Main Assay I (-S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.
Main Assay III (-S9, 24 hour treatment): 4.00, 2.00, 1.00, 0.500, 0.250 and 0.125 mg/mL.
Vehicle / solvent:
Test item solutions were prepared using complete medium (RPMI 5%).
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Mutation assay
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Preparation of test cell cultures was performed as described in section 4.4. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10^5 cells/mL.
Plating for 5-trifluorothymidine resistance: After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2×103 cells were plated in each well of four 96-well plates. Plateswere incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored. During the scoring of one culture
treated at 0.625mg/mL, one of the four mutation plates was accidentally overturned. This test point was thus excluded from the statistical analysis.
Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Survival after treatment:
In the first experiment, in the absence of S9 metabolic activation, moderate toxicity, reducing relative total growth (RTG) to 35% of the concurrent negative control, was noted at 5.00 mg/mL, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, test item treatment at 5.00 mg/mL yielded slight toxicity reducing RTG to 69% of the concurrent negative control value, no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the absence of S9 metabolic activation using a long treatment time, dose-related toxicity was observed from 0.500 mg/mL onwards, reducing the RTG values from 61% to 10%. No toxicity was observed at the two remaining concentrations, 0.250 and 0.125mg/mL.

Mutation results:
InMain Assay I, no relevant increases in mutant frequency were observed at any concentration, in the presence of S9 metabolism. A slight increase in mutant frequency was noted in the absence of metabolic activation at the highest concentration tested. However, the observed increase was lower than the Global Evaluation Factor. In addition, no dose-effect relationship was observed, both in the absence and presence of S9 metabolism.InMain Assay II, an increase in mutant frequency was observed at the highest concentration tested and the observed increase was higher than the Global Evaluation Factor. However,
the corresponding RTG value was 10% and it is acknowledged that a result cannot be considered positive if the increase in MF occurs only at 10% RTG. In fact, no increases were observed at the other concentrations tested where RTG values were between 31% and 61%.
Furthermore, since the test item is freely soluble at the concentrations tested and is not related to specific chemical classes, positive only by using the long term treatment, the overall results are considered adequate to exclude any mutagenic effect.
Moreover, in the first experiment, it should be noted that unacceptable heterogeneity, due to the absence of one mutation plate (see section 4.6.3), was observed between replicate mutation cultures at 0.625 mg/mL in the presence of S9 metabolism, therefore this concentration was excluded from the statistical analysis.
For the negative and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. Results are presented in Table 4. An adequate recovery of small colony mutants was observed following treatment with the positive controls.
Conclusions:
It is concluded that REACTIVE YELLOW F01-0555 does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

Since the test item does not consist of a single active ingredient, the concentration of 5.00 mg/mL was selected as the maximum dose level to be tested as indicated in the Study Protocol. The test item was found to be soluble in complete medium (RPMI 5%).

A cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, where the test item was assayed at a maximum dose level of 5.00 mg/mL and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mg/mL.

No precipitation of the test item was noted upon addition of the test item to the cultures and at the end of the 3 and 24 hour treatment periods.

Using the 3 hour treatment time, both in the absence and presence of S9 metabolic activation, no toxicity was observed.

Using the 24 hour treatment time, dose dependent cytotoxicity was noted starting from the highest concentration tested down to 0.625 mg/mL; the Relative Survival was in the range from 11% to 57%. No relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Main Assay I (+S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.

Main Assay I (-S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.

Main Assay III (-S9, 24 hour treatment): 4.00, 2.00, 1.00, 0.500, 0.250 and 0.125 mg/mL.

In the mutation test with the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at the highest concentration tested reducing the Relative Total Growth (RTG) to 35% of the concurrent negative control. A less pronounced toxic effect was observed in the presence of S9 metabolism, where the test item yielded 69% RTG at the top concentration. Using the long treatment time in the absence of S9 metabolic activation,

dose-related toxicity was observed from 0.500 mg/mL onwards, reducing the RTG values from 61% to 10%. No toxicity was observed at the two remaining concentrations tested.

At adequate levels of cytotoxicity, no relevant increases in mutant frequencieswere observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.

Negative and positive control treatments were included in each mutation experiment inthe absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments both as an absolute increase in total mutation frequency (MF) and as an increase in the small colony MF over the concurrent negative control value, indicating the correct functioning of the assay system.

It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

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

Genetic toxicity in vivo

Description of key information

The test item did not show any cytogenic effects in the micronucleus assay in rats.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 October 2011 to 6 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
Hygienic level: SPF at the supplier; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group
At the completion of the study, the spare animals were returned to CiToxLAB Hungary Ltd. spare colony, as their use was not required (no replacements with spare animals were performed)
Age of animals: Young adult rats, approximately 11-12 weeks old at starting and 13-14 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 320-404 g, Females: 205 g- 259 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 6 days (6 days from animal arrival to pre-treatment ophthalmoscopy examination, 12 days to onset of treatment)

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.7-23.8°C
Relative humidity: 34 - 55%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

The temperature and humidity were measured twice daily; no deviations from the target ranges were noted during the study.

Food and water supply

Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).

The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification

Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section.

The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.

The new-borns (Offspring, F1 Generation) were identified by cutting off digit-tips up to one day after birth.

Randomization
All parental/adult (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.
Route of administration:
oral: gavage
Vehicle:
The test item was formulated in distilled, sterile water for injection at 6.25, 25 and 100 mg/mL concentrations without correction for purity, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared and stored refrigerated at 2-8ºC pending use within 4 days. Stability tests (CiToxLAB Hungary Ltd. study code 11/174-316AN) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated a 1-day stability at room temperature and 4-day stability while stored refrigerated at 2-8ºC, when the recovery range was 100%-103%, which lies within the acceptance range of 100 ± 10%.

Vehicle
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 2170511, 2190511
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, May 2014, respectively
Storage: Room temperature


Details on exposure:
Dosing procedure


Main animals

Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Dosing of both sexes began after at least 6 days acclimation (A) and 12 days after the animal arrival; the animals were dosed for 2 weeks before mating, during the mating/post-mating, and were continued up to and including the day of necropsy.

Males were dosed for at least 28 days (14 days pre-mating, 14 days mating/post-mating period and on the day of necropsy), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.

Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to and including the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 26-27 days after the end of the mating period.





Duration of treatment / exposure:
Males were dosed for at least 28 days (14 days pre-mating, 14 days mating/post-mating period and on the day of necropsy), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.

Females were dosed for 14 days pre-mating, for up to 6 days mating period, through gestation and up to and including the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 27-28 days after the end of the mating period.

Positive Control MNT animals
Group 5 animals were mated and females allowed to deliver, similarly to the Main animals. All animals were treated with 20 mg/kg bw/day Cyclophosphamide, administered by intraperitoneal injection approximately 24 h prior to scheduled necropsy (males, on Day 27 for necropsy on Day 28; females, on PND4 for necropsy on PND5).

Recovery animals
Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 56.

Frequency of treatment:
Once daily, 7 days per week.
Post exposure period:
Main animals were treated with test item up to euthanasia.
Recovery animals were kept for at least 14 days without treatment prior to euthanasia.
Positive control animals were euthanised approximately 24 hours after administration of cyclophosphamide.

Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group
Control animals:
yes, concurrent vehicle
other: Positive control: cyclophosphamide
Positive control(s):
Positive Control Micronucleus Test (MNT) animals
Group 5 animals were mated and females allowed to deliver, similarly to the Main animals. All animals were treated with 20 mg/kg bw/day Cyclophosphamide, administered by intraperitoneal injection approximately 24 h prior to scheduled necropsy (males, on Day 27 for necropsy on Day 28; females, on PND4 for necropsy on PND5).

Positive Control Name: Cyclophosphamide monohydrate
Lot number: 079K1569
Supplier: Sigma-Aldrich Co.
Retest/Expiry date: July 2012
Storage condition: Refrigerated (2-8 °C)
Purpose of use: Positive Control item Group 5

Name: Physiological saline (0.9% NaCl solution)
Lot number: 6880610
Supplier: TEVA Gyógyszergyár ZRT
Retest/Expiry date: June 2013
Storage condition: Room temperature
Purpose of use: Preparation of Positive Control Group 5 solution
Tissues and cell types examined:
Four sets of bone marrow smears for MNT were prepared from the animals, including the Vehicle Control (water) and the Positive Control (Cyclophosphamide) groups. According to the study plan and/or subsequent amendment(s), the bone marrow was collected from the right femur of the rats immediately after euthanasia (the left femur of Main and Recovery group animals was used for routine histopathology, the left femur of Positive Control animals was discarded) and flushed with foetal bovine serum (5 mL) using a syringe and needle.
Details of tissue and slide preparation:
Cells were concentrated by a gentle centrifugation. Smears of the cell pellet were made on standard microscope slides and the slides were then air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for at least 5 minutes and allowed to air-dry.

One set of Giemsa-stained slides was given unique code numbers for blinded evaluation (the code labels covered all unique identification markings on the slides to ensure that they were scored without bias). All slides were blinded; only those of the Control (Gr. 1), Positive Control (Gr. 5) and High dose (Gr. 4) Main animals were sent for evaluation.

2000 polychromatic (immature) erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated (MN) cells. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei was recorded in both types of erythrocytes.

Criteria for Identification of Micronucleated Erythrocytes

A micronucleus is defined in following way:

- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

The Micronucleus Test is considered acceptable/valid in the conditions of this study, as it met the following criteria:

-the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls fell within the range of historical laboratory control data.
-the positive control item produced biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
-each treated and control group included at least 5 analysable animals.

Evaluation criteria:
Criteria for a positive response: The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related.

Criteria for a negative response: The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.

Equivocal response: It may be necessary to perform further investigations or to score additional cells if equivocal results are obtained which do not meet the criteria for a positive or negative response. In this study there were no equivocal results, therefore no additional scoring was required.
Statistics:
Data were collected by completing a pre-prepared sheet by hand. The data were tabulated using appropriate forms for reporting. The frequencies of micronucleated polychromatic erythrocytes in animals in the test groups were compared to the values found in the corresponding negative control group. Statistical analysis was performed using Kruskal Wallis Non Parametric ANOVA test (level of significance 5%) except where the frequency in treated animals was less than that in the negative control animals. As some of the positive control frequencies were less than 12 micronuclei in 2000 PCEs, the positive control data were also tested statistically to verify the overall positive response in these groups.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
See Chapter 7.5.1 for details
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No statistical analysis of the frequencies of micronuclei in the animals treated with the high dose in either males or females was appropriate as the average number of micronuclei was lower than the corresponding negative controls in both cases. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, thus, no further slide examination was considered required.

The frequencies in the high dose animals were compared with the control animals; in the males, H = 0.244 and in the females H = 0.143 which are both substantially below statistical significance. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, thus, no further slide examination was considered required. The positive and negative control results were also compared, and both males and females gave a significant response, with values of H = 4.841 (p<0.05) and H = 14.817 (p<0.001) respectively.

The individual and mean data are presented in Tables 1 – 6 below.

  

 

TABLE 1: DOSE GROUP -           CONTROL MALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1001

15

2

448

1002

02

4

414

1003

29

1.5a

464

1004

34

7

401

1005

39

3

374

1006

21

9

469

1007

07

7

440

1008

47

4

422

1009

27

7

434

1010

51

11.5a

521

1011

13

1

429

1012

44

4

465

Mean

 

5.083

440.08

SD

 

3.225

37.78

                                    aNo. of micronuclei assessed in 4000 PCE

TABLE 2: DOSE GROUP - HIGH DOSE 1000 mg/kg bw/day MALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

4001

18

18a

418

4002

25

8

416

4003

43

3

530

4004

05

0

395

4005

41

15a

499

4006

56

2

397

4007

36

3

452

4008

55

1

354

4009

10

1

490

4010

58

4

384

4011

30

8

446

4012

54

3

423

Mean

 

5.500

433.67

SD

 

5.745

51.82

                                   aNo. of micronuclei assessed in 4000 PCE

 

 

TABLE 3: DOSE GROUP - CYCLOPHOSPHAMIDE MALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5001

06

5

315

5002

42

13

315

5003

11

7

367

5004

31

2

325

5005

49

2a

206

5006

04

7

265

5007

48

29

425

5008

17

24

315

5009

24

19a

331

5010

52

18

348

5011

16

16

380

5012

35

23

319

Mean

 

13.750

325.92

SD

 

9.137

55.14

                   aNo. of micronuclei assessed in 4000 PCE

TABLE 4: DOSE GROUP - CONTROL FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

1501

100

4

448

1502

106

3

460

1503

115

5

486

1504

75

10

497

1505

111

5

465

1506

90

7

437

1507

63

5

418

1508

118

6

419

1509

78

7

501

1510

84

4

458

1511

66

2

515

1512

94

1

486

Mean

 

4.917

465.83

SD

 

2.429

31.86

 

 

TABLE 5: DOSE GROUP - HIGH DOSE 1000 mg/kg bw/day FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

4501

65

11

536

4502

98

12

519

4503

120

2

455

4504

77

5

523

4505

113

4

419

4506

83

1

446

4507

103

2

451

4508

88

13

510

4509

69

11

531

4510

107

2

365

4511

93

6

461

4512

116

5

481

Mean

 

6.167

474.75

SD

 

4.407

51.88

 

TABLE 6: DOSE GROUP - CYCLOPHOSPHAMIDE FEMALES

 

Animal code

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 PCE+NCE

5501

70

33

470

5502

108

51

425

5503

117

14

396

5504

87

5

323

5505

112

68

435

5506

64

25

353

5507

102

17

405

5508

76

61

359

5509

82

66

476

5510

92

36

436

5511

71

36

477

5512

97

28

452

Mean

 

36.667

417.25

SD

 

20.821

51.12

 

 

 

Conclusions:
No induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE YELLOW F01-0555 to Wistar rats daily by oral gavage to the High dose Main animals at 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
Executive summary:

The objective of this study was to assess the potential genotoxic effect of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals. 

This study was conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.

Male and female rats were treated with the test item at dose levels of 62.5, 250, and 1000 mg/kg bw/day for a minimum of 28 days.

The frequencies of micronuclei in the high dose animals were compared with the control animals in the males and in the females which

are both substantially below statistical significance. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, hence, no further slide examination was considered required.  The positive and negative control results were also compared, and both males and females showed a statistically significant increase in micronuclei.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of the test item to Wistar rats daily by oral gavage at 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

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

Additional information

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. Hence, the test item had no mutagenic activity on the growth of the bacterium tester strainsunder the test conditions used in this study.

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

Since the test item does not consist of a single active ingredient, the concentration of 5.00 mg/mL was selected as the maximum dose level to be tested as indicated in the Study Protocol. The test item was found to be soluble in complete medium (RPMI 5%).

A cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, where the test item was assayed at a maximum dose level of 5.00 mg/mL and at a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 mg/mL.

No precipitation of the test item was noted upon addition of the test item to the cultures and at the end of the 3 and 24 hour treatment periods. Using the 3 hour treatment time, both in the absence and presence of S9 metabolic activation, no toxicity was observed. Using the 24 hour treatment time, dose dependent cytotoxicity was noted starting from the highest concentration tested down to 0.625 mg/mL; the Relative Survival was in the range from 11% to 57%. No relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:

Main Assay I (+S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.

Main Assay I (-S9, 3 hour treatment): 5.00, 2.50, 1.25, 0.625 and 0.313 mg/mL.

Main Assay III (-S9, 24 hour treatment): 4.00, 2.00, 1.00, 0.500, 0.250 and 0.125 mg/mL.

In the mutation test with the short treatment time in the absence of S9 metabolism, moderate toxicity was noticed at the highest concentration tested reducing the Relative Total Growth (RTG) to 35% of the concurrent negative control. A less pronounced toxic effect was observed in the presence of S9 metabolism, where the test item yielded 69% RTG at the top concentration. Using the long treatment time in the absence of S9 metabolic activation,

dose-related toxicity was observed from 0.500 mg/mL onwards, reducing the RTG values from 61% to 10%. No toxicity was observed at the two remaining concentrations tested.

At adequate levels of cytotoxicity, no relevant increases in mutant frequencieswere observed following treatment with the test item, in the absence or presence of S9 metabolism, in any experiment.

Negative and positive control treatments were included in each mutation experiment inthe absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments both as an absolute increase in total mutation frequency (MF) and as an increase in the small colony MF over the concurrent negative control value, indicating the correct functioning of the assay system.

It is concluded that the test item does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

The potential cytogenic effect of the test item was assessed by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals. Male and female rats were treated with the test item at dose levels of 62.5, 250, and 1000 mg/kg bw/day for a minimum of 28 days.

The frequencies of micronuclei in the high dose animals were compared with the control animals in the males and in the females which are both substantially below statistical significance. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, hence, no further slide examination was considered required.  The positive and negative control results were also compared, and both males and females showed a statistically significant increase in micronuclei.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of the test item to Wistar rats daily by oral gavage at 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. 

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