Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Bacterial reverse mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July - 22 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Test item: Yellow LF 6911
Batch No.: UGe-RS Kilo 1
Appearance: Yellow powder
Expiration date: 12 December 2018
Storage: Room temperature

Method

Target gene:
In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. 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 (WP2uvrA) is also defective in DNA excision repair.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 fraction
Test concentrations with justification for top dose:
The Yellow LF 6911 concentrations examined in the Initial and Confirmatory Mutation Tests:
5000, 1600, 500, 160, 50 and 16 µg/plate.
Vehicle / solvent:
ultrapure water (ASTM Type 1)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
congo red
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, NPD; 2-aminoanthracene, 2AA;
Details on test system and experimental conditions:
Origin of the Bacterial Strains:
The tester strains arrived to the test facility in a form of disc cultures. The origin of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA:
Supplier: Trinova Biochem GmbH; (Rathenau Str. 2; D-35394 Giessen, Germany);
Manufacturer: MOLTOX INC., (P.O. BOX 1189; BOONE, NC 28607 USA).

Storage of Tester Strains:
The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO is added as a cryoprotective agent

Confirmation of Phenotypes of Tester Strains:
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. [1][2].
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. Historical control data are included in the test report.

Procedure for Bacterial Cultures:
The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 (Section: 5.4.2) for the overnight cultures in the assay. The cultures were incubated for approximately 10-14 hours in a 37oC Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures:
The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media
The Minimal Glucose Agar (MGA) Plates:
Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis1) were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
1) Batch No.:138386; Expiry date: 07 September 2016; (used in the Informatory Toxicity Test)
Batch No.:139234; Expiry date: 24 October 2016; (used in the Initial and Confirmatory Mutation Tests)

Nutrient Broth No. 2
Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

utrient Agar
Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes 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
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth by 5.4.2 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by 5.4.4 947.5 mL

Metabolic Activation System

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

Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier1). The copies of the corresponding Quality Control & Production Certificates of the S9 fractions used are attached as Appendix VII., the originals are stored in the Laboratory of TOXI-COOP ZRT.
1) Lot Number: 3453; Expiry date: April 29, 2017; Protein content: 41.8 mg/mL
(used in the Initial Mutation Test);
Lot Number: 3582; Expiry date: February 03, 2018; Protein content: 33.7 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3577; Expiry date: January 21, 2018; Protein content: 38.4 mg/mL
(used in the Informatory Toxicity and Initial Mutation Tests).

The S9 Mix (with Rat Liver S9)
Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm 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 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix was kept in an ice bath before it was added to the culture medium.

Hamster Liver S9 Fraction
The S9 fraction of uninduced hamster liver was also provided by Trinova Biochem GmbH (Section: 5.5.1.). Certificate of Analysis was obtained from the supplier2). The copy of the corresponding Quality Control & Production Certificate of the S9 fraction used is attached as Appendix VII., the original is stored in the Laboratory of TOXI-COOP ZRT.
2) Lot Number: 3525; Expiry date: September 17, 2017; Protein content: 39.7 mg/mL
(used in the Confirmatory Mutation Test);

The S9 Mix (with Hamster Liver S9)
Salt solution for S9 Mix: Final concentration in S9 Mix:
NADP Na 15.31 g 4 mM
NADH Na2 x H2O 7.27 g 2 mM
FMN (Riboflavine-5’-phosphate-sodium salt) x H2O 4.96 g 2 mM
D-glucose-6 phosphate Na 28.20 g 20 mM
MgCl2 3.80 g 8 mM
KCl 12.31 g 33 mM
Ultrapure water ad 1000 mL

Sterilization was performed by filtration through a 0.22 µm membrane filter.

The complete S9 Mix was freshly prepared containing components as follows (per 1000 mL):
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Hamster liver homogenate (S9) 300 mL
Salt solution for S9 Mix 200 mL
D-glucose-6-phosphate-dehydrogenase 2800 U
Before adding to the culture medium the S9 Mix was kept in an ice bath.

Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL

Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.



Rationale for test conditions:
Justification of concentrations:
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test).
Based on the solubility test, the stock solution with a concentration of 50 mg/mL was prepared in the vehicle and diluted in at least 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
Evaluation criteria:
The colony numbers on the controls (untreated, vehicle, positive) and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the Initial and Confirmatory Mutation Tests the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix were within the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

Any other information on results incl. tables

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichiacoli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

16.7

0.86

28.0

0.82

96.7

0.95

142.0

0.90

15.3

0.75

14.7

1.16

10.0

1.15

7.7

1.21

24.0

0.85

33.0

0.93

DMSO Control

20.0

1.00

31.0

1.00

142.0

1.00

20.3

1.00

8.0

1.00

7.3

1.00

27.3

1.00

Ultrapure Water Control

19.3

1.00

34.0

1.00

102.0

1.00

157.0

1.00

20.3

1.00

12.7

1.00

8.7

1.00

6.3

1.00

28.3

1.00

35.7

1.00

5000

20.0

1.03

29.7

0.87

110.0

1.08

109.0

0.69

13.0

0.64

11.0

0.87

9.7

1.12

8.7

1.37

28.0

0.99

27.7

0.78

1600

21.7

1.12

26.0

0.76

106.7

1.05

95.3

0.61

10.7

0.52

13.3

1.05

9.7

1.12

7.3

1.16

30.7

1.08

35.3

0.99

500

24.0

1.24

26.0

0.76

100.0

0.98

111.0

0.71

14.3

0.70

13.0

1.03

8.0

0.92

6.3

1.00

22.7

0.80

33.3

0.93

160

28.7

1.48

32.0

0.94

114.7

1.12

105.7

0.67

16.0

0.79

12.3

0.97

8.0

0.92

10.0

1.58

27.3

0.96

36.3

1.02

50

20.0

1.03

30.3

0.89

112.7

1.10

126.3

0.80

15.3

0.75

11.0

0.87

7.7

0.88

6.3

1.00

27.7

0.98

27.7

0.78

16

18.0

0.93

29.3

0.86

109.3

1.07

123.0

0.78

17.7

0.87

13.3

1.05

7.0

0.81

10.3

1.63

25.7

0.91

27.7

0.78

NPD (4mg)

273.0

13.65

SAZ (2mg)

692.7

6.79

741.3

36.46

9AA (50mg)

326.7

40.83

MMS (2mL)

590.0

20.82

2AA (2mg)

1584.0

51.10

2184.0

15.38

156.7

7.70

106.3

14.50

2AA (50mg)

216.7

7.93

MR:Mutation Rate

Remarks:           Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

19.0

0.97

29.7

1.25

108.0

1.04

115.3

1.07

11.7

1.00

11.3

1.03

4.3

1.08

5.3

0.73

25.7

1.10

23.0

0.96

DMSO Control

19.3

1.00

99.0

1.00

10.0

1.00

3.7

1.00

6.3

1.00

24.0

1.00

Ultrapure Water Control

19.7

1.00

23.7

1.00

104.0

1.00

107.7

1.00

11.7

1.00

11.0

1.00

4.0

1.00

7.3

1.00

23.3

1.00

24.0

1.00

5000

18.3

0.93

32.3

1.37

108.7

1.04

125.3

1.16

11.3

0.97

14.3

1.30

5.0

1.25

7.3

1.00

24.3

1.04

24.7

1.03

1600

19.3

0.98

33.3

1.41

101.0

0.97

154.7

1.44

15.3

1.31

12.7

1.15

6.7

1.67

5.0

0.68

24.3

1.04

24.3

1.01

500

18.7

0.95

29.0

1.23

97.3

0.94

146.0

1.36

10.0

0.86

13.0

1.18

5.0

1.25

6.3

0.86

22.0

0.94

26.7

1.11

160

24.0

1.22

27.7

1.17

116.0

1.12

133.3

1.24

10.7

0.91

11.7

1.06

4.3

1.08

8.3

1.14

23.0

0.99

17.0

0.71

50

23.0

1.17

26.0

1.10

108.0

1.04

124.7

1.16

12.7

1.09

9.3

0.85

3.3

0.83

7.0

0.95

23.7

1.01

20.0

0.83

16

18.0

0.92

27.3

1.15

110.7

1.06

121.3

1.13

9.0

0.77

10.7

0.97

5.0

1.25

8.0

1.09

27.3

1.17

17.7

0.74

NPD (4mg)

342.0

17.69

SAZ (2mg)

680.7

6.54

778.0

66.69

9AA (50mg)

690.7

188.36

MMS (2mL)

780.0

33.43

Congo Red (250mg)

294.0

10.02

2AA (2mg)

1162.0

11.74

319.3

31.93

120.3

19.00

2AA (50mg)

166.3

6.93

MR:Mutation Rate

Remarks:           Ultrapure water was applied as vehicle of the test item and the positive control substances: Congo Red, SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Applicant's summary and conclusion

Conclusions:
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 Yellow LF 6911 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test itemYellow LF 6911was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The test item belongs to azo-dyes therefore in the Confirmatory Mutation Test (Pre-Incubation Test) a modified protocol (using uninduced hamster liver S9, modified co-factor and S9 mixture, longer pre-incubation) proposed by Prival and Mitchell [10] was applied.

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9). In the plate incorporation test (Initial Mutation Test) S9 prepared from livers of phenobarbital/b-naphthoflavone-induced rats was used, in the pre-incubation test S9 prepared from uninduced hamster liver was used.

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

Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity; furthermore appropriate historical control database is available in the testing laboratory.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000; 1600; 500; 160; 50 and 16 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withYellow LF 6911at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The reported data of this mutagenicity assay show (see Appendix I to IV) that under the experimental conditions applied, the test itemdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test itemYellow LF 6911has no mutagenic activity on the applied bacterium tester strainsunder the test conditions used in this study.