Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2015 to 18 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Study report is a translation from Japanese to English.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: - see below
Principles of method if other than guideline:
- “Standards for Mutagencity Tests using Microorganisms” (Notification No. 77, September 1, 1988 & partial revision: Notification No. 67, June 2, 1997, Ministry of Labour, Japan and Notification No. 120, December 25, 2000, , Ministry of Labour, Japan) and the “Amendment of the Reporting Form of the Results of the Mutagenicity Tests using Microorganisms” (Notification No. 653, September 29, 1997, Labour Standards Bureau, Ministry of Labour, Japan)
- “Guidelines for Toxicity Testings of New Chemical Substances” (Notification No. 7 of 0331 Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 5 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, & No. 110331009 of Environmental Policy Bureau, Ministry of the Environment, Japan, March 31, 2011)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Purple powder
- Storage conditions: Room temperature in the dark

Method

Target gene:
Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
- Reason for choice of the strains: These strains are very sensitive to mutagens and are the most commonly used in bacterial reverse mutation assays.
- Type and identity of media: The bacterial suspension and DMSO were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided in 0.3 mL aliquots, and then frozen and stored at -85 to -80 °C.
- Properly maintained: Yes
- Characterisation: The number of viable cell, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.
Conditions of pre-culture
- Nutrient Broth: Nutrient Broth No. 2
- Pre-culture procedure
A bacterial suspension of each strain (20 µL of S. typhimurium TA strains, 5 µL of E. coli WP2 uvrA) was inoculated into an L-form culture tube (35 mL capacity) containing 10 mL of Nutrient Broth. This culture tube was left at 4 °C until starting incubation, and then incubated while shaking (100 rpm) in a water bath at 37°C for 8 hours. After incubation, the optical density was measured and the number of viable cells was calculated by growth curve for each strain. The bacterial cultures were stored at room temperature until starting the test.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate
Mutation Test Experiments 1 and 2: 0, 313, 625, 1250, 2500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Water for injection
- Justification for choice of solvent/vehicle: Based on information from the sponsor that the test material was soluble at >100 g/L in water; water has been chosen as the vehicle
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
furylfuramide
other: 2-Methoxy-6-chloro-9-[3-(2-choroethyl)aminopropylamino]acridine.2HCl (ICR-191), 10Aminoanthracene (2AA)
Remarks:
Negative control same as solvent/vehicle control: water
Details on test system and conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation
For tests without metabolic activation: 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution.
For tests with metabolic activation: 0.5 mL of the S-9 mix was added to each tube instead of the 0.1M Na-phosphate buffer. The mixture was pre-incubated in a water bath at 37 °C for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate; 0.1 mL of the positive control solution was carried out equally.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in two main tests, which were performed at the same doses.

COUNTING PROCEDURE
The number of revertant colonies was counted visually due to the colour of the test material on the plates. But the revertant colonies of positive controls were counted with a colony counter.
Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The growth inhibition by the test material and the precipitate of the test material was not observed in any strains either with or without metabolic activation.

DEFINITIVE TESTS:
In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.
The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean +/- 3SD) in historical data, indicating that this study was performed correctly.
The test material has been determined to be negative in respect to gene mutagenicity.
The growth inhibition of the test strains by the test material was not observed. And the precipitate of the test material on the plates was not observed either with or without metabolic activation.
In the sterility test on the test material and the S9 mix, no growth of bacteria was observed.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

313

625

1250

2500

5000

122

106

114

114

98

99

10

11

9

10

11

10

32

28

29

30

25

27

24

21

20

24

27

28

14

9

8

7

8

9

17

+

Solvent

313

625

1250

2500

5000

129

108

132

121

115

112

10

8

10

11

8

7

29

31

29

26

30

23

34

33

37

37

33

32

17

17

14

13

16

13

Positive Controls

-

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1.0

Mean no. colonies/plate

539

430

144

551

1703

+

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Concentration (µg/plate)

5.0

2.0

10.0

5.0

5.0

Mean no. colonies/plate

936

286

360

228

83

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (Furylfuramide)

NaN3 = Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine∙2HCl

B[a]P = Benzo[a]pyrene

2AA = 2-Aminoanthracene

 

Table 2: Summary of Experiment 2

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

313

625

1250

2500

5000

100

98

94

100

103

97

11

12

9

10

8

9

32

24

26

24

27

22

21

21

19

19

17

23

13

9

9

8

11

8

+

Solvent

313

625

1250

2500

5000

110

101

102

107

92

96

9

10

8

9

7

8

31

29

31

29

28

26

30

28

25

26

23

24

17

14

15

13

17

13

Positive Controls

-

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1.0

Mean no. colonies/plate

574

377

127

520

1984

+

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Concentration (µg/plate)

5.0

2.0

10.0

5.0

5.0

Mean no. colonies/plate

904

323

396

237

86

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (Furylfuramide)

NaN3 = Sodium azide

ICR-191  2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine∙2HCl

B[a]P = Benzo[a]pyrene

2AA = 2-Aminoanthracene

Applicant's summary and conclusion

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

Under the conditions of the study, the test material has been determined to be negative in respect to gene mutagenicity.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities.

Furthermore, the study has been performed under GLP conditions and this particular study report is a translation from Japanese to English.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five dose levels, both with and without metabolic activation. The dose levels assessed were 313, 625, 1250, 2500 and 5000 µg/plate.

A dose selection test was performed, plus two main tests.

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls in historical data, indicating that this study was performed correctly.

The test material has been determined to be negative in respect to gene mutagenicity.

The growth inhibition of the test strains by the test material was not observed. And the precipitate of the test material on the plates was not observed either with or without metabolic activation.

In the sterility test on the test material and the S9 mix, no growth of bacteria was observed.

The test material was considered to be non-mutagenic under the conditions of this test.