Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 August - 21 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Reference substance name:
Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized
Test material form:
liquid: viscous
Details on test material:
Appearance: Off white paste

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix was prepared immediately before use and kept on ice.
Test concentrations with justification for top dose:
5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate

5000 µg/plate is the maximum concentration prescribed in the OECD 471 guidance document for non-cytotoxic test items.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test item soluble in ethanol and compatible with the survival of the bacteria and S9 activity.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
Bacteria preparation

Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Agar plates

Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Sigma).

Top agar

Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) Sodium Chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

S-9 fraction

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch was characterised with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S-9 mix

S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Range finder test

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA strain, both with and without 5% (v/v) S9-mix. Seven concentrations, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assay was the level at which the test item inhibited bacterial growth or exhibited limited solubility, or up to 5000 µg/plate.

Mutation assay- main test

At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98, and in tester strain TA100 in the presence of S9-mix. In an additional experiment, to complete the data of the first mutation experiment, the test item was tested in the tester strains TA1535 and TA1537 in the absence of S9-mix, and in all Salmonella typhimurium strains in the presence of S9-mix. In a follow-up experiment with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. An additional experiment, to compete the data of the second mutation experiment, was performed with the tester strains TA1535 (presence S9-mix) and TA100 (absence and presence of S9-mix). The vehicle (negative control) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.05 ml of a dilution of the test item in ethanol. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony counting

The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.









Rationale for test conditions:
The test conditions allowed the determination of the test items ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.

b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.

In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Range finding test results

Dose

(µg/plate)

Mean number of revertant colonies/3 replicates plates (± S.D.) with one strain ofSalmonella typhimuriumand oneEscherichia colistrain

TA100

WP2uvrA

Without S-9 mix

Positive Control

908 ± 25

242 ± 40

Solvent Control

104 ± 6

24 ± 7

5.4

68 ± 52

24 ± 1

17

111 ± 6

29 ± 4

52

115 ± 13n NP

23 ± 2

164

105 ± 71m NP

23 ± 3

512

0 ± 0a SP

16 ± 9

1600

0 ± 0a SP

20 ± 3NP

5000

0 ± 0a SP

18 ± 7n SP

With S-9 mix1

Positive Control

1604 ± 277

624 ± 55

Solvent Control

103 ± 19

51 ± 40

5.4

108 ± 14n

37 ± 3

17

0 ± 0a

29 ± 5

52

0 ± 0a

28 ± 8

164

0 ± 0a

30 ± 7

512

e SP MC

26 ± 6

1600

e SP MC

23 ± 10NP

5000

e SP MC

43 ± 23n SP

1      The S9 -mix contained 5 % (v/v) S9 fraction

2      Mean of 2 plates

MC   Microcolonies

NP    No precipitate

a      Bacterial lawn absent

e      Bacterial lawn extremely reduced

m     Bacterial lawn moderately reduced

n      Normal bacterial background lawn

 

Table 2: Experiment 1 - Mutagenic response in theSalmonella typhimuriumreverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicates plates (± S.D.) with different strains ofSalmonella typhimurium

TA1535

TA1537

TA98

TA100

Without S-9 mix

Positive Control

951 ± 43

103 ± 10

1473 ± 244

 

Solvent Control

11 ± 5

7 ± 4

19 ± 5

 

0.55

10 ± 6

6 ± 2

23 ± 3

 

1.7

8 ± 3

5 ± 3

14 ± 2

 

5.4

7 ± 4n

3 ± 3

21 ± 4

 

17

e MC

e MC

15 ± 5n

 

52

e MC

e MC

25 ± 6m

 

164

e NP MC

e NP MC

16 ± 4m NP

 

512

e SP MC

0 ± 0

e SP MC

 

With S-9 mix1

Positive Control

160 ± 16

244 ± 47

558 ± 93

854 ± 129

Solvent Control

12 ± 2

6 ± 2

33 ± 4

123 ± 22

0.017

12 ± 5

7 ± 0

25 ± 1

129 ± 9

0.055

12 ± 7

4 ± 3

32 ± 5

131 ± 7

0.17

11 ± 4

7 ± 4

27 ± 11

113 ± 10

0.55

9 ± 3

8 ± 3

22 ± 14

103 ± 16

1.7

13 ± 6

8 ± 4

24 ± 3

109 ± 11

5.4

10 ± 2

10 ± 5

33 ± 3

112 ± 9

17

9 ± 2n NP

6 ± 2n NP

30 ± 7n NP

112 ± 22n NP

1      The S9 -mix contained 5 % (v/v) S9 fraction

MC   Microcolonies

NP    No precipitate

SP    Slight precipitate

a      Bacterial lawn absent

e      Bacterial lawn extremely reduced

m     Bacterial lawn moderately reduced

n      Normal bacterial background lawn

 

Table 3: Experiment 1A- Mutagenic response in theSalmonella typhimuriumreverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicates plates (± S.D.) with different strains ofSalmonella typhimurium

TA1535

TA1537

TA98

TA100

Without S-9 mix

Positive Control

856 ± 10

79 ± 5

 

 

Solvent Control

16 ± 6

11 ± 7

 

 

0.055

17 ± 4i

5 ± 2

 

 

0.17

21 ± 6

8 ± 5

 

 

0.55

16 ± 6i

8 ± 2

 

 

1.7

11 ± 4

7 ± 4

 

 

5.4

14 ± 1n

9 ± 6n

 

 

17

e NP MC

e NP MC

 

 

With S-9 mix1

Positive Control

162 ± 13

291 ± 14

840 ± 36

408 ± 129

Solvent Control

22 ± 6

13 ± 4

32 ± 5

103 ± 22

5.4

23 ± 3

7 ± 4

18 ± 3

122 ± 9

17

2

5 ± 3

23 ± 10

104 ± 7

52

21 ± 11n

7 ± 3

35 ± 5n

107 ± 10n

164

e MC

6 ± 2n

26 ± 4s i

e MC

512

e NP MC

e NP MC

20 ± 4m NP

e NP MC

1      The S9 -mix contained 5 % (v/v) S9 fraction

2      Plates could not be scored due to a technical error

MC   Microcolonies

NP    No precipitate

a      Bacterial lawn absent

e      Bacterial lawn extremely reduced

m     Bacterial lawn moderately reduced

n      Normal bacterial background lawn

 

Table 4: Experiment 2- Mutagenic response in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicates plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S-9 mix

Positive Control

805 ± 25

93 ± 6

1767 ± 233

680 ± 45

72 ± 5

Solvent Control

7 ± 3

5 ± 1

9 ± 5

80 ± 14

22 ± 3

0.055

11 ± 4

6 ± 1

-

-

-

0.17

12 ± 3

5 ± 3

-

-

-

0.55

10 ± 3

3 ± 2

-

-

-

1.7

6 ± 4

2 ± 2

13 ± 5

92 ± 10

-

5.4

8 ± 3n

3 ± 2n NP

11 ± 1

71 ± 7n

-

17

e NP MC

2 ± 1s SP

11 ± 1n

e MC

-

52

-

-

9 ± 4s NP

0 ± 0a

23 ± 7

164

-

-

6 ± 1s NP

0 ± 0a

21 ± 8

512

-

-

e NP MC

e NP MC

22 ± 2NP

1600

-

-

-

-

24 ± 14SP

5000

-

-

-

-

18 ± 5n SP

With S-9 mix1

Positive Control

55 ± 11

97 ± 17

250 ± 17

1082 ± 89

343 ± 44

Solvent Control

11 ± 1

10 ± 2

20 ± 4

74 ± 8

30 ± 6

0.55

11 ± 4

-

-

80 ± 10

-

1.7

11 ± 7

6 ± 4

-

72 ± 17

-

5.4

12 ± 7

9 ± 4

29 ± 3

68 ± 10

-

17

11 ± 7

9 ± 3

24 ± 4

69 ± 4

-

52

10 ± 2

7 ± 2

26 ± 4

72 ± 17

43 ± 12

164

7 ± 3n NP

5 ± 2n NP

23 ± 6n

68 ± 6n NP

30 ± 4

512

 

e SP MC

19 ± 4s NP

-

33 ± 8NP

1600

 

 

15 ± 2s SP

-

29 ± 3SP

5000

 

 

-

-

19 ± 10n SP

1      The S9 -mix contained 10 % (v/v) S9 fraction

MC   Microcolonies

NP    No precipitate

SP    Slight precipitate

a      Bacterial lawn absent

e      Bacterial lawn extremely reduced

m     Bacterial lawn moderately reduced

n      Normal bacterial background lawn

-     Not tested

 

Table 5: Experiment 2A- Mutagenic response in theSalmonella typhimuriumreverse mutation assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicates plates (± S.D.) with different strains ofSalmonella typhimurium

TA1535

TA100

Without S-9 mix

Positive Control

 

749 ± 35

Solvent Control

 

124 ± 22

0.09

 

99 ± 10

0.28

 

106 ± 12

0.85

 

95 ± 3

2.7

 

104 ± 10n

8.5

 

58 ± 22m NP

With S-9 mix1

Positive Control

115 ± 57

1365 ± 110

Solvent Control

11 ± 4

106 ± 8

8.5

5 ± 5

110 ± 7

26

11 ± 1

117 ± 38

82

10 ± 4n

100 ± 12n

256

e MC

56 ± 1m

800

e SP MC

e SP MC

1      The S9 -mix contained 10 % (v/v) S9 fraction

MC   Microcolonies

NP    No precipitate

e      Bacterial lawn extremely reduced

m     Bacterial lawn moderately reduced

n      Normal bacterial background lawn

 

Applicant's summary and conclusion

Conclusions:
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The genotoxicity potential of the test item was established in a bacteria reverse mutation assay in accordance with OECD guideline 471 and GLP.

The objective of this study was to evaluate the test item for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

In the dose range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards in tester strain TA100 and at 5000 µg/plate in tester strain WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, and/or a reduction in the bacterial background lawn was observed in tester strain TA100 at dose levels of 164 µg/plate and above in the absence of S9-mix and at 17 µg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 0.55 to 512 µg/plate in the absence of S9-mix in tester strains TA1535, TA1537 and TA98 and at 0.017 to 17 µg/plate in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98 and TA100. The test item precipitated on the plates at the dose level of 512 μg/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, the presence of microcolonies and/or a decrease in the number of revertants was observed in all three tester strains in the absence of S9-mix. In the presence of S9-mix, no toxicity was observed at any of the dose levels tested.

Since in the first mutation test, only three analysable dose levels were present in the tester strains TA1535 and TA1537 in the absence of S9-mix, and no toxicity or precipitate on the plates was observed up to the top dose of 17 µg/plate in all four tester strains in the presence of S9-mix, an additional mutation assay was performed. In this additional experiment (1A),the test item was tested at a concentration range of 0.055 to 17 µg/plate in the absence of S9-mix and at 5.4 to 512 µg/plate in the presence of 5% (v/v) S9-mix. The test item did not precipitate on the plates up to the top concentration of 512 µg/plate. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation assay, the test item was tested in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitate was observed in some tester strains. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in the tester strains TA1535 and TA100 in the presence of S9-mix.

Since in the second mutation test, only two analysable dose levels were present in tester strain TA100 in the absence of S9-mix, and no toxicity or precipitate on the plates was observed up to the top dose of 164 µg/plate in the tester strains TA1535 and TA100 in the presence of S9-mix, an additional mutation assay was performed. In this additional mutation experiment (2A), the test item was tested at concentration ranges of 0.09 to 8.5 µg/plate and 8.5 to 800 µg/plate in the absence and presence of 10% (v/v) S9-mix, respectively. The test item did not precipitate on the plates up to the top concentration of 800 µg/plate. Toxicity was observed at the top dose of 8.5µg/plate in the absence of S9-mix and at 256 and 800µg/plate in the presence of S9-mix.

The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.