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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2019 - 18 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
No. 440/2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene
EC Number:
830-582-9
Cas Number:
1951440-04-2
Molecular formula:
C32H50O2
IUPAC Name:
1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene
Test material form:
liquid: viscous
Specific details on test material used for the study:
Test item information

Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date) (taken from label)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light
Test item handling: No specific handling conditions required

Method

Target gene:
Strain: Histidine mutation: Mutation type:
TA1537 hisC3076 F Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
WP2uvrA - Excision repair system deletion

*: R-factor = plasmid pKM101 (increases error-prone DNA repair).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: rfa : deep rough (defective lipopolysaccharide cellcoat) gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Metabolic activation:
with and without
Metabolic activation system:
- source of S9 : Rat liver microsomal enzymes
- method of preparation of S9 mix: Per 10mL, S9-mix contains 30mg NDP and 15.2 mg glucose-6-phosphate in 5.5ml or 5.0 ml Milli-Q water (first and second experiment respectively), 2 mL 0.5M sodium phosphate buffer pH 7.4, 1 mL 0.08 M MgCl2 solution, and 1 mL 0.33 M KCl solution. The solution was filter (0.22 micrometre)-sterilised.
- concentration or volume of S9 mix and S9 in the final culture medium: First experiment - 9.5 mL S9-mix, 0.5 mL S9-fraction (5% (v/v) S9-fraction); Second experiment - 9.0 mL S9-mix, 1.0 mL S9-fraction (10% (v/v) S9-fraction)
Test concentrations with justification for top dose:
Dose-range finding study: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix.
The following doses for experiment 1 were based on the dose-range finding study results:
Experiment 1: 0, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix.
Experiment 2: 492, 878, 1568, 2800 and 5000 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
Ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 and 4-nitroquinoline N-oxide (4-NQO; Solvent: DMSO)
Remarks:
Without S9 activation system
Untreated negative controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
With S9 activation system. Solvent: DMSO in all cases
Details on test system and experimental conditions:
The Salmonella typhimurium strains are checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
Stock cultures of the five strains were stored in the freezer (-150°C).

Cell Culture.

Preparation of bacterial cultures.
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 testing.

Agar plates.
Agar plates (ø 9 cm) containing 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, Bad Homburg, Germany). 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.

Environmental conditions.
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 31.9 - 39.1°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System.
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 is characterized with the mutagens benzo-(a)-pyrene 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 S9-Mix.
S9-mix was prepared immediately before use and kept refrigerated. 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.

Mutation Assay.

At least five different doses (increasing with approximately half-log steps) of the substance (chosen on the basis of range-finding test) 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 substance was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) 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 successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the substance in ethanol and 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). 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 substance precipitate to interfere with automated colony counting were counted manually. Evidence of substance 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:
Recommended test system in international guidelines (OECD, EC).
Evaluation criteria:
A substance is considered negative (not mutagenic) in the test if:

1) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
2) The negative response should be reproducible in at least one follow-up experiment.

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

1) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
2) In case a follow up 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.

Any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

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:
no cytotoxicity, but tested up to precipitating concentrations
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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, but tested up to precipitating concentrations
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: negative
Remarks:
up to 5000 ug/plate

Any other information on results incl. tables

Dose-Range Finding Test: Mutagenic Response of the substance in the Salmonella typhimurium and in the Escherichia coli Reverse Mutation Assay.

Without S9-mix: Mean number of revertant colonies/3 replicate plates (±S.D.).

Dose

(µg/plate)

TA100

       ± SD

    WP2uvrA

± SD

Positive control

1109

15

1636

155

Solvent control

110

15

56

5

1.7

103

2

48

6

5.4

106

16

56

9

17

118

4

64

4

52

131

11

53

4

164

120

4

75

4

512

99

12

55

10

1600

120

21 NP

60

17 NP

5000

125

4 nSP

72

6 nSP


With S9-mix1: Mean number of revertant colonies/3 replicate plates (±S.D.).

Dose

(µg/plate)

TA100

       ± SD

    WP2uvrA

± SD

Positive control

1941

85

538

11

Solvent control

131

10

62

7

1.7

111

22

67

7

5.4

105

13

64

5

17

109

8

59

6

52

112

18

56

4

164

123

11

63

5

512

114

3

61

10

1600

115

6  NP

63

6  NP

5000

104

27 nSP

75

4  nSP

Experiment 1: Mutagenic Response of the substance in the Salmonella typhimurium Reverse Mutation Assay.

Without S9-mix: Mean number of revertant colonies/3 replicate plates (±S.D.).

Dose

(µg/plate)

TA1535

   ± SD (TA1535)

     TA1537

    

± SD (TA1537)

  TA98

 

± SD  (TA98)

Positive control

892

102

903

78

1131

217

Solvent control

14

3

3

2

11

4

52

13

5

4

3

13

6

164

7

4

5

3

9

3

512

10

3  NP

3

2 NP

12

5 NP

1600

7

3 SP

4

0 SP

9

4 SP

5000

7

0  nSP

3 

2 nSP

10

2 nSP

With S9-mix1 : Mean number of revertant colonies/3 replicate plates (±S.D).

Dose

(µg/plate)

TA1535

  ± SD (TA1535)

       TA1537

    

± SD (TA1537)

  TA98

 

± SD (TA98)

Positive control

335

7

300

24

1165

172

Solvent control

13

3

5

0

14

2

52

10

2

3

3

10

5

164

10

4

1

0

13

4

512

12

3 NP

3

2 NP

18

8 NP

1600

9

3 SP

4

3 SP

15

1 SP

5000

14

2 nSP

3

2 nSP

20

6  nSP

Experiment 2: Mutagenic Response of the substance in the Salmonella typhimurium and in the Escherichia coli Reverse Mutation Assay.

 

Without S9-mix:Mean number of revertant colonies/3 replicate plates (±S.D).

Dose

(µg/plate)

TA1535

   ± SD      (TA1535)

   TA1537

    

± SD (TA1537)

 TA98

 

±SD       (TA98)

TA100

±SD        (TA100)

WP2uvrA

± SD     (WP2uvr)

Positive control

1022

20

1111

60

1290

88

872

98

1937

61

Solvent control

12

2

5

2

17

4

116

11

41

12

492

12

2

6

2

14

4

88

7

45

12

878

10

4

8

4

11

1

96

17

48

11

1568

12

2 NP

5

2 NP

15

4 NP

105

7 NP

40

7 NP

2800

11

6  SP

6

1 SP

12

4 SP

101

7 SP

44

6 SP

5000

7

1 nSP

7

4 nSP

19

2 nSP

103

22 nSP

52

13 nSP

With S9-mix1:Mean number of revertant colonies/3 replicate plates (±S.D).

Dose

(µg/plate)

TA1535

   ± SD      (TA1535)

  TA1537

    

± SD (TA1537)

 TA98

 

± SD        (TA98)

TA100

± SD         (TA100)

WP2uvrA

± SD       (WP2uvrA)

Positive control

312

43

279

33

495

102

1476

240

478

33

Solvent control

10

3

10

5

27

7

79

8

43

12

492

8

2

7

4

26

7

78

9

45

11

878

14

3

9

5

24

6

86

5

49

13

1568

12

3 NP

7

3 NP

26

1 NP

94

9 NP

53

13 NP

2800

16

6  SP

5

2  SP

28

9  SP

81

2  SP

38

1  SP

5000

10

1 nSP

8

7 nSP

28

4 nSP

95

14 nSP

60

5 nSP

1

Plate incorporation assay (5% S9)

NP

No precipitate

SP

Slight Precipitate

n

Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in the presence and absence of metabolic activation.
Executive summary:

The potential of the substance to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium: TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA was evaluated in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was conducted in accordance with OECD TG 471 ‘Bacterial Reverse Mutation Assay’.

Prior to the main study a dose-range finding test was conducted on S. typhimurium TA100 and E.coli WP2urvA strains, which were exposed to the following concentrations of the substance: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (in triplicate). The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The first mutation assay was performed using the following concentrations of the substance 0, 52, 164, 512, 1600 and 5000 µg/plate (in triplicate) in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The second mutation assay was performed using the following concentrations of the substance: 0, 492, 878, 1568, 2800 and 5000 µg/plate (in triplicate) in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in the presence and absence of metabolic activation.  

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