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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYI/38593-5/2012
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
rel-(6R,7R,15R,16R,24R,25R,33R,34R,42R,43R,59R,64R,65R,69R,70R,75R)-41,44,46,48,50,52,54,56,58,1,61,63,66,68,71,39,37,35,32,30,28,26,23,21,19,17,14,12,10,8,5,3-dotriacontaazapentacosacyclo[39.18.13.1^(5,17).1^(8,58).1^(14,26).1^(23,35).1^(32,44).1^(56,59).1^(63,66).1^(68,71).0^(7,3).0^(10,6).0^(16,12).0^(19,15).0^(25,21).0^(28,24).0^(34,30).0^(37,33).0^(42,46).0^(43,39).0^(48,70).0^(50,69).0^(52,65).0^(54,64).0^(61,75)]octacontane-45,49,53,57,60,40,36,31,27,22,18,13,9,4,73,74-hexadecone hydrochloride hydrate
EC Number:
700-970-1
Molecular formula:
C48N32H48O16xH2O
IUPAC Name:
rel-(6R,7R,15R,16R,24R,25R,33R,34R,42R,43R,59R,64R,65R,69R,70R,75R)-41,44,46,48,50,52,54,56,58,1,61,63,66,68,71,39,37,35,32,30,28,26,23,21,19,17,14,12,10,8,5,3-dotriacontaazapentacosacyclo[39.18.13.1^(5,17).1^(8,58).1^(14,26).1^(23,35).1^(32,44).1^(56,59).1^(63,66).1^(68,71).0^(7,3).0^(10,6).0^(16,12).0^(19,15).0^(25,21).0^(28,24).0^(34,30).0^(37,33).0^(42,46).0^(43,39).0^(48,70).0^(50,69).0^(52,65).0^(54,64).0^(61,75)]octacontane-45,49,53,57,60,40,36,31,27,22,18,13,9,4,73,74-hexadecone hydrochloride hydrate
Test material form:
solid

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:
cofactor-supplemented post-mitochondrial S9 fraction
Test concentrations with justification for top dose:
Based on the results of the preliminary experiment, the examined test concentrations in the main tests were 2500, 791, 250, 79.1, 25, 7.91, 2.5 and 0.791 μg/plate with and without metabolic activation. At the top dose precipitation and cytotoxicity were observed in all strains.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide, water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine; 2-aminoanthracene

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
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item: CUCURBIT[8]URIL

Strain: Salmonella typhimurium TA98

Cell count (overnight culture): 2.01 x 109CFU/mL

Concentration: (μg/plate)

 

Revertant colony number

Concentration: (μg/plate)

 

Revertant colony number

-S9

+S9

-S9

 

+S9

25000

 

2 SR, SP

2 SR, SP

6 SR, SP

4 R, SP

4 R, SP

8 R, SP

Untreated control

 

19

21

28

 

29

31

35

Mean

SD

MF

3.3

2.31

0.19

5.3

2.31

0.19

Mean

SD

MF

22.7

4.73

1.28

 

31.7

3.06

1.10

791

 

12 SR

9 SR

6 SR

10 R

10 R

10 R

DMSO control

 

16

17

20

 

27

29

30

Mean

SD

MF

9.0

3.00

0.51

10.0

0.00

0.35

Mean

SD

MF

17.7

2.08

1.00

 

28.7

1.53

1.00

250

 

11 SR

13 SR

22SR

31

25

27

Positive control

NPD (4 μg)

 

293

312

302

2AA

(2 μg)

2366

2380

2374

Mean

SD

MF

15.3

5.86

0.87

27.7

3.06

0.97

Mean

SD

MF

302.3

9.50

17.11

 

2373.3

7.02

82.79

79.1

 

18

24

18

37

39

40

SP: Slight precipitate

R: Reduced background lawn

SR: Slightly reduced background lawn

MF: Mutation factor

SD: Standard deviation

+S9: with S9 mix

-S9: without S9 mix

 

                  Mean revertants (test item)

Mutation factor = -------------------------------------

                   Mean revertants (solvent control)

Mean

SD

MF

20.0

3.46

1.13

38.7

1.53

1.35

25

 

23

30

19

34

32

33

Mean

SD

MF

24.0

5.57

1.36

33.0

1.00

1.15

7.91

 

30

27

22

34

35

25

Mean

SD

MF

26.3

4.04

1.49

31.3

5.51

1.09

2.5

 

17

30

18

31

23

36

Mean

SD

MF

21.7

7.23

1.23

30.0

6.56

1.05

0.791

 

21

19

17

21

19

28

Mean

SD

MF

19.0

2.00

1.08

22.7

4.73

0.79

 

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item: CUCURBIT[8]URIL

Strain: Salmonella typhimurium TA100

Cell count (overnight culture): 2.09 x 109CFU/mL

Concentration: (μg/plate)

 

Revertant colony number

Concentration: (μg/plate)

 

Revertant colony number

-S9

+S9

-S9

 

+S9

25000

 

22 SP, SR

37 SP, SR

19 SP, R

26 SP, SR

28 SP, SR

13 SP, SR

Untreated control

 

102

96

95

 

81

102

109

Mean

SD

MF

26.0

9.64

0.27

22.3

8.14

0.24

Mean

SD

MF

97.7

3.79

1.00

 

100.7

9.07

1.09

791

 

63 SR

88 SR

82 SR

97 SR

66 SR

86 SR

DMSO control

 

96

102

95

 

87

97

92

Mean

SD

MF

77.7

13.05

0.80

83.0

15.72

0.90

Mean

SD

MF

97.7

3.79

1.00

 

92.0

5.00

1.00

250

 

84 SR

81 SR

115 SR

106

100

87

Distilled water control*

 

103

97

94

 

92

93

109

Mean

SD

MF

93.3

18.82

0.96

97.7

9.71

1.06

Mean

SD

MF

98.0

4.58

1.00

 

98.0

9.54

1.07

79.1

 

113

85

109

101

67

106

Positive control

SAZ

(2 μg)

 

1196

1212

1184

2AA (2 μg)

2412

2388

2420

Mean

SD

MF

102.3

15.14

1.05

91.3

21.22

0.99

Mean

SD

MF

1197.3

14.05

12.22

2406.7

16.65

26.16

25

 

94

87

93

97

107

95

SP:Slight precipitate

R:Reduced background lawn

SR:Slightly reduced background lawn

MF:Mutation factor

SD:Standard deviation

+S9:with S9 mix

-S9:without S9 mix

 

                        Mean revertants (test item)

Mutation factor = --------------------------------------

                   Mean revertants (solvent control)

 

 

 

 

*Distilled water control was used because of SAZ

Mean

SD

MF

91.3

3.79

0.94

99.7

6.43

1.08

7.91

 

99

98

97

94

109

86

Mean

SD

MF

98.0

1.00

1.00

96.3

11.68

1.05

2.5

 

95

92

93

110

84

108

Mean

SD

MF

93.3

1.53

0.96

100.7

14.47

1.09

0.791

 

108

103

85

66

95

104

Mean

SD

MF

98.7

12.10

1.01

88.3

19.86

0.96

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item: CUCURBIT[8]URIL

Strain: Salmonella typhimurium TA1535

Cell count (overnight culture): 2.67 x 109CFU/mL

Concentration: (μg/plate)

 

Revertant colony number

Concentration: (μg/plate)

 

Revertant colony number

-S9

+S9

-S9

 

+S9

25000

 

15 SP, SR

16 SP, SR

10 SP, SR

5 P, SR

13 P, SR

11 P, SR

Untreated control

 

8

10

11

 

9

14

15

Mean

SD

MF

13.7

3.21

1.46

9.7

4.16

0.88

Mean

SD

MF

9.7

1.53

1.04

 

12.7

3.21

1.15

791

 

13 SR

12 SR

12 SR

11 SR

15 SR

8 SR

DMSO control

 

7

10

11

 

10

11

12

Mean

SD

MF

12.3

0.58

1.32

11.3

3.51

1.03

Mean

SD

MF

9.3

2.08

1.00

 

11.0

1.00

1.00

250

 

14 SR

15 SR

14 SR

10

15

8

Distilled water control*

 

13

14

16

 

11

15

15

Mean

SD

MF

14.3

0.58

1.54

11.0

3.61

1.00

Mean

SD

MF

14.3

1.53

1.54

 

13.7

2.31

1.24

79.1

 

14

19

8

10

12

17

Positive control

SAZ

(2 μg)

 

1204

1176

1184

2AA (2 μg)

267

277

283

Mean

SD

MF

13.7

5.51

1.46

13.0

3.61

1.18

Mean

SD

MF

1188.0

14.42

82.88

275.7

8.08

25.06

25

 

13

13

18

13

10

10

P:Precipitate

SP:Slight precipitate

SR:Slightly reduced background lawn

MF:Mutation factor

SD:Standard deviation

+S9:with S9 mix

-S9:without S9 mix

 

                      Mean revertants (test item)

Mutation factor = --------------------------------------

                  Mean revertants (solvent control)

 

 

 

 

*Distilled water control was used because of SAZ

Mean

SD

MF

14.7

2.89

1.57

11.0

1.73

1.00

7.91

 

13

13

11

15

12

15

Mean

SD

MF

12.3

1.15

1.32

14.0

1.73

1.27

2.5

 

11

10

14

16

19

10

Mean

SD

MF

11.7

2.08

1.25

15.0

4.58

1.36

0.791

 

14

16

15

14

19

15

Mean

SD

MF

15.0

1.00

1.61

13.0

2.65

1.18

Confirmatory Mutation Test (Pre-Incubation Method)

Test Item: CUCURBIT[8]URIL

Strain: Salmonella typhimurium TA1537

Cell count (overnight culture): 2.26 x 109CFU/mL

Concentration: (μg/plate)

 

Revertant colony number

Concentration: (μg/plate)

 

Revertant colony number

-S9

+S9

-S9

 

+S9

25000

 

1 P,R

1 P,R

2 P,R

0 P,R

1 P,R

2 P,R

Untreated control

 

7

8

11

 

3

6

7

Mean

SD

MF

1.3

0.58

0.19

1.0

1.00

0.14

Mean

SD

MF

8.7

2.08

1.24

 

5.3

2.08

0.76

791

 

3 SP,SR

5 SP,SR

6 SP,SR

5 SR

8 SR

11 SR

DMSO control

 

5

7

9

 

6

7

8

Mean

SD

MF

4.7

1.53

0.67

8.0

3.00

1.14

Mean

SD

MF

7.0

2.00

1.00

 

7.0

1.00

1.00

250

 

5 SR

5 SR

8 SR

5

10

17

Positive control

9AA

(50 μg)

 

404

426

408

2AA

(2 μg)

198

204

220

Mean

SD

MF

6.0

1.73

0.86

10.7

6.03

1.52

Mean

SD

MF

412.7

11.72

58.95

 

207.3

11.37

29.62

79.1

 

6

7

9

4

11

12

P:Precipitate

SP:Slight precipitate

R:Reduced background lawn

SR:Slightly reduced background lawn

MF:Mutation factor

SD:Standard deviation

+S9:with S9 mix

-S9:without S9 mix

 

                  Mean revertants (test item)

Mutation factor = ------------------------------------

                  Mean revertants (solvent control)

Mean

SD

MF

7.3

1.53

1.05

9.0

4.36

1.29

25

 

7

11

14

9

11

14

Mean

SD

MF

10.7

3.51

1.52

11.3

2.52

1.62

7.91

 

6

7

11

7

10

12

Mean

SD

MF

8.0

2.65

1.14

9.7

2.52

1.38

2.5

 

4

6

17

10

12

13

Mean

SD

MF

9.0

7.00

1.29

11.7

1.53

1.67

0.791

 

5

8

11

10

10

11

Mean

SD

MF

8.0

3.00

1.14

10.3

0.58

1.48

  

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

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 CUCURBIT[8]URIL had no mutagenic activity in the examined bacterial strains under the test conditions of this study.
Executive summary:

The purpose of this study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimuriumand at the tryptophan locus of Escherichia coli WP2uvrA strain in the presence and absence of activated rat liver S9 fraction. Study was performed to – Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", 21 July 1997 – EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA

712-C-98-247, August 1998 – Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria", 30 May 2008 The method described in the above mentioned guidelines conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

In conclusion 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 CUCURBIT[8]URIL had no mutagenic activity in the examined bacterial strains under the test conditions of this study.