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Diss Factsheets

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:
The experimental phase of this study was performed between 24 December 2008 and 03 February 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted 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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-bis(methoxymethyl)-1,1'-biphenyl
EC Number:
700-008-0
Cas Number:
3753-18-2
Molecular formula:
C16 H18 O2
IUPAC Name:
4,4'-bis(methoxymethyl)-1,1'-biphenyl
Constituent 2
Reference substance name:
4,4-bis(methoxymethyl)-biphenyl
IUPAC Name:
4,4-bis(methoxymethyl)-biphenyl

Method

Target gene:
Histidine operon for Salmonella
Tryptophan operon for Escherichia
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone
Test concentrations with justification for top dose:
Salmonella strains (with and without S9): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
E.coli strain (with and without S9): 50, 150, 500, 1500 and 5000 µg/plate.
Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO - dimethul sulphoxide

- Justification for choice of solvent/vehicle: The test material was soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. The Sponsor indicated that the test material would be insoluble in water, therefore, this solvent was not evaluated.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix

Migrated to IUCLID6: (ENNG)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): not appliacble
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 - 72 hours


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS: triplicate plating


NUMBER OF CELLS EVALUATED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction


OTHER EXAMINATIONS: none
Evaluation criteria:
Acceptance criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.


Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
tested up to the maximum recommended dose level of 5000 µg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
tested up to the maximum recommended dose level of 5000 µg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated in report
- Effects of osmolality: not stated in report
- Evaporation from medium: not stated in report
- Water solubility: insoluble
- Precipitation: no
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:
The test material induced toxicity as weakened bacterial background lawns to TA100 from 1500 µg/plate and was non-toxic to WP2uvrA-. The test material formulation and S9-mix used in this experiment were both shown to be sterile.


COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.



ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the Salmonella strains both with and without metabolic activation at and above 1500 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied slightly between strain type, exposures with and without S9-mix and experiment number. The test material caused no visible reduction in the growth of the bacterial background lawns of Escherichia colistrain WP2uvrA-at any dose level. A precipitate (greasy in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1      Test Results: Range-Finding Test– Without Metabolic Activation

Test Period

From: 20 January 2009

To: 23 January 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

99

96

96

(97)

1.7#

22

25

24

(24)

1.5

32

31

28

(30)

2.1

15

20

16

(17)

2.6

6

5

12

(8)

3.8

-

5

75

100

91

(89)

12.7

25

24

20

(23)

2.6

N/T

11

16

22

(16)

5.5

6

5

10

(7)

2.6

-

15

79

79

108

(89)

16.7

20

19

20

(20)

0.6

N/T

11

21

15

(16)

5.0

8

15

6

(10)

4.7

-

50

87

100

104

(97)

8.9

25

15

22

(21)

5.1

26

34

27

(29)

4.4

12

22

15

(16)

5.1

7

8

8

(8)

0.6

-

150

105

85

102

(97)

10.8

22

21

19

(21)

1.5

22

32

33

(29)

6.1

11

14

28

(18)

9.1

16

12

4

(11)

6.1

-

500

93

85

75

(84)

9.0

22

19

31

(24)

6.2

37

37

29

(34)

4.6

21

22

11

(18)

6.1

5

8

13

(9)

4.0

-

1500

48 P

43 P

33 P

(41)

7.6

1 *P

2 *P

3 *P

(2)

1.0

16 P

28 P

20 P

(21)

6.1

5 P

7 P

8 P

(7)

1.5

3 *P

3 *P

3 *P

(3)

0.0

-

5000

1 *P

2 *P

0 *P

(1)

1.0

0 *P

0 *P

0 *P

(0)

0.0

27 P

21 P

14 P

(21)

6.5

3 *P

3 *P

4 *P

(3)

0.6

2 *P

2 *P

1 *P

(2)

0.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

567

590

588

(582)

12.7

104

163

168

(145)

35.6

153

139

170

(154)

15.5

197

186

168

(184)

14.6

1178

1375

1157

(1237)

120.3

 

 


Table 2            Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From: 20 January 2009

To: 23 January 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

106

95

101

(101)

5.5#

14

8

8

(10)

3.5

36

25

36

(32)

6.4

26

19

19

(21)

4.0

8

6

6

(7)

1.2

+

5

106

94

93

(98)

7.2

12

12

15

(13)

1.7

N/T

29

26

20

(25)

4.6

9

10

8

(9)

1.0

+

15

76

99

91

(89)

11.7

13

13

11

(12)

1.2

N/T

22

25

27

(25)

2.5

8

10

11

(10)

1.5

+

50

100

93

81

(91)

9.6

13

10

9

(11)

2.1

24

33

32

(30)

4.9

30

27

14

(24)

8.5

5

5

3

(4)

1.2

+

150

94

112

75

(94)

18.5

9

11

12

(11)

1.5

24

29

35

(29)

5.5

15

15

20

(17)

2.9

7

3

7

(6)

2.3

+

500

105

98

79

(94)

13.5

14

15

14

(14)

0.6

31

26

31

(29)

2.9

20

24

24

(23)

2.3

6

8

8

(7)

1.2

+

1500

27 P

30 P

34 P

(30)

3.5

3 P

5 P

2 P

(3)

1.5

27 P

27 P

15 P

(23)

6.9

15 P

6 P

4 P

(8)

5.9

2 P

1 P

1 P

(1)

0.6

+

5000

2 *P

0 *P

1 *P

(1)

1.0

0 P

1 P

0 P

(0)

0.6

29 P

29 P

14 P

(24)

8.7

3 *P

6 *P

3 *P

(4)

1.7

0 *P

0 *P

0 *P

(0)

0.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

3037

3133

3206

(3125)

84.8

154

221

209

(195)

35.7

771

775

719

(755)

31.2

213

194

190

(199)

12.3

445

555

488

(496)

55.4

 

 


Table 3          Test Results: Main Test– Without Metabolic Activation

Test Period

From: 31 January 2009

To: 03 February 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

106

101

119

(109)

9.3#

20

20

25

(22)

2.9

22

20

31

(24)

5.9

19

19

22

(20)

1.7

12

12

14

(13)

1.2

-

5

114

90

100

(101)

12.1

14

20

21

(18)

3.8

N/T

18

25

21

(21)

3.5

8

8

13

(10)

2.9

-

15

96

89

102

(96)

6.5

23

34

21

(26)

7.0

N/T

19

18

19

(19)

0.6

12

10

12

(11)

1.2

-

50

103

88

100

(97)

7.9

16

22

19

(19)

3.0

24

30

18

(24)

6.0

22

16

18

(19)

3.1

11

10

13

(11)

1.5

-

150

99

93

93

(95)

3.5

23

21

23

(22)

1.2

31

25

21

(26)

5.0

19

18

23

(20)

2.6

15

11

9

(12)

3.1

-

500

112

100

115

(109)

7.9

24

19

19

(21)

2.9

30

24

25

(26)

3.2

23

15

19

(19)

4.0

9

11

9

(10)

1.2

-

1500

69 *P

69 *P

66 *P

(68)

1.7

5 *P

5 *P

4 *P

(5)

0.6

26 P

25 P

29 P

(27)

2.1

13 P

13 P

9 P

(12)

2.3

5 *P

2 *P

2 *P

(3)

1.7

-

5000

9 *P

5 *P

4 *P

(6)

2.6

0 *P

0 *P

0 *P

(0)

0.0

26 P

25 P

30 P

(27)

2.6

4 *P

3 *P

3 *P

(3)

0.6

1 *P

0 *P

0 *P

(0)

0.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

1183

1514

1387

(1361)

167.0

1680

1967

1918

(1855)

153.5

1864

1830

1710

(1801)

80.9

196

206

228

(210)

16.4

1070

1184

1389

(1214)

161.6

 


Table 4        Test Results: Main Test– With Metabolic Activation

Test Period

From: 31 January 2009

To: 03 February 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

82

95

95

(91)

7.5#

14

15

11

(13)

2.1

26

29

36

(30)

5.1

31

26

25

(27)

3.2

13

13

9

(12)

2.3

+

5

97

95

89

(94)

4.2

15

11

15

(14)

2.3

N/T

27

26

29

(27)

1.5

10

11

9

(10)

1.0

+

15

88

85

95

(89)

5.1

10

16

18

(15)

4.2

N/T

22

23

19

(21)

2.1

11

10

11

(11)

0.6

+

50

97

92

110

(100)

9.3

15

13

11

(13)

2.0

26

30

31

(29)

2.6

23

24

23

(23)

0.6

15

10

10

(12)

2.9

+

150

84

91

91

(89)

4.0

16

11

7

(11)

4.5

29

26

26

(27)

1.7

24

24

20

(23)

2.3

12

13

9

(11)

2.1

+

500

93

84

93

(90)

5.2

10

11

11

(11)

0.6

29

31

31

(30)

1.2

21

23

20

(21)

1.5

8

10

14

(11)

3.1

+

1500

48 *P

47 *P

59 *P

(51)

6.7

4 *P

3 *P

3 *P

(3)

0.6

30 P

27 P

32 P

(30)

2.5

14 *P

12 *P

15 *P

(14)

1.5

4 P

5 P

4 P

(4)

0.6

+

5000

7 *P

3 *P

4 *P

(5)

2.1

0 *P

0 *P

0 *P

(0)

0.0

27 P

32 P

22 P

(27)

5.0

5 *P

3 *P

3 *P

(4)

1.2

0 *P

1 *P

0 *P

(0)

0.6

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

3606

3248

3213

(3356)

217.5

125

140

155

(140)

15.0

617

530

493

(547)

63.7

379

224

245

(283)

84.1

474

361

420

(418)

56.5

BP2AAN/TP*#

 



ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

N/T     Not tested at this dose level

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co‑factors). The dose range was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using the same dose ranges as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the revertant colony frequency of all of the Salmonella strains both with and without metabolic activation at and above 1500 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied slightly between strain type, exposures with and without S9-mix and experiment number. The test material caused no visible reduction in the growth of the bacterial background lawns of Escherichia coli strain WP2uvrA-at any dose level. A precipitate (greasy in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

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