Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 27 - Nov 01, 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:
other: Japanese Guidelines for this test system
Version / remarks:
1993
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis-biphenyl-4-yl-(9,9‘-spirobifluoren-4-yl)-amine
Cas Number:
1450933-43-3
Molecular formula:
C49 H33 N
IUPAC Name:
Bis-biphenyl-4-yl-(9,9‘-spirobifluoren-4-yl)-amine
Test material form:
solid

Method

Target gene:
HIS operon (S. thyphimurium)
TRP operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor (pKM101)
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor (pKM101)
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
trp-, uvrA
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from beta-Naphthoflavone/Phenobarbital-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
The test material concentrations used were selected according to the OECD and Japanese guidelines for this test system. The test material showed best solubility in THF and 5000 µg/plate was chosen as the appropriate maximum concentration due to limited solubility.
1. Series: 1.58, 5.00, 15.8, 50.0, 158, 500, and 1580 µg/plate
2. Series: 1.58, 5.00, 15.8, 50.0, and 158 µg/plate
Vehicle / solvent:
THF
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
other: 4-Nitro-o-phenylendiamine
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with beta-naphthoflavone/phenobarbital was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 20% S9 in the 2nd series.
Rationale for test conditions:
according to Guideline
Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the con-current negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed.

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
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:
not examined
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:
not examined
Positive controls validity:
valid
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:
not examined
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:
not examined
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:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Summary 1st Series



Metabolic Activation

Test
Material

Concentr. [µg/plate]

 

Revertants per plate (Mean ± SD)

 

 

 

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without

activation

THF

 

37 ± 9

118 ± 10

34 ± 3

10 ± 3

36 ± 8

 

Test material

1.58

50 ± 6

116 ± 4

39 ± 5

7 ± 1

40 ± 8

 

 

5.00

41 ± 2

118 ± 10

32 ± 8

10 ± 2

37 ± 8

 

 

15.8

45 ± 10

120 ± 26

34 ± 4

10 ± 3

42 ± 9

 

 

50.0

39 ± 8

114 ± 9

33 ± 7

12 ± 4

40 ± 1

 

 

158

32 ± 10 M E

107 ± 4 M E

28 ± 4 M E

5 ± 3 M E

36 ± 2 M E

 

 

500

28 ± 1 M E

96 ± 12 M E

30 ± 7 M E

6 ± 5 M E

39 ± 2 M E

 

 

1580

37 ± 2 M E

100 ± 7 M E

35 ± 3 M E

5 ± 1 M E

35 ± 3 M E

 

NaN3

2.00

 

1774 ± 100

876 ± 41

 

 

 

NQO

2.00

 

 

 

 

1150 ± 138

 

4-NOPD

60.0

 

 

 

86 ± 5

 

 

4-NOPD

20.0

512 ± 63

 

 

 

 

 

With

activation

THF

 

50 ± 7

132 ± 15

29 ± 6

11 ± 3

37 ± 6

 

Test material

1.58

66 ± 8

139 ± 4

26 ± 4

7 ± 3

38 ± 2

 

 

5.00

51 ± 8

137 ± 5

29 ± 6

7 ± 2

51 ± 5

 

 

15.8

52 ± 6

135 ± 4

28 ± 6

12 ± 2

31 ± 3

 

 

50.0

54 ± 2

140 ± 24

23 ± 7

13 ± 2

46 ± 11

 

 

158

40 ± 8 M E

128 ± 19 M E

16 ± 5 M E

4 ± 1 M E

41 ± 6 M E

 

 

500

43 ± 16 M E

133 ± 13 M E

19 ± 2 M E

8 ± 4 M E

29 ± 3 M E

 

 

1580

37 ± 5 M E

127 ± 12 M E

17 ± 1 M E

5 ± 0 M E

36 ± 5 M E

 

2-AA

2.00

1873 ± 162

3653 ± 446

 

 

 

 

2-AA

10.0

 

 

 

 

319 ± 8

 

2-AA

5.00

 

 

139 ± 31

625 ± 24

 

 

 

Key to Positive controls

Key to Plate Postfix Codes

NQO

4-Nitroquinoline-N-oxide

2-AA

2-Aminoanthracene

4-NOPD

4-Nitro-o-phenylendiamin

NaN3

Sodium azide

E

Precipitation until end of experiment

M

manual count

Summary 2nd Series

Metabolic Activation

Test
Material

Concentr. [µg/plate]

 

Revertants per plate (Mean ± SD)

 

 

 

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without

activation

THF

 

45 ± 13

138 ± 11

31 ± 3

14 ± 3

43 ± 2

 

Test Material

1.58

52 ± 0

126 ± 19

27 ± 7

12 ± 7

42 ± 2

 

 

5.00

41 ± 6

138 ± 18

30 ± 3

15 ± 2

49 ± 4

 

 

15.8

44 ± 7

134 ± 8

25 ± 6

14 ± 6

40 ± 6

 

 

50.0

37 ± 9

129 ± 18

35 ± 8

13 ± 1

43 ± 3

 

 

158

38 ± 9 E

150 ± 11 E

32 ± 6 E

20 ± 4 E

55 ± 7 E

 

NaN3

2.00

 

1880 ± 226

736 ± 23

 

 

 

NQO

2.00

 

 

 

 

1320 ± 201

 

4-NOPD

60.0

 

 

 

111 ± 15

 

 

4-NOPD

20.0

726 ± 19

 

 

 

 

 

With

activation

THF

 

45 ± 11

139 ± 15

26 ± 8

14 ± 5

45 ± 5

 

Test Material

1.58

40 ± 7

135 ± 14

27 ± 2

19 ± 7

40 ± 7

 

 

5.00

55 ± 6

151 ± 19

30 ± 8

19 ± 3

41 ± 14

 

 

15.8

47 ± 12

115 ± 10

27 ± 9

17 ± 3

41 ± 11

 

 

50.0

48 ± 6

123 ± 21

36 ± 1

13 ± 6

47 ± 2

 

 

158

45 ± 4 E

145 ± 21 E

33 ± 6 E

31 ± 5 E

49 ± 10 E

 

2-AA

2.00

537 ± 76

859 ± 221

 

 

 

 

2-AA

10.0

 

 

 

 

269 ± 13

 

2-AA

5.00

 

 

114 ± 23

161 ± 48

 

 

 

Key to Positive controls

Key to Plate Postfix Codes

NQO

4-Nitroquinoline-N-oxide

2-AA

2-Aminoanthracene

4-NOPD

4-Nitro-o-phenylendiamin

NaN3

Sodium azide

E

Precipitation until end of experiment

 

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.