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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:
2018-11-23 till 2018-11-30
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2’-Phenyl-biphenyl-2-yl-(9,9-dimethyl-9H-fluoren-2-yl)-(9,9’-spirobifluoren-2-yl)-amine
EC Number:
949-957-4
Molecular formula:
C58H41N
IUPAC Name:
2’-Phenyl-biphenyl-2-yl-(9,9-dimethyl-9H-fluoren-2-yl)-(9,9’-spirobifluoren-2-yl)-amine
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I & Experiment II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
strain TA 1535 in exp.1 at 5000 µg/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: none
Water solubility: not tested
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in strain TA1535 at the highest tested concentration in the presence of metabolic activation in experiment I.
Remarks on result:
other: reverse mutation assay migrated from the field Test System

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1929000

Study Code: Envigo 1929000

Experiment: 1929000 VV Plate

Date Plated: 23.11.2018

Assay Conditions:

Date Counted: 26.11.2018

Metabolic

Activation

Test

Group

Dose Level

(per plate)

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

12 ± 3

11 ± 1

32 ± 3

115 ± 8

47 ± 8

 

Untreated

 

13 ± 2

13 ± 2

34 ± 10

123 ± 11

45 ± 7

 

test item

3 µg

12 ± 3

8 ± 2

32 ± 5

127 ± 9

34 ± 1

 

10 µg

11 ± 4

13 ± 3

28 ± 9

131 ± 11

40 ± 7

 

33 µg

13 ± 3

13 ± 3

34 ± 8

121 ± 13

40 ± 6

 

 

100 µg

11 ± 4

14 ± 3

39 ± 6

111 ± 20

46 ± 7

 

 

333 µg

11 ± 1P

12 ± 5P

29 ± 2P

114 ± 11P

41 ± 1P

 

 

1000 µg

12 ± 2P M

8 ± 2P M

20 ± 3P M

112 ± 9P M

37 ± 8P

 

 

2500 µg

9 ± 1P M

8 ± 2P M

23 ± 6P M

92 ± 4P M

26 ± 2P M

 

 

5000 µg

9 ± 3P M

8 ± 2P M

26 ± 8P M

86 ± 3P M

23 ± 6P M

 

NaN3

10 µg

1158 ± 62

 

 

1912 ± 57

 

 

4-NOPD

10 µg

 

 

494 ± 18

 

 

 

4-NOPD

50 µg

 

86 ± 20

 

 

 

 

MMS

2.0 µL

 

 

 

 

953 ± 36

 

 

 

 

 

 

 

 

With Activation

DMSO

 

15 ± 3

10 ± 3

38 ± 5

149 ± 17

51 ± 9

 

Untreated

 

17 ± 2

10 ± 2

53 ± 4

144 ± 10

51 ± 8

 

test item

3 µg

12 ± 6

14 ± 2

51 ± 5

140 ± 6

54 ± 7

 

10 µg

13 ± 1

11 ± 1

51 ± 3

143 ± 8

50 ± 10

 

33 µg

13 ± 1

9 ± 3

40 ± 7

138 ± 8

47 ± 8

 

 

100 µg

11 ± 2

12 ± 3

54 ± 8

142 ± 13

53 ± 8

 

 

333 µg

12 ± 1P

9 ± 3P

45 ± 4P

168 ± 14P

42 ± 1P

 

 

1000 µg

8 ± 2P M

11 ± 4P M

35 ± 3P M

154 ± 10P

45 ± 7P

 

 

2500 µg

10 ± 3P M

10 ± 2P M

30 ± 10P M

138 ± 3P M

36 ± 7P M

 

 

5000 µg

5 ± 1P M

9 ± 1P M

24 ± 2P M

122 ± 5P M

29 ± 1P M

 

2-AA

2.5 µg

445 ± 26

226 ± 12

3707 ± 160

4228 ± 130

 

 

2-AA

10.0 µg

 

 

 

 

342 ± 16

 

 

 

 

 

 

 

 

Summary of Experiment II

Study Name: 1929000

Study Code: Envigo 1929000

Experiment: 1929000 HV2 Pre

Date Plated: 27.11.2018

Assay Conditions:

Date Counted: 30.11.2018

Metabolic

Activation

Test

Group

Dose Level

(per plate)

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

13 ± 4

12 ± 2

26 ± 3

128 ± 13

49 ± 9

 

Untreated

 

13 ± 3

9 ± 2

29 ± 6

147 ± 8

54 ± 16

 

Test material

3 µg

12 ± 3

10 ± 2

30 ± 9

118 ± 11

44 ± 3

 

10 µg

14 ± 3

11 ± 5

31 ± 2

131 ± 4

48 ± 2

 

33 µg

10 ± 2

12 ± 3

29 ± 3

125 ± 11

52 ± 8

 

 

100 µg

13 ± 5

8 ± 2

33 ± 3

122 ± 11

46 ± 10

 

 

333 µg

10 ± 1P

8 ± 2P

29 ± 7P

123 ± 13P

47 ± 6P

 

 

1000 µg

10 ± 4P M

10 ± 1P

22 ± 8P

127 ± 2P

51 ± 3P

 

 

2500 µg

8 ± 2P M

12 ± 4P M

22 ± 5P M

103 ± 3P M

40 ± 3P M

 

 

5000 µg

9 ± 3P M

7 ± 2P M

16 ± 4P M

78 ± 14P M

24 ± 4P M

 

NaN3

10 µg

1073 ± 22

 

 

1725 ± 14

 

 

4-NOPD

10 µg

 

 

502 ± 18

 

 

 

4-NOPD

50 µg

 

80 ± 12

 

 

 

 

MMS

2.0 µL

 

 

 

 

799 ± 50

 

 

 

 

 

 

 

 

With Activation

DMSO

 

12 ± 2

11 ± 5

40 ± 7

145 ± 18

50 ± 11

 

Untreated

 

14 ± 4

12 ± 2

52 ± 6

151 ± 14

52 ± 5

 

Test material

3 µg

9 ± 1

12 ± 5

45 ± 3

139 ± 16

54 ± 1

 

10 µg

9 ± 4

10 ± 3

47 ± 14

134 ± 5

55 ± 8

 

33 µg

9 ± 2

10 ± 2

39 ± 4

162 ± 19

55 ± 8

 

 

100 µg

13 ± 2

13 ± 2

41 ± 6

151 ± 20

49 ± 3

 

 

333 µg

11 ± 1P

14 ± 3P

52 ± 10P

138 ± 8P

54 ± 2P

 

 

1000 µg

11 ± 4P M

11 ± 3P M

39 ± 7P

133 ± 9P M

58 ± 5P

 

 

2500 µg

11 ± 4P M

9 ± 2P M

25 ± 3P M

141 ± 7P M

43 ± 1P M

 

 

5000 µg

7 ± 3P M

7 ± 1P M

27 ± 7P M

139 ± 7P M

43 ± 6P M

 

2-AA

2.5 µg

408 ± 55

192 ± 13

4371 ± 397

4082 ± 130

 

 

2-AA

10.0 µg

 

 

 

 

329 ± 9

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test material was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in strain TA1535 at the highest tested concentration in the presence of metabolic activation in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.