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Toxicological information

Genetic toxicity: in vitro

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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:
2017-09-19 to 2017-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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:
4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid
EC Number:
929-209-3
Cas Number:
1123312-95-7
Molecular formula:
C14 H13 B F2 O2
IUPAC Name:
4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

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 (experiment I) non-induced hamster liver (experiment II)
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
Experiment IIa:
Strain TA 100 without S9 mix: 0.3; 1; 3; 10; 33; 100; and 333 µg/plate

No correction for purity was made.
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
congo red
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 exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding 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
Key result
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:
in all strains used
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in experiment I, from 1000 to 5000 µg/plate in experiment II and at 333 µg/plate in experiment IIa. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and II and from 100 to 333 µg/plate in experiment IIa. The undissolved particles had no influence on the data recording.
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 indication factor of 0.5), occurred in all strains used.

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1862000

Study Code: Envigo 1862000

Experiment: 1862000 VV Plate

Date Plated: 19.09.2017

Assay Conditions:

Date Counted: 22.09.2017

Metabolic

Activation

Test

Group

 

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

DMSO

 

13 ± 4

10 ± 2

22 ± 3

151 ± 7

31 ± 2

Activation

Untreated

 

22 ± 8

10 ± 4

20 ± 4

150 ± 6

31 ± 6

 

Test item

3 µg

12 ± 4

7 ± 1

26 ± 5

132 ± 15

30 ± 8

 

10 µg

13 ± 6

8 ± 2

19 ± 3

135 ± 6

31 ± 6

 

33 µg

11 ± 1

9 ± 3

24 ± 4

107 ± 7

31 ± 7

 

 

100 µg

9 ± 2

8 ± 3

19 ± 6

42 ± 9

41 ± 10

 

 

333 µg

7 ± 2P

8 ± 1P

13 ± 3P M

9 ± 2P M

25 ± 9P

 

 

1000 µg

5 ± 2P M

4 ± 1P M

5 ± 1P M

4 ± 1P M

13 ± 2P M

 

 

2500 µg

4 ± 1P M

3 ± 1P M

3 ± 2P M

2 ± 1P M

13 ± 1P M

 

 

5000 µg

1 ± 1P M

1 ± 1P M

2 ± 1P M

1 ± 1P M

8 ± 2P M

 

NaN3

10 µg

1181 ± 33

 

 

2113 ± 150

 

 

4-NOPD

10 µg

 

 

293 ± 29

 

 

 

4-NOPD

50 µg

 

78 ± 11

 

 

 

 

MMS

2.0 µL

 

 

 

 

994 ± 85

 

 

 

 

 

 

 

 

With

DMSO

 

15 ± 3

13 ± 3

27 ± 6

130 ± 1

38 ± 2

Activation

Untreated

 

9 ± 2

14 ± 4

35 ± 7

126 ± 13

41 ± 10

 

Test item

3 µg

14 ± 3

16 ± 6

28 ± 10

118 ± 10

32 ± 11

 

10 µg

11 ± 5

15 ± 5

29 ± 3

123 ± 17

48 ± 6

 

33 µg

14 ± 4

11 ± 2

31 ± 10

127 ± 6

34 ± 6

 

 

100 µg

11 ± 1

12 ± 6

27 ± 4

132 ± 8

48 ± 12

 

 

333 µg

8 ± 2P

18 ± 3P

23 ± 4P

42 ± 11P

30 ± 7P

 

 

1000 µg

5 ± 1P M

7 ± 1P M

12 ± 3P M

7 ± 2P M

19 ± 4P M

 

 

2500 µg

4 ± 1P M

4 ± 1P M

5 ± 1P M

3 ± 1P M

14 ± 1P M

 

 

5000 µg

2 ± 1P M

2 ± 1P M

3 ± 1P M

2 ± 1P M

12 ± 0P M

 

2-AA

2.5 µg

410 ± 30

166 ± 26

3187 ± 195

4245 ± 136

 

 

2-AA

10.0 µg

 

 

 

 

415 ± 59

 

 

 

 

 

 

 

 


Summary of Experiment II

Study Name: 1862000

Study Code: Envigo 1862000

Experiment: 1862000 HV2 Pre

Date Plated: 04.10.2017

Assay Conditions:

Date Counted: 11.10.2017

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

DMSO

 

9 ± 1

12 ± 3

30 ± 7

155 ± 9

32 ± 5

Activation

Untreated

 

9 ± 3

13 ± 2

32 ± 6

162 ± 1

39 ± 8

 

Test item

3 µg

7 ± 2

15 ± 3

23 ± 5

139 ± 5

36 ± 10

 

10 µg

5 ± 2

15 ± 6

29 ± 5

130 ± 7

34 ± 11

 

33 µg

6 ± 1

12 ± 4

20 ± 3

61 ± 5

32 ± 6

 

 

100 µg

6 ± 1

10 ± 3

12 ± 2

36 ± 11

25 ± 2

 

 

333 µg

6 ± 1P

9 ± 3P M

2 ± 1P M

2 ± 1P M

18 ± 4P

 

 

1000 µg

3 ± 2P M

4 ± 1P M

0 ± 1P M

0 ± 1P M

18 ± 4P

 

 

2500 µg

2 ± 1P M

3 ± 1P M

0 ± 1P M

0 ± 0P M

10 ± 2P M

 

 

5000 µg

 

 

 

 

6 ± 2P M

 

NaN3

10 µg

1213 ± 71

 

 

1961 ± 62

 

 

4-NOPD

10 µg

 

 

314 ± 6

 

 

 

4-NOPD

50 µg

 

89 ± 9

 

 

 

 

MMS

2.0 µL

 

 

 

 

858 ± 90

 

 

 

 

 

 

 

 

With

DMSO

 

14 ± 5

17 ± 3

33 ± 3

140 ± 13

45 ± 7

Activation

Untreated

 

11 ± 5

17 ± 7

40 ± 8

193 ± 35

54 ± 12

 

Test item

3 µg

15 ± 4

16 ± 8

36 ± 10

144 ± 17

47 ± 2

 

10 µg

14 ± 3

20 ± 2

35 ± 7

145 ± 14

39 ± 5

 

33 µg

18 ± 4

14 ± 6

39 ± 4

127 ± 12

44 ± 8

 

 

100 µg

13 ± 4

15 ± 6

41 ± 8

100 ± 9

42 ± 6

 

 

333 µg

10 ± 4P

15 ± 5P

29 ± 8P

8 ± 3P M

26 ± 9P

 

 

1000 µg

9 ± 3P

6 ± 2P M

7 ± 2P M

2 ± 1P M

26 ± 5P

 

 

2500 µg

7 ± 3P M

6 ± 1P M

1 ± 1P M

0 ± 1P M

15 ± 4P M

 

 

5000 µg

 

 

 

 

13 ± 1P M

 

2-AA

2.5 µg

378 ± 11

104 ± 17

4405 ± 188

3595 ± 36

 

 

2-AA

10.0 µg

 

 

 

 

532 ± 25

 

 

 

 

 

 

 

 

Summary of Experiment IIa

Study Name: 1862000

Study Code: Envigo 1862000

Experiment: 1862000 HV2a Pre

Date Plated: 19.10.2017

Assay Conditions:

Date Counted: 23.10.2017

Metabolic

Activation

Test

Group

Dose Level

(per plate)

TA 100

Revertant Colony Counts (Mean ±SD)

 

 

 

 

Without

DMSO

 

145 ± 17

Activation

Untreated

 

192 ± 14

 

Test item

0.3 µg

137 ± 7

 

1 µg

145 ± 12

 

3 µg

152 ± 6

 

 

10 µg

130 ± 9

 

 

33 µg

71 ± 9

 

 

100 µg

1 ± 1P M

 

 

333 µg

0 ± 0P M

 

NaN3

10 µg

2092 ± 34

 

 

 

 

 

 

 

 

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 item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

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

The assay was performed in three independent experiments with and without liver microsomal activation (S9 mix). 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:

Strain WP2uvrA:                              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The remaining strains:                       3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment IIa:

Strain TA 100 without S9 mix:         0.3; 1;3; 10; 33; 100; and 333 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in experiment I, from 1000 to 5000 µg/plate in experiment II and at 333 µg/plate in experiment IIa. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and II and from 100 to 333 µg/plate in experiment IIa. The undissolved particles had no influence on the data recording.

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), were observed at the following concentrations (µg/plate):

Strain

Experiment I without S9 mix

Experiment I with S9 mix

Experiment II without S9 mix

Experiment II with S9 mix

 

Experiment IIa without S9 mix

TA 1535

1000 – 5000

1000 – 5000

1000 – 2500

/

-

TA 1537

1000 – 5000

1000 – 5000

1000 – 2500

1000 – 2500

-

TA 98

1000 – 5000

2500 – 5000

100 – 2500

1000 – 2500

-

TA 100

100 – 5000

333 – 5000

33 – 2500

333 – 2500

100 – 333

WP2uvrA

1000 – 5000

2500 – 5000

2500 – 5000

2500 – 5000

-

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

- = not performed

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.