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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 October 2017 - 20 November 2017
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 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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
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-bromobenzyl alcohol
EC Number:
212-851-7
EC Name:
4-bromobenzyl alcohol
Cas Number:
873-75-6
Molecular formula:
C7H7BrO
IUPAC Name:
(4-bromophenyl)methanol

Method

Target gene:
his/trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
uvrA
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: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:
Strains TA 1535 and TA 98 (+/- S9 mix): 33; 100; 333; 1000; and 2500; and 5000 µg/plate
Strains TA 1537, TA100 and WP2 uvrA (+/- S9 mix): 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment IIa:
Strains TA 1537 (+S9 mix) and WP2 uvrA (-S9 mix): 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 min
- Exposure duration: 72 h

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 resultsopen allclose all
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:
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:
valid
Positive controls validity:
valid
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:
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:
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:
valid
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 at 5000 µg/plate under all experimental conditions. However, no precipitation of the test item occurred up to the highest investigated dose in the overlay agar on the incubated agar plates in this study.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviation

ADDITIONAL INFORMATION ON CYTOTOXICITY: Bacteriotoxicity indicated by reduced background growth on the plates was observed from about 2500 µg/plate onward depending on the experimental condition. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in almost all strains from about 2500 µg/plate onward.

Any other information on results incl. tables

Table 1: Summary of Experiment I

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

 

11 ± 4

8 ± 1

26 ± 4

155 ± 7

41 ± 5

Activation

Untreated

 

10 ± 4

8 ± 3

31 ± 3

163 ± 7

41 ± 9

 

Test item

3 µg

9 ± 3

9 ± 2

20 ± 3

133 ± 13

45 ± 14

 

10 µg

7 ± 3

8 ± 3

20 ± 3

133 ± 6

40 ± 8

 

33 µg

9 ± 5

11 ± 1

23 ± 4

137 ± 3

47 ± 10

 

 

100 µg

10 ± 1

9 ± 3

31 ± 10

141 ± 14

45 ± 11

 

 

333 µg

8 ± 1

10 ± 1

19 ± 6

142 ± 12

40 ± 7

 

 

1000 µg

12 ± 1

11 ± 2

20 ± 2

137 ± 15

29 ± 2

 

 

2500 µg

10 ± 3

5 ± 1R

15 ± 4R

74 ± 8

22 ± 7

 

 

5000 µg

7 ± 2R

1 ± 1M R

13 ± 2M R

21 ± 2R

11 ± 2

 

NaN3

10 µg

1290 ± 30

 

 

2471 ± 93

 

 

4-NOPD

10 µg

 

 

318 ± 26

 

 

 

4-NOPD

50 µg

 

125 ± 12

 

 

 

 

MMS

2.0 µL

 

 

 

 

1025 ± 28

 

 

 

 

 

 

 

 

With

DMSO

 

12 ± 4

11 ± 4

32 ± 7

94 ± 19

44 ± 8

Activation

Untreated

 

12 ± 3

9 ± 3

34 ± 7

127 ± 14

54 ± 5

 

Art. 841647

3 µg

12 ± 5

11 ± 4

33 ± 3

97 ± 10

52 ± 5

 

(4-bromobenzyl

10 µg

11 ± 5

10 ± 4

30 ± 7

124 ± 11

52 ± 9

 

alcohol)

33 µg

14 ± 5

10 ± 3

33 ± 8

120 ± 4

58 ± 13

 

 

100 µg

9 ± 2

10 ± 1

37 ± 5

125 ± 8

51 ± 1

 

 

333 µg

13 ± 4

15 ± 5

32 ± 10

121 ± 20

43 ± 4

 

 

1000 µg

11 ± 5

13 ± 3

35 ± 6

128 ± 22

36 ± 1

 

 

2500 µg

10 ± 3

9 ± 2R

28 ± 3

44 ± 12R

25 ± 5

 

 

5000 µg

3 ± 2

4 ± 1R M

9 ± 2M R

9 ± 2M R

11 ± 3

 

2-AA

2.5 µg

464 ± 8

187 ± 13

4795 ± 440

3459 ± 236

 

 

2-AA

10.0 µg

 

 

 

 

524 ± 5

 

 

 

 

 

 

 

 

Table 2: Summary of Experiment II

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

 

11 ± 1

11 ± 5

29 ± 11

116 ± 23

27 ± 9

Activation

Untreated

 

12 ± 5

9 ± 4

24 ± 8

212 ± 11

37 ± 4

 

Art. 841647

10 µg

 

10 ± 5

 

134 ± 15

28 ± 2

 

(4-bromobenzyl

33 µg

11 ± 3

8 ± 3

24 ± 7

137 ± 4

28 ± 2

 

alcohol)

100 µg

8 ± 2

9 ± 4

24 ± 4

137 ± 5

25 ± 8

 

 

333 µg

12 ± 2

9 ± 4

26 ± 6

121 ± 6

22 ± 6

 

 

1000 µg

11 ± 1

6 ± 1

23 ± 1

104 ± 9

19 ± 6

 

 

2500 µg

11 ± 5

4 ± 2R

18 ± 3

26 ± 9R

15 ± 4

 

 

5000 µg

7 ± 2R

 

6 ± 3M R

 

 

 

NaN3

10 µg

1051 ± 33

 

 

1676 ± 82

 

 

4-NOPD

10 µg

 

 

366 ± 22

 

 

 

4-NOPD

50 µg

 

177 ± 4

 

 

 

 

MMS

2.0 µL

 

 

 

 

812 ± 120

 

 

 

 

 

 

 

 

With

DMSO

 

10 ± 3

11 ± 1

36 ± 6

104 ± 1

37 ± 4

Activation

Untreated

 

14 ± 2

13 ± 2

41 ± 2

180 ± 23

43 ± 5

 

Test item

10 µg

 

9 ± 4

 

95 ± 12

40 ± 13

 

33 µg

7 ± 3

12 ± 5

37 ± 5

108 ± 6

37 ± 5

 

100 µg

7 ± 4

10 ± 4

34 ± 10

116 ± 9

44 ± 11

 

 

333 µg

6 ± 3

11 ± 1

27 ± 1

106 ± 23

33 ± 11

 

 

1000 µg

7 ± 2

10 ± 1

40 ± 7

58 ± 11R

38 ± 11

 

 

2500 µg

4 ± 1M R

8 ± 3R

6 ± 2

4 ± 1M R

16 ± 4R

 

 

5000 µg

0 ± 1M R

 

1 ± 1M R

 

 

 

2-AA

2.5 µg

374 ± 38

116 ± 7

2296 ± 674

1785 ± 123

 

 

2-AA

10.0 µg

 

 

 

 

421 ± 26

 

 

 

 

 

 

 

 

Table 3: Summary of Experiment IIa

Metabolic Activation

Test Group

Dose Level
(per plate)

TA 1537

Revertant Colony Counts (Mean ± SD)

WP2 uvrA

Revertant Colony Counts (Mean ± SD)

 

 

 

 

 

Without

DMSO

 

 

27 ± 4

Activation

Untreated

 

 

36 ± 6

 

Test item

33 µg

 

36 ± 4

 

100 µg

 

31 ± 11

 

 

333 µg

 

34 ± 2

 

 

1000 µg

 

27 ± 6

 

 

2500 µg

 

17 ± 4

 

 

5000 µg

 

7 ± 1R

 

MMS

2.0 µL

 

911 ± 32

 

 

 

 

 

With

DMSO

 

13 ± 3

 

Activation

Untreated

 

12 ± 3

 

 

Test item

33 µg

11 ± 2

 

 

100 µg

11 ± 2

 

 

 

333 µg

13 ± 2

 

 

 

1000 µg

12 ± 2

 

 

 

2500 µg

4 ± 2

 

 

 

5000 µg

1 ± 1

 

 

2-AA

2.5 µg

123 ± 14

 

 

 

 

 

 

Key to Plate Postfix Codes:              

M: Manuel Count

R:  Reduced Background growth

Key to positive controls:

NaN3: sodium azide

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

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Table 4: Bacteriotoxicity

Strain

Exp.I

without S9 mix

Exp. I
with S9 mix

Exp. II without
S9 mix

Exp. II
with S9 mix

Exp. IIa without
S9 mix

Exp. IIa with
S9 mix

TA 1535

/

5000

/

2500 - 5000

-

-

TA 1537

5000

5000

2500

/

-

2500 - 5000

TA 98

/

5000

5000

2500 - 5000

-

-

TA 100

5000

5000

2500

2500

-

-

WP2 uvrA

5000

5000

/

2500

5000

-

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

- = not performed

Applicant's summary and conclusion

Conclusions:
In an in vitro bacterial reverse mutation assay according to OECD 471 the test item did not induce gene mutations.

Executive summary:

The potential of the test item to induce gene mutations was assessed according to OECD Guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in three independent experiments with and without liver microsomal activation (phenobarbital/beta-naphthoflavone induced rat liver S9 mix). Each concentration and the controls were tested in triplicate. The test item was tested at the concentration 3 - 5000 µg/plate (pre-Experiment/Experiment I), 33 - 5000 µg/plate (Experiment II, Strains TA 1535, TA 98), 10 - 2500 µg/plate (Experiment II, Strains TA 1537, TA 100, E.coli WP2uvrA) and 33 - 5000 µg/plate (Experiment IIa). The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate under all experimental conditions. However, no precipitation of the test item occurred up to the highest investigated dose in the overlay agar on the incubated agar plates in this study. The plates incubated with the test item showed reduced background growth as indication of bacteria toxicity from about 2500 µg/plate onward depending on the experimental condition. 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. Positive, vehicle and negative controls were valid. 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.