Registration Dossier

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:
2018-10-02 till 2018-10-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
guideline-conform study under GLP without deviations

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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 S9 (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: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
Solvent used:
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: 30 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 resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
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
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: soluble, solvent
Precipitation: No precipitation of the test item occurred up to the highest investigated dose, but an intense color in the overlay agar of the plates incubated with the test item was observed from 2500 to 5000 µg/plate in both experiments.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, In experiment II, the data in solvent control of strain TA 98 with S9 mix were slightly above our historical control range (60 versus 56 colonies). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no detrimental impact on the outcome of the study.
ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1922401

Study Code: Envigo 1922401

Experiment: 1922401 VV Plate

Date Plated: 02.10.2018

Assay Conditions:

Date Counted: 05.10.2018

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

Deionised water

 

11 ± 4

11 ± 4

34 ± 3

114 ± 19

43 ± 5

Activation

Untreated

 

7 ± 3

9 ± 2

35 ± 9

135 ± 9

47 ± 7

 

Reactive

3 µg

12 ± 4

11 ± 3

34 ± 8

114 ± 14

42 ± 7

 

Blue

10 µg

13 ± 3

10 ± 3

29 ± 3

115 ± 3

40 ± 10

 

F07-0195

33 µg

9 ± 3

12 ± 5

35 ± 4

126 ± 7

45 ± 6

 

 

100 µg

12 ± 3

10 ± 3

27 ± 5

128 ± 23

42 ± 7

 

 

333 µg

12 ± 4

11 ± 5

28 ± 9

131 ± 15

44 ± 2

 

 

1000 µg

9 ± 1

7 ± 2

27 ± 6

112 ± 16

35 ± 7

 

 

2500 µg

9 ± 3D M

8 ± 2D M

19 ± 1D M

107 ± 6D M

45 ± 4D M

 

 

5000 µg

8 ± 1D M

8 ± 2D M

16 ± 3D M

98 ± 8D M

52 ± 6D M

 

NaN3

10 µg

1101 ± 46

 

 

1699 ± 102

 

 

4-NOPD

10 µg

 

 

508 ± 14

 

 

 

4-NOPD

50 µg

 

91 ± 4

 

 

 

 

MMS

2.0 µL

 

 

 

 

939 ± 54

 

 

 

 

 

 

 

 

With

Deionised water

 

15 ± 6

11 ± 4

33 ± 3

154 ± 3

53 ± 17

Activation

Untreated

 

10 ± 4

15 ± 4

40 ± 3

150 ± 6

51 ± 3

 

Reactive

3 µg

13 ± 6

16 ± 6

38 ± 6

139 ± 38

54 ± 7

 

Blue

10 µg

16 ± 1

11 ± 2

46 ± 5

151 ± 22

52 ± 5

 

F07-0195

33 µg

15 ± 4

18 ± 2

35 ± 9

144 ± 7

52 ± 3

 

 

100 µg

14 ± 4

17 ± 4

38 ± 5

146 ± 5

54 ± 2

 

 

333 µg

12 ± 5

16 ± 2

37 ± 11

144 ± 9

51 ± 5

 

 

1000 µg

15 ± 3

11 ± 5

31 ± 5

128 ± 12

38 ± 11

 

 

2500 µg

10 ± 3D M

6 ± 2D M

20 ± 2D M

96 ± 6D M

44 ± 3D M

 

 

5000 µg

7 ± 1D M

5 ± 2D M

17 ± 4D M

94 ± 5D M

45 ± 9D M

 

2-AA

2.5 µg

445 ± 25

188 ± 22

3579 ± 273

4001 ± 23

 

 

2-AA

10.0 µg

 

 

 

 

347 ± 16

 

 

 

 

 

 

 

 


Summary of Experiment II

Study Name: 1922401

Study Code: Envigo 1922401

Experiment: 1922401 HV2 Pre

Date Plated: 10.10.2018

Assay Conditions:

Date Counted: 17.10.2018

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

Deionised water

 

12 ± 4

9 ± 1

30 ± 5

118 ± 24

52 ± 9

Activation

Untreated

 

15 ± 5

14 ± 7

33 ± 4

128 ± 7

48 ± 3

 

Reactive

33 µg

15 ± 5

11 ± 1

36 ± 1

113 ± 3

47 ± 10

 

Blue

100 µg

13 ± 2

12 ± 4

43 ± 9

136 ± 10

58 ± 6

 

F07-0195

333 µg

12 ± 3

12 ± 2

30 ± 4

152 ± 18

57 ± 8

 

 

1000 µg

12 ± 3

9 ± 3

28 ± 9

135 ± 13

52 ± 9

 

 

2500 µg

9 ± 2D M

8 ± 3D M

23 ± 5D M

109 ± 11D M

47 ± 7D M

 

 

5000 µg

9 ± 2D M

6 ± 1D M

17 ± 7D M

107 ± 24D M

56 ± 9D M

 

NaN3

10 µg

1229 ± 54

 

 

1810 ± 26

 

 

4-NOPD

10 µg

 

 

571 ± 44

 

 

 

4-NOPD

50 µg

 

87 ± 8

 

 

 

 

MMS

2 µL

 

 

 

 

1054 ± 46

 

 

 

 

 

 

 

 

With

Deionised water

 

16 ± 4

22 ± 3

60 ± 6

156 ± 11

48 ± 8

Activation

Untreated

 

16 ± 4

19 ± 5

47 ± 1

143 ± 15

53 ± 6

 

Reactive

33 µg

19 ± 1

28 ± 5

62 ± 6

156 ± 12

55 ± 5

 

Blue

100 µg

14 ± 3

27 ± 3

62 ± 9

152 ± 19

42 ± 6

 

F07-0195

333 µg

17 ± 5

24 ± 4

46 ± 12

150 ± 6

45 ± 4

 

 

1000 µg

13 ± 3

21 ± 5

41 ± 5

171 ± 6

55 ± 4

 

 

2500 µg

15 ± 2D M

18 ± 1D M

37 ± 6D M

138 ± 15D M

47 ± 7D M

 

 

5000 µg

12 ± 3D M

14 ± 3D M

27 ± 9D M

99 ± 4D M

53 ± 6D M

 

2-AA

2.5 µg

 

 

 

4276 ± 423

 

 

2-AA

2.5 µg

333 ± 11

528 ± 9

 

 

 

 

2-AA

10 µg

 

 

 

 

976 ± 47

 

Congo red

500 µg

 

 

252 ± 51

 

 

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes:              

M: Manuel Count

D:  Densely Colored Plate

Key to positive controls:

NaN3: sodium azide

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

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Congo Red

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:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration, including the controls, was 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:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose, but an intense color in the overlay agar of the plates incubated with the test item was observed from 2500 to 5000 µg/plate in both experiments.

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.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with the test item at any dose 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 and showed a distinct increase of induced revertant colonies.

In experiment II, the data in solvent control of strain TA 98 with S9 mix were slightly above our historical control range (60 versus 56 colonies). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no detrimental impact on the outcome of the study.