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:
2017-01-17 till 2017-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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
Details on test material:
UVCB

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: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
Solvent used: deionised water
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 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:
no 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: soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
Remarks on result:
other:
Remarks:
the test item did not induce gene mutations

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1802902

Study Code: Envigo 1802902

Experiment: 1802902 VV Plate

Date Plated: 17.01.2017

Assay Conditions:

Date Counted: 20.01.2017

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

Without

A. deion.

 

13 ± 6

10 ± 3

24 ± 3

135 ± 7

44 ± 3

Activation

Untreated

 

15 ± 4

9 ± 2

28 ± 2

144 ± 9

37 ± 6

 

Test item

3 µg

12 ± 6

9 ± 3

27 ± 2

137 ± 11

35 ± 7

 

10 µg

12 ± 4

9 ± 0

27 ± 6

116 ± 5

34 ± 8

 

33 µg

11 ± 1

9 ± 1

23 ± 7

143 ± 13

39 ± 6

 

 

100 µg

9 ± 5

10 ± 1

23 ± 7

141 ± 23

35 ± 5

 

 

333 µg

10 ± 2

8 ± 2

27 ± 5

132 ± 23

44 ± 3

 

 

1000 µg

13 ± 1

8 ± 2

21 ± 2

115 ± 10D M

42 ± 2

 

 

2500 µg

15 ± 3D M

8 ± 2D M

21 ± 2D M

109 ± 11D M

34 ± 4D M

 

 

5000 µg

7 ± 1D M P

6 ± 1D M P

21 ± 2D M P

102 ± 10D M P

34 ± 4D M P

 

NaN3

10 µg

1346 ± 108

 

 

2133 ± 98

 

 

4-NOPD

10 µg

 

 

424 ± 31

 

 

 

4-NOPD

50 µg

 

80 ± 5

 

 

 

 

MMS

2.0 µL

 

 

 

 

978 ± 38

 

 

 

 

 

 

 

 

With

A. deion.

 

11 ± 6

10 ± 4

32 ± 7

122 ± 12

47 ± 6

Activation

Untreated

 

12 ± 5

11 ± 3

45 ± 4

123 ± 5

49 ± 8

 

Test item

3 µg

12 ± 6

10 ± 1

39 ± 8

122 ± 7

47 ± 5

 

10 µg

11 ± 5

12 ± 3

32 ± 5

119 ± 6

53 ± 5

 

33 µg

11 ± 1

10 ± 3

32 ± 7

117 ± 12

51 ± 5

 

 

100 µg

9 ± 2

13 ± 2

40 ± 5

124 ± 6

53 ± 11

 

 

333 µg

12 ± 2

11 ± 4

45 ± 3

121 ± 9

42 ± 9

 

 

1000 µg

14 ± 6

11 ± 1

42 ± 8

131 ± 6

44 ± 5

 

 

2500 µg

11 ± 1

12 ± 3

37 ± 3

114 ± 14D M

49 ± 9

 

 

5000 µg

13 ± 3D M P

9 ± 1D M P

25 ± 3D M P

113 ± 9D M P

43 ± 7D M P

 

2-AA

2.5 µg

469 ± 21

120 ± 13

4399 ± 760

4016 ± 110

 

 

2-AA

10.0 µg

 

 

 

 

357 ± 48

 

 

 

 

 

 

 

 


Summary of Experiment II

Study Name: 1802902

Study Code: Envigo 1802902

Experiment: 1802902 HV2 Pre

Date Plated: 25.01.2017

Assay Conditions:

Date Counted: 01.02.2017

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

Revertant Colony Counts (Mean ±SD)

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

Without

A. deion.

 

13 ± 4

10 ± 4

29 ± 6

209 ± 1

34 ± 3

Activation

Untreated

 

11 ± 4

10 ± 5

28 ± 4

204 ± 9

39 ± 4

 

Test item

10 µg

12 ± 3

9 ± 4

26 ± 5

214 ± 18

39 ± 4

 

33 µg

15 ± 4

10 ± 0

24 ± 2

192 ± 13

36 ± 3

 

100 µg

16 ± 5

10 ± 3

27 ± 3

178 ± 39

41 ± 7

 

 

333 µg

16 ± 1

10 ± 3

26 ± 5

149 ± 16

33 ± 12

 

 

1000 µg

11 ± 1D

11 ± 4D

23 ± 8D

187 ± 8M D

28 ± 8D

 

 

2500 µg

11 ± 2M D

9 ± 4M D

27 ± 3M D

181 ± 12M D

27 ± 8M D

 

 

5000 µg

11 ± 4P M D

8 ± 2P M D

23 ± 5P M D

175 ± 19P M D

25 ± 1P M D

 

NaN3

10 µg

1355 ± 101

 

 

1873 ± 158

 

 

4-NOPD

10 µg

 

 

525 ± 34

 

 

 

4-NOPD

50 µg

 

99 ± 7

 

 

 

 

MMS

2.0 µL

 

 

 

 

742 ± 32

 

 

 

 

 

 

 

 

With

A. deion.

 

12 ± 5

11 ± 2

33 ± 3

198 ± 25

46 ± 6

Activation

Untreated

 

12 ± 5

9 ± 4

41 ± 11

193 ± 29

41 ± 6

 

Test item

10 µg

14 ± 4

11 ± 1

44 ± 6

206 ± 3

48 ± 6

 

33 µg

10 ± 3

13 ± 3

29 ± 2

198 ± 14

50 ± 3

 

100 µg

11 ± 1

9 ± 4

39 ± 12

182 ± 3

48 ± 9

 

 

333 µg

15 ± 5

10 ± 5

51 ± 9

206 ± 19

48 ± 6

 

 

1000 µg

15 ± 6D

12 ± 2D

35 ± 8D

189 ± 22D

47 ± 8D

 

 

2500 µg

17 ± 7D

10 ± 6D

31 ± 5D

189 ± 7M D

36 ± 10D

 

 

5000 µg

10 ± 3P M D

8 ± 2P M D

24 ± 5P M D

190 ± 24P M D

35 ± 3P M D

 

2-AA

2.5 µg

374 ± 18

137 ± 16

3336 ± 515

3451 ± 343

 

 

2-AA

10.0 µg

 

 

 

 

347 ± 8

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes:              

P: Precipitate

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

Applicant's summary and conclusion

Conclusions:
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) using Salmonella 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:                            10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. 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.

 

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

 

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.