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:
2016
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:
2016

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:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / 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 / experiment IIa
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: best suitable solvent
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
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, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: not soluble
- Precipitation: The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 µg/plate with and without S9 mix, in experiment II from 333 to 5000 µg/plate in the presence S9 mix and from 1000 to 5000 µg/plate in the absence of S9 mix, and in experiment IIa from 333 to 5000 µg/plate in the absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed I from 333 to 5000 µg/plate with and without S9 mix in experiment, from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix in experiment II, and from 333 to 5000 µg/plate without S9 mix in experiment IIa. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: The number of colonies did not quite reach the lower limit of our historical control data In experiment I in the negative control of strain WP2 uvrA with S9 mix and the data in the solvent control of strain TA 100 with S9 mix were slightly above our historical control range in experiment II. Since these deviations are rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.
ADDITIONAL INFORMATION ON CYTOTOXICITY: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 Experiment II Experiment IIa
without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix
TA 1535 / / 5000 / n.p
TA 1537 / 5000 2500 – 5000 / n.p.
TA 98 5000 5000 5000 5000 2500 - 5000.
TA 100 5000 5000 333 – 5000 333 – 5000 n.p
WP2 uvrA 5000 2500 – 5000 5000 / n.p
/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)
n.p. = not performed
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1743704

Study Code: Envigo 1743704

Experiment: 1743704 VV Plate

Date Plated: 15/01/2016

Assay Conditions:

Date Counted: 18/01/2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

13 ± 5

9 ± 4

24 ± 2

193 ± 8

47 ± 4

Untreated

 

 

10 ± 5

13 ± 1

36 ± 5

204 ± 4

50 ± 2

Savinyl-Gelb

3 µg

 

13 ± 6

9 ± 3

30 ± 8

222 ± 13

44 ± 14

2GLS 01

10 µg

 

13 ± 4

12 ± 4

22 ± 2

217 ± 9

41 ± 7

trocken

33 µg

 

15 ± 2

12 ± 3

28 ± 9

223 ± 2

44 ± 1

 

100 µg

 

12 ± 0

11 ± 3

30 ± 6

226 ± 4

41 ± 4

 

333 µg

 

14 ± 3P

13 ± 4P

30 ± 7P

195 ± 13P

42 ± 3P

 

1000 µg

 

12 ± 3P

16 ± 3P

34 ± 6P

183 ± 27P

33 ± 3P

 

2500 µg

 

13 ± 5P

12 ± 3P M

31 ± 3P

188 ± 21P

27 ± 4P

 

5000 µg

 

12 ± 3P M

4 ± 2P M

5 ± 2P M

28 ± 8P

8 ± 2P M

NaN3

10 µg

 

1145 ± 83

 

 

2013 ± 9

 

4-NOPD

10 µg

 

 

 

316 ± 20

 

 

4-NOPD

50 µg

 

 

79 ± 7

 

 

 

MMS

2.0 µL

 

 

 

 

 

1021 ± 114

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

12 ± 0

11 ± 1

25 ± 6

206 ± 10

52 ± 7

Untreated

 

 

20 ± 6

9 ± 2

30 ± 4

207 ± 14

64 ± 7

Savinyl-Gelb

3 µg

 

14 ± 5

11 ± 4

24 ± 3

218 ± 21

47 ± 12

2GLS 01

10 µg

 

12 ± 6

9 ± 4

36 ± 2

223 ± 18

51 ± 15

trocken

33 µg

 

14 ± 0

9 ± 5

32 ± 5

226 ± 18

64 ± 8

 

100 µg

 

13 ± 4

11 ± 2

28 ± 7

221 ± 7

50 ± 4

 

333 µg

 

11 ± 2P

13 ± 4P

25 ± 4P

207 ± 13P

44 ± 8P

 

1000 µg

 

7 ± 2P

14 ± 1P

31 ± 4P

220 ± 17P

44 ± 4P

 

2500 µg

 

7 ± 2P

8 ± 1P M

12 ± 3P M

174 ± 28P

18 ± 4P

 

5000 µg

 

6 ± 2P M

4 ± 1P M

2 ± 2P M

87 ± 13P

9 ± 2P M

2-AA

2.5 µg

 

516 ± 21

133 ± 35

3246 ± 818

5206 ± 183

 

2-AA

10.0 µg

 

 

 

 

 

332 ± 8

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

 

 

Table2            Summary of Experiment II

Study Name: 1743704

Study Code: Envigo 1743704

Experiment: 1743704 HV2 Pre

Date Plated: 28/01/2016

Assay Conditions:

Date Counted: 03/02/2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

9 ± 3

11 ± 2

19 ± 8

158 ± 5

33 ± 5

Untreated

 

 

7 ± 3

7 ± 1

23 ± 6

200 ± 3

37 ± 6

Savinyl-Gelb

3 µg

 

8 ± 2

8 ± 1

20 ± 6

138 ± 8

33 ± 3

2GLS 01

10 µg

 

11 ± 2

8 ± 2

25 ± 3

141 ± 10

30 ± 6

trocken

33 µg

 

8 ± 3

8 ± 5

20 ± 1

145 ± 4

30 ± 3

 

100 µg

 

11 ± 2

12 ± 3

25 ± 5

132 ± 8

36 ± 12

 

333 µg

 

12 ± 3

8 ± 1

33 ± 7

70 ± 8

26 ± 3

 

1000 µg

 

9 ± 3P

11 ± 2P

41 ± 7P

63 ± 12P

27 ± 6P

 

2500 µg

 

9 ± 3P

5 ± 1P M

18 ± 4P M

10 ± 3P

16 ± 4P

 

5000 µg

 

1 ± 1P M

2 ± 1P

1 ± 1P M

2 ± 1P

13 ± 1P

NaN3

10 µg

 

1046 ± 92

 

 

1697 ± 99

 

4-NOPD

10 µg

 

 

 

362 ± 15

 

 

4-NOPD

50 µg

 

 

102 ± 14

 

 

 

MMS

2.0 µL

 

 

 

 

 

640 ± 14

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

12 ± 4

11 ± 1

28 ± 8

133 ± 7

40 ± 4

Untreated

 

 

10 ± 6

11 ± 1

31 ± 4

174 ± 8

37 ± 2

Savinyl-Gelb

3 µg

 

10 ± 2

14 ± 2

24 ± 4

119 ± 8

45 ± 2

2GLS 01

10 µg

 

12 ± 3

15 ± 1

30 ± 10

126 ± 6

40 ± 6

trocken

33 µg

 

7 ± 2

10 ± 4

32 ± 6

128 ± 5

47 ± 10

 

100 µg

 

9 ± 2

11 ± 4

33 ± 7

120 ± 7

40 ± 8

 

333 µg

 

8 ± 2P

11 ± 4P

34 ± 7P

41 ± 6P

35 ± 11P

 

1000 µg

 

10 ± 3P

12 ± 1P

38 ± 10P

24 ± 5P

34 ± 5P

 

2500 µg

 

6 ± 1P

11 ± 2P

38 ± 3P

9 ± 3P

35 ± 12P

 

5000 µg

 

12 ± 4P

12 ± 3P

4 ± 2P M

1 ± 1P M

27 ± 6P

2-AA

2.5 µg

 

383 ± 46

169 ± 12

4199 ± 453

3167 ± 228

 

2-AA

10.0 µg

 

 

 

 

 

351 ± 11

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

 

Table3            Summary of Experiment IIa

Study Name: 1743704

Study Code: Envigo 1743704

Experiment: 1743704 HV2a Pre

Date Plated: 10/02/2016

Assay Conditions:

Date Counted: 16/02/2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

TA 98

 

 

 

 

 

Without Activation

DMSO

 

 

22 ± 2

Untreated

 

 

31 ± 4

Savinyl-Gelb

3 µg

 

25 ± 3

2GLS 01

10 µg

 

31 ± 10

trocken

33 µg

 

31 ± 5

 

100 µg

 

26 ± 8

 

333 µg

 

29 ± 2P

 

1000 µg

 

35 ± 5P

 

2500 µg

 

5 ± 2P M

 

5000 µg

 

0 ± 1P

4-NOPD

10 µg

 

447 ± 4

 

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

4-NOPD

4-nitro-o-phenylene-diamine

P

M

Precipitate

Manual count

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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.
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) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA. Since a minor increase in revertant colony numbers was observed in experiment II in strain TA 98 without S9 mix, this part was repeated (reported as experiment IIa).

 

The assay was performed in three independent experiments with and without liver microsomal activation. 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:                                3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment IIa with TA98             3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 µg/plate with and without S9 mix, in experiment II from 333 to 5000 µg/plate in the presence S9 mix and from 1000 to 5000 µg/plate in the absence of S9 mix, and in experiment IIa from 333 to 5000 µg/plate in the absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed I from 333 to 5000 µg/plate with and without S9 mix in experiment, from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix in experiment II, and from 333 to 5000 µg/plate without S9 mix 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

Experiment II

Experiment IIa

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

without S9 mix

TA 1535

/

/

5000

/

n.p

TA 1537

/

5000

2500 – 5000

/

n.p.

TA 98

5000

5000

5000

5000

2500 - 5000.

TA 100

5000

5000

333 – 5000

333 – 5000

n.p

WP2uvrA

5000

2500 – 5000

5000

/

n.p

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

n.p. = not performed

No relevant increase of the number of revertant colonies occurred in any of the strains up to the maximal concentration tested, neither in the presence nor in the absence of metabolic activation. In experiment II, an isolated increase (2.1) in revertant colony numbers slightly exceeding the threshold of twice the number of the corresponding solvent control, was observed in strain TA 98 in the absence of metabolic activation. This borderline effect was considered as biologically irrelevant since it was not reproduced in Experiment IIa.

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

 

The number of colonies did not quite reach the lower limit of our historical control data In experiment I in the negative control of strain WP2uvrAwith S9 mix and the data in the solvent control of strain TA 100 with S9 mix were slightly above our historical control range in experiment II. Since these deviations are rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.